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POLITECNICO DI MILANO
Department of Physics
Doctoral Programme in Physics
Mechanisms of cellular photostimulation in hybrid
interfaces based on organic semiconductors
Supervisor: Dr. Maria Rosa ANTOGNAZZA
Tutor: Prof. Guglielmo LANZANI
PhD Programme Coordinator: Prof. Paola TARONI
Doctoral Dissertation of:
Nicola MARTINO
2015 – PhD Cycle XXVI
i
Abstract
Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in
vitro and in vivo applications, bearing a huge potential, from basic researches to clinical
applications. In particular, light sensitive conjugated polymers can be exploited as a new approach
for optical modulation of cellular activity. This thesis is focused on the study of the functioning
mechanisms of these interfaces, both from the physical point of view and from their ability to
stimulate biological cells. In particular, we are interested in understanding how photoexcitation of
the active material in the device is able to modulate the membrane potential of cells. First, we
review the current strategies used for measuring and controlling bioelectrical activity, with a
particular attention paid to optical techniques, and we introduce the biophysical mechanisms behind
the instauration of a potential across the plasma membrane of cells. We present a thorough
experimental characterization of the hybrid polymer/electrolyte interfaces, in which their
spectroscopic, electrical and thermal properties are investigated, delineating the main phenomena
that occur at the device surface upon illumination. The possibility of growing HEK-293 cells on
these hybrid interfaces is the investigated, and we study the different effects that the device
photoexcitation has on the cell membrane potential via patch-clamp analysis. We conclude by
wrapping up the results in the context of existing techniques for cell stimulation and by pointing out
to future developments, towards the creation of a multi-functional platform for light-controlled cell
manipulation, with possible applications in different fields of neuroscience and medicine.
ii
Table of contents
Chapter 1 - Bioelectricity ............................................................................................................... 1
1.1 Electrical stimulation and recording ....................................................................... 1
1.2 Optical techniques .................................................................................................. 4
1.2.1 Optical measurement of bioelectric activity ............................................................. 4
1.2.2 Direct optical stimulation ......................................................................................... 5
1.2.3 Molecular-based stimulation .................................................................................... 7
1.2.4 Nano-/micro-particle stimulation .............................................................................. 8
1.2.5 Device-based stimulation .......................................................................................... 9
1.3 Organic semiconductors for biological applications ............................................... 9
1.4 Photoactive bio-polymer interfaces ...................................................................... 11
1.4.1 The hybrid solid-liquid organic photovoltaic cell ................................................... 12
1.4.2 Poly(3-hexylthiophene) .......................................................................................... 13
1.4.3 Photostimulation of primary cells ........................................................................... 14
1.4.4 Ex-vivo experiments on blind retinas ...................................................................... 15
Chapter 2 – The plasma membrane ............................................................................................ 16
2.1 The structure of the plasma membrane ................................................................. 17
2.2 Ion channels ......................................................................................................... 19
2.2.1 Ion channel structure and selectivity ...................................................................... 20
2.2.2 Gating mechanisms ................................................................................................. 21
2.2.3 Channel conductance and temperature dependence ............................................... 22
2.3 The membrane potential ....................................................................................... 23
2.3.1 Electrochemical equilibrium in biological membranes .......................................... 24
2.3.2 Action potentials in excitable cells ......................................................................... 26
2.3.3 Maintenance of ion concentrations ......................................................................... 27
2.3.4 Electrical equivalent of a cell membrane ................................................................ 28
Chapter 3 – Hybrid interfaces characterization ........................................................................ 32
3.1 Standard organic photovoltaic devices .................................................................. 32
3.2 Hybrid devices structure ....................................................................................... 34
iii
3.3 Spectroscopic characterization ............................................................................. 35
3.3.1 Absorption and fluorescence .................................................................................. 35
3.3.2 Pump and probe spectroscopies .............................................................................. 36
3.3.3 Femtosecond transient absorption spectroscopy ..................................................... 37
3.3.4 Nanosecond transient absorption spectroscopy ...................................................... 40
3.3.5 CW photoinduced absorption spectroscopy ........................................................... 41
3.4 Electrical characterization .................................................................................... 42
3.4.1 Photovoltage measurements ................................................................................... 43
3.4.2 Photocurrent measurements .................................................................................... 48
3.4.3 Surface potential measurements ............................................................................. 50
3.4.4 Measurements on P3HT:PCBM ............................................................................. 52
3.5 Thermal characterization ...................................................................................... 53
3.5.1 Local temperature measurements ........................................................................... 53
3.5.2 Numerical simulations ............................................................................................ 56
Chapter 4 – Coupling hybrid interfaces with cells .................................................................... 59
4.1 Human Embryonic Kidney (HEK) 293 cells ......................................................... 59
4.1.1 Cultures of HEK-293 cells on polymeric substrates ............................................... 61
4.1.2 Basic electrophysiology of HEK-293 cells ............................................................. 62
4.2 Measurements on different substrates ................................................................... 65
4.3 Analysis of thermal effects ................................................................................... 67
4.3.1 Transient depolarization ......................................................................................... 68
4.3.2 Gradual hyperpolarization ...................................................................................... 70
4.3.3 Time evolution of membrane properties ................................................................. 73
4.3.4 Numerical modeling ............................................................................................... 77
4.4 Considerations on capacitive charging .................................................................. 80
Chapter 5 – Discussion and perspectives .................................................................................... 87
5.1 Discussion ............................................................................................................ 87
5.1.1 Capacitive stimulation ............................................................................................ 88
5.1.2 Thermal stimulation ................................................................................................ 90
5.1.3 Comparison with previous works ........................................................................... 91
5.2 Perspectives ......................................................................................................... 93
Appendix A .................................................................................................................................... 97
A.1 Optical Measurements .............................................................................................. 97
iv
A.1.1 Femtosecond spectroscopy ........................................................................................... 97
A.1.2 Nanosecond spectroscopy ............................................................................................. 98
A.1.3 CW Photoinduced Absorption ...................................................................................... 99
A.2 Electrical and thermal characterization ................................................................... 100
A.2.1 Photovoltage measurements ....................................................................................... 100
A.2.2 Photocurrent measurements ........................................................................................ 101
A.2.3 Surface potential and temperature measurements ....................................................... 101
A.3 Electrophysiology measurements ............................................................................ 103
A.3.1 Electrophysiology setup .............................................................................................. 103
A.3.2 Electrolytic solutions and cell growth medium .......................................................... 104
Appendix B .................................................................................................................................. 106
B.1 Astrocyte cultures and electrophysiological properties ............................................ 107
B.2 Photostimulation of astrocytes membrane conductances ......................................... 109
B.3 Experimental methods ............................................................................................. 112
Bibliography ................................................................................................................................ 115
Acknowledgements ..................................................................................................................... 130
List of publications ..................................................................................................................... 133
1
Chapter 1 - Bioelectricity
Bioelectricity, i.e. the arising of electrical potentials and currents in living systems, is a fundamental
process at the basis of many biological functions.1 Central in the formation of such electrical
phenomena are the biomembranes2 that enclose the different compartments of cells.
Electrochemical gradients arise across these selectively permeable membranes due to asymmetric
ion distributions,3 leading to the instauration of potential differences usually in the range from few
to hundreds of millivolts. The prototypical example of a bioelectrical phenomenon is the action
potential,4 i.e. the rapid variation of the plasma membrane potential that is able to rapidly propagate
along neurons transporting information in the nervous system. The same mechanism is also
responsible for muscular contraction in myocytes5,6
and release of hormones in endocrine cells.7
However, transmembrane potentials are present in non-excitable cells too, and are for example
involved in regulating the diffusion of ions and metabolites inside cells and organelles,8 in driving
the production of ATP in mitochondria9 and in controlling the fertilization process of oocytes.
10
Moreover, electric fields and associated currents are present also at the tissue scale and it has been
demonstrated that they play a pivotal role for example in the process of wound healing and in
establishing the left-right organ asymmetry during embryonic development.11
Understanding the
origin of bioelectrical phenomena and the ability of monitoring and controlling them represent thus
a fundamental aspect of biological sciences, with immense implications in medicine.12,13
In this chapter, the main techniques currently available for modulating and recording electrical
signals in biological systems will be presented; in particular, after a first introduction on standard,
purely electrical methods (Section 1.1), different strategies based on optical stimulation and
recording will be discussed (Section 1.2). In Section 1.3 the class of organic semiconductors and
their application in bioelectronics will be presented. Finally, in Section 1.4 photoactive bio-polymer
interfaces, which are the main topic of this thesis, will be introduced.
1.1 Electrical stimulation and recording
The study and exploitation of bioelectricity actually dates back to ancient times and has gone in
parallel with the discovery of electricity by mankind. Historical records show that in ancient Egypt
and Greece discharges from electrical catfishes and eels were used as treatments for pain relief and
2
to improve blood circulation.11
However, the foundation of bioelectricity as a science is usually
dated to the pioneering experiments of the Italian physicist Luigi Galvani in the late XVIII century.
Around 1780 he began to conduct a series of experiments to prove that electric discharges from
different sources applied to preparations of frog legs were able to induce muscle contractions.14
His
observations were collected in the essay “De Viribus Electricitatis in Motu Musculari
Commentarius” (Commentary on the Effect of Electricity on Muscular Motion) published in 1791.15
To describe these phenomena he coined the term “animal electricity”, indicating a form of energy,
similar but different from natural electricity, that was generated by the tissues themselves. Galvani’s
work was then carried on and clarified by other scientists of that period, including Alessandro
Volta, Galvani’s cousin Giovanni Aldini and the German naturalist Alexander von Humboldt.14
Fifty years later, the German scientists Emil Heinrich Du Bois-Reymond and Hermann von
Helmholtz were able for the first time to record with a galvanometer action potentials (which they
actually called “action currents”) in frog nerves and to measure their propagation velocity. Du Bois-
Reymond’s book “Untersuchungen über thierische Elektricität” (Researches on Animal Electricity)
of 184816
is actually considered the beginning of scientific electrophysiology. In the following
decades the nature and properties of the nervous signals were investigated by many scientists,
culminating in the work of Alan Hodgkin and Andrew Huxley,17,18
who in 1952 published their
theory on the propagation of action potentials, one of the earliest and most famous models in
computational biochemistry.
The next big advancement in electrophysiology came in the late ‘70s with the invention of the
patch-clamp technique by Erwin Neher and Bert Sakmann.19–21
Recordings of electrical activity in
cells and tissues can be performed extracellularly, by placing the electrodes in proximity of one or
more cells, or intracellularly by accessing the cytoplasm to record the internal potential.
Intracellular recordings have the great advantage of giving information on the actual variations of
the membrane potential and the currents flowing through the cell membrane, thus allowing a deeper
understanding of the biophysical properties of the cell behavior. Before the introduction of the
patch-clamp technique, however, intracellular recordings were performed by impaling the cell with
metal electrodes or thin glass pipettes filled with an electrolyte solution;11
in order not to stress
excessively the cell, such a pipette needed to be very small (with sub-micrometer tip dimension)
and thus possessed a very high electrical impedance, making recording of small currents very noisy.
In a patch-clamp experiment, instead, a bigger pipette is used (usually with 1-2 μm tips) and it is not
inserted into the cell, but just put in contact with the cell membrane; after the formation of the so-
called gigaseal, the interior of the cell becomes electrically accessible, with much smaller
impedance with respect to intracellular electrodes.21
With this technique Neher and Sakmann were
able to isolate and record the conductances of single ion channels in the cell membrane for the first
3
time, opening the way to the study of their fundamental influence on cell physiology and
pathophysiology.22,23
Patch-clamp rapidly became the gold standard in electrophysiology and allowed to decipher the
mechanisms by which neurons compute information and communicate between each other.
However, it has some intrinsic limitations, especially the possibility of measuring just one (or at
best very few) cells at a time and the short time the patched cell remains viable (usually less than
one hour). Thus, while it is an invaluable tool to study the functioning of single cells, this technique
is not suited for the investigation of the complex interactions between cells in large ensembles like
neural circuits, whose understanding is regarded as one of the major challenge of modern science.24–
26 In contrast, extracellular recording and stimulation techniques allow accessing larger populations
of cells and for longer times (even months), since they usually do not damage the cell membrane,
enabling to investigate the long-term properties of plasticity and learning in neural circuits.
Extracellular recordings can be performed with single electrodes (insulated metal electrodes or glass
pipettes) that record the activity of the cells in their proximity or with multi-electrode systems that
allow adding spatial resolution to the neural activity investigation.27,28
For in vitro experiments,
Multi Electrode Arrays (MEAs) exceeding 10000 electrodes are currently available;27–29
in vivo,
polytrodes30
with more than 100 electrodes have been developed. The main limitation in these
systems is the increasing impedance for smaller electrodes, which degrades the signal-to-noise
ration in recording and can lead to excessive heating in stimulation. Another strategy used for
extracellular recording is based on the field effect transistor (FET) architecture;31
in this case, the
cell extracellular potential basically acts as the gate signal of the transistor, modulating the current
flowing from source to drain. Both for electrodes and for FETs, the adhesion of cells to the active
surface and the electrical properties of the thin cleft between the basal membrane of the cell and the
device interface are fundamental in determining an efficient electrical coupling.32,33
In any case, all extracellular recording methods do not have access to the actual membrane potential
of the cell, but measure the so-called “(local) field potential”, in which the different electrical
signals related to neural (and glial) activity (action potentials, synaptic currents, calcium waves, …)
are superimposed in a complex spatiotemporal-dependent manner.34
Thus, retrieving relevant
information from extracellular recordings usually requires a great computational power and a
detailed previous knowledge of the system under study. To overcome the different limitations of
these techniques, considerable effort is now being undertaken in the development of devices, both
for multi-electrode and for transistor architectures, with capabilities of intracellular recording. These
systems usually consist in arrays of nanostructured surfaces which are engulfed or even partly
internalized by the basal cell membrane, allowing a very good electrical coupling between the
device and the intracellular compartment of the cell.26,27,35,36
This approach offers the advantages of
4
both intracellular and extracellular techniques, being able to spatially address multiple
stimulating/recording sites and, at the same time, offering sensitivities comparable to intracellular
recordings.
1.2 Optical techniques
The obvious advantage of using electrodes to measure and/or excite biological tissue is that they
deal with signal of the same nature of bioelectrical phenomena, namely electrical current and
potentials. However, they need physical contact with the cell or tissue and their geometry is fixed
by design, so it cannot be adapted in real-time to the actual morphology of the system under
investigation. Moreover, it is usually difficult to obtain inhibition of neural activity instead of
excitation with purely electrical stimulation methods. Tools complementary to electrical means
have thus been developed in order to address these and other limitations; optical techniques have in
particular attracted a lot of interest.37
Since light can, to a certain extent and depending on the
wavelength, propagate through different biological tissues, no physical contact is required, thus
decreasing the risk of mechanical stress and damage. Moreover, light can be readily shaped in
desired pattern that can be adjusted to address specific regions of the field under study, with spatial
resolutions on the subcellular scale and a flexibility that pre-fabricated electrodes cannot achieve.
However, to modulate and monitor bioelectrical signal with light, some kind of transduction
mechanism is required. One of the main drawbacks is that quantitative analysis is not
straightforward and careful calibrations need to be performed. Also, at the moment there is no
single optical technique that is able to provide at the same time both stimulation and recording
capabilities. For stimulation, many architectures have been developed, which can be classified
based on the nature of the transducer: intrinsic absorbers, molecular probes, nano/micro-particles,
solid-state devices. As for recordings, the great majority of the research has focused on fluorescent
probes, even if few different approaches have been proposed.38,39
1.2.1 Optical measurement of bioelectric activity
Fluorescence is a ubiquitous tool in life science research and a wide variety of fluorescent probes
are nowadays available to detect virtually every molecule or investigate many biological processes.
To optically detect and record membrane potentials, the main strategy is to use the so-called voltage
sensitive dyes (VSDs).40,41
Standard VSDs are fluorescent molecules that get incorporated into the
cell membrane and have a fluorescence yield modulated by the external electric field; variation in
the membrane potential are thus reflected in a modulation of their fluorescence intensities. The main
problem of standard VSDs is that they generally stain unspecifically every membrane in the cell,
5
reducing the effect of variations in the plasma membrane potential on the total modulation of
fluorescence. A very promising approach is thus the development of genetically encoded voltage
indicators (GEVIs)42,43
gene that can be genetically targeted to subpopulations of cells and to
subcompartments of the cell itself (like the cell membrane); these systems are usually composed of
a fluorescent protein and a voltage sensitive element able to modulate its emission. Both VSDs and
GEVIs allow the simultaneous recording of electrical activity in large populations of cells, both in-
vitro and in-vivo, with the possibility of having subcellular resolution, in a manner that is not
achievable with electrical recordings. However, they still face some issues with respect to the
sensitivity they can reach, especially when single-shot measurements need to be taken without
averaging (for example, when investigating spontaneous activity).37
Cell electrical activity can be investigated with fluorescence-based methods also by detecting
indirectly its effects. As one of the major signaling molecules, calcium ions have long been used to
assess cell activity.41,44
Calcium-sensitive dyes exploit the high transmembrane gradient in calcium
ions concentration and have been demonstrated to be sensitive enough to detect the variations due to
the opening of a single calcium channel in a synaptic spine. Other strategies involve the detection of
neurotransmitters released by synapses during neural activity or the activation of transmitter-gated
channels.
Optical monitoring of membrane potential has also been achieved without the need of fluorescent
probes, by exploiting intrinsic variation in the optical properties of cells, like changes in light
scattering, in birefringence or in optical dichroism, mainly due to local variations in refractive index
near the membrane upon osmotic changes associated with ion fluxes.45,46
However, the small signals
involved in these processes usually require extensive averaging and obtaining high-resolution
imaging with single-cell precision is still a challenge.
1.2.2 Direct optical stimulation
Except for some notable exception, like the retina and photosynthetic units, light usually does not
specifically interact with biological systems. To optically modulate cellular activity are thus
generally employed some photosensitive transducers brought into the cell or in its close proximity.
However, some examples of direct stimulation, i.e. without the use of exogenous absorbers, of
biological systems with light are present in literature. Actually, already in 1891 the French scientist
Jacques-Arséne d’Arsonval reported the capability of exciting muscular fibers with light.47
Later,
Arvanitaki and Chalazonitis published, starting from the ‘40s, different works in which both
excitation and inhibition of neural activity could be obtained optically in different neural
preparations.48,49
While many of these preparations were actually stained with vital dyes to obtain
6
photosensitivity, other systems, like some large neurons of the marine mollusk Aplysia Californica,
showed the presence of endogenous fluorophores. This intrinsic pigmentation was related to heme
or carotenoid molecules. Their work was expanded by Fork in his paper of 1971,50
where he showed
that blue or green laser light can be used to stimulate Aplysia neurons and map cellular
interconnections. In all these reports, however, the mechanism of transduction was never clearly
identified. In 2008, Reece et al. reported that inhibition of C1 neurons of Helix Aspersa following
irradiation by 532 nm laser light is mediated by the activation of chloride currents;51
also in this
case, however, the primary photoabsorber could not be identified.
Another strategy for direct optical stimulation of neurons is based on the use of ultrashort laser
pulses (in the femtosecond to nanosecond regime) of near-infrared light. Transduction of the optical
signal is based in this case on non-linear processes due to the high peak intensity of the pulses.52–54
Two regimes of stimulation have actually been identified, depending on the laser intensity. At low
intensities, production of reactive oxygen species is reported to mainly mediate the firing of action
potentials, probably due to two-photon absorption by some endogenous fluorophore; at higher
intensities, spiking activity is a consequence of membrane depolarization due to transient poration
of the plasma membrane.
A more interesting approach to optically modulate cellular activity without the use of external
sensitizers is Infrared Neural Stimulation (INS), which is based on water absorption of pulses of IR
light. It has been proposed by Wells et al. in 2005,55
when they reported the successful in vivo
stimulation of compound nerve and muscle potentials in frogs and rats. Following this first
demonstration, the biophysical mechanism of transduction was investigated by different groups.56–60
In 2012 Shapiro et al.58
demonstrated that the local rise in temperature following light absorption by
water results in a variation of the plasma membrane capacitance in different kinds of cells, which is
reflected in a transient depolarization of the cell with values compatible with the firing of action
potentials in neurons. However, Albert et al.57
showed that infrared laser-evoked stimulation of
sensory neurons is mediated by the opening of temperature sensitive ion channels (in particular the
Transient Receptor Potential Vanilloid channel 4,61–64
TRPV4, which is expressed in different
neuron families). Also, while short pulses (on the time scale of milliseconds) of IR light have been
demonstrated to promote neural activity, for longer illumination suppression of action potential
formation and the blocking of spikes transmission along nerve fibres have been observed.65
This
opposite behaviour is attributed to the effect of the increasing baseline temperature on the Hodgkin-
Huxley gating mechanism of action potentials.66
7
1.2.3 Molecular-based stimulation
Although, as reported above, cells can exhibit some intrinsic sensitivity to light, the efficiency of
such processes is quite low, especially for visible illumination (where intensities up to several
W/mm2 can be necessary), and usually not fully controllable. Researchers have thus generally relied
on exogenous (i.e. not present in the system in natural conditions) transducers with more efficient
absorption of optical radiation. A first approach is based on photoactive molecules or
macromolecules, i.e. molecular systems that upon illumination are able to modulate some relevant
function in the cell.39
Photoisomerizable compounds (also known as photoswitches) exploit the variation in functionality
of a molecule between two different isomeric forms; upon light irradiation in a specific wavelength
range the original inert system, usually based on an azobenzene unit, undergoes a cis-trans
isomerization that results in a biologically active compound. The active form can be stable for hours
or days, and inactivation, i.e. the reverse isomerization, can be usually obtained by irradiation with a
different wavelength. This principle has been applied for light-mediated pharmacological control of
different targets, like enzymes, ion channels and G-protein-coupled receptors.67,68
In particular,
targeting of ion channels can be exploited to control cell membrane potential and thus promote or
inhibit activity in neurons. As an example, acrylamide-azobenzene-quaternary ammonium (AAQ) is
a photoswitch inert in its cis form, but acts as a potassium channels blocker in its trans form,
increasing neuron excitability.69,70
This molecule has been successfully employed to restore light
sensitivity in blind rat retinas both in vitro and in vivo with intensities of few tens of μw/mm2.
A similar strategy is that of photocleavable compounds, in which the functional molecule is
inactivated by blocking it inside a molecular cage; this cage presents photolabile bonds that are
broken upon illumination, releasing the trapped molecule, for example a neurotransmitter, a
secondary messenger or an enzyme.71–73
With these systems it is possible to control with unique
spatiotemporal resolution the release of a molecule in specific regions inside or outside the cell and
they represent an invaluable tool to investigate cellular physiology and patophysiology. However,
they still suffer from some limitations, especially the inability to reverse the activation process and
thus decrease the local concentration of the active molecule, which can also diffuse to other regions.
Moreover, uncaging usually requires illumination with UV light, which has a limited penetration
depth in tissues.
In 2005 Boyden et al.74
reported the possibility to express in mammalian neurons channelrhodopsin-
2, a light-sensitive ion channel, to control neural activity with millisecond precision. This work
opened the way to the optogenetic revolution in neuroscience;75
over the next decade a wide variety
of optically controlled systems to modulate cellular activity have been developed: ion channels for
8
both excitation74
and inhibition76,77
of neural activity, G-protein-coupled receptors to control
biochemical signaling pathways,78
transcriptional effectors to influence gene expression.79
The main
advantage of optogenetics, apart for the high spatiotemporal resolution achievable with optical
stimulation, is the possibility to genetically target the expression of the light-sensitive proteins to
specific subpopulation of cells, allowing the investigation of specific functions of different types of
cells in complex biological tissues.
1.2.4 Nano-/micro-particle stimulation
The use of nanoparticles (NPs) in biology has developed so much in the last few decades that the
term “nanomedicine”80,81
has been introduced to describe this broad field of research. The interest in
these systems stems from the combination of several properties: (i) they can be used as scaffolds to
transport different functional molecules; (ii) they possess a great surface-to-volume ratio; (iii) they
are easily controlled in shape and dimension; (iv) they present unique optical properties. NPs have
been employed both for diagnostic and therapeutic means as contrast agents for functional imaging,
carriers for drug delivery and actuators for photodynamic and photothermal therapies.
Their use as transducers for direct modulation of cell membrane potential is a relatively new and
less explored field. In a first attempt, Winter et al.82
proposed in 2001 to bind semiconducting
quantum dots (QDs) to the plasma membrane of different cell types and to exploit their electrical
dipole upon illumination to optically stimulate the cell, but they could not obtain any reliable
photoactivation. A more successful approach has been that of using NPs in form of thin films, both
as a functional substrate for cell growth83
or as a coating for patch micropipettes.84,85
In these
reports, apart from the capability of inducing a local electric field upon illumination, also the
possibility to have net Faradaic currents due to charge transfer reactions has been investigated.
Recently, semiconducting nanoparticles have also been used as sensitizers for thermal stimulation
based on the principle of Infrared Neural Stimulation.86,87
The advantage of using exogenous
sensitizers with respect to rely on water absorption is in the possibility of using light in the near-IR
(around 800 nm), while water absorbs efficiently light at higher wavelengths. As for INS, also in
this case both excitation and inhibition of neural activity could be observed, based on the timescales
of the stimulation protocol. A similar approach has been followed by other researchers using bigger
particles, on the scale of few microns, of iron oxide59,88
or carbon, again as photoabsorbers both in
the visible and the near-IR.
9
1.2.5 Device-based stimulation
Solid-state electronic devices have been largely investigated in the past as bidirectional platforms
for electrical interfaces with neural tissues, starting with the pioneering work with silicon-based
transistors of Fromherz and coworkers in the ’90s.89–91
Coupling of electronic devices with optical
excitation has been proposed in 2001 by Colicos et al.92
in order to overcome the problem of poor
spatial resolution of electrode-based stimulation. Their approach relies on the variation in
conductivity of silicon under illumination;93
a passivated silicon chip is used as a substrate for the
cell culture; while an electric bias is applied to the device, a pulse of light is shone on the desired
area, producing a current in the semiconductor that is capacitively coupled to the cell layer on top,
resulting in a local excitation.94,95
This method allows thus to have an electrical capacitive
stimulation but with a flexible geometry defined by the patterned illumination that can be varied
during the experiment. Beyond crystalline silicon, also thin-film devices based on amorphous
hydrogenated silicon or titanium dioxide have been demonstrated.96,97
Recently, Palanker and his collaborator have realized a wireless retinal prosthesis based on the
photovoltaic effect in silicon. In this case, however, an array of discrete photodiodes driving
discrete electrodes for cell stimulation has been employed.98
Light is used here not to improve
spatial resolution, but to avoid the need of wiring to power the electrode array in the eye.
1.3 Organic semiconductors for biological applications
The devices described in the previous section are all based on inorganic semiconductors and metals;
indeed, these materials have been widely applied in the field of bioelectronics, i.e. the coupling of
solid-state electronic devices with biological systems. The continuous evolution of silicon
electronics has lead in the past decades to the development of many technologies now ubiquitous in
medical research and practice, from stimulating devices like pacemakers and cochlear implants to
recording instruments such electrocardiographs and electroencephalographs. However, problems
like the rigid nature of inorganic crystalline materials and their purely electronic conduction
properties have always posed difficulties in realizing efficient direct interfacing with biological
tissues.
Organic semiconductors99
are materials based on conjugated carbon atoms with sp2 hybridization;
these materials show semiconducting properties since the π-electrons easily delocalize along the
conjugated system. In the last thirty years this class of materials has gained a great deal of attention
in the scientific community due to their unique properties: they can be chemically modified to fine
tune their optoelectronic properties or to add functional groups; they can be usually processed with
10
solution-based technologies that allow relatively simple and cheap fabrication processes and fast
prototyping; they generally posses, especially conjugated polymers, a “soft” nature that permits the
realization of flexible and conformable devices; they can, in some cases, conduct both electronic
and ionic charges. These properties have led to the development, and in some cases
commercialization, of different technologies, especially organic light emitting diodes (OLEDs),
organic photovoltaic cells (OPVCs) and organic thin film transistors (OTFTs).
Organic semiconductors, and in particular conjugated polymers, have been extensively used as
coating materials for inorganic electrodes in bioelectronic applications.100
Since their “soft” nature
closely matches the mechanical properties of biological tissues and their carbon-based chemical
structure resembles that of basic compounds in living matter, these materials generally present
better biocompatibility properties with respect to standard inorganic metals and semiconductors.101
Moreover, the possibility to have both ionic and electronic conduction represents a bridge between
the typical transport mechanisms in biological systems, which is based on ionic species, and the
electron-based conduction in standard electronic devices.102
All these properties have allowed the
achievement of better interfacing between electrodes and biological matter, decreasing both
electrical impedance of the contact and inflammatory responses from the tissue.
In the past decade, however, organic semiconductors have started to emerge in life sciences not only
as passive elements in coating layers, but as active functional materials for novel technologies. This
field of research, named organic bioelectronics by Berggren and Dahlfors in a seminal review paper
of 2007,103
has been greatly expanding in the last years; it now encompasses a number of different
applications that exploit the peculiar optoelectronic and mechanical properties of organic
semiconductors,101
from sensing of biomolecules104,105
to functional substrates for cellular
growth.106,107
Among these, technologies for interacting with bioelectrical signals in living systems
have attracted considerable attention.
The preferred architecture proposed for sensing bioelectrical signals, and in particular neural
activity, is the transistor, where the local potential variation to be detected acts as a gate signal for
the device. The most common design for an organic transistor is the one based on the field effect
(OFET, organic field effect transistor), in which the electric field generated by the gate potential
modulates the conductivity of the semiconductor. Muccini and his group have realized in 2013 an
organic thin film transistor capable not only of recording, but also of stimulating and inhibiting
neural activity;108
interestingly, they showed in recording configuration a signal-to-noise ratio 6 to
16 times better than the one of standard commercially available MEAs devices. This increase in
performance was tentatively attributed to a more efficient capacitive coupling between the organic
interface and the biological tissue. This device was based on a perylene-based small molecule as the
11
organic semiconductor, but similar OFET architectures were also proposed by Biscarini and
coworkers with pentacene molecules.109
Another example where the peculiar properties of organic semiconductors are fully exploited is the
organic electrochemical transistor (OECT).110
In these devices the electrolyte, acting as a gate
electrode, is in direct contact with the organic semiconductor and ions can migrate into the material
modulating its bulk conductivity. This 3D modulation of the semiconductor conduction, with
respect to a standard field effect transistor where charges are transported along a 2D channel at the
interface with the gate dielectric, gives these devices a very high sensitivity, at expense of the
switching speed. Based on the OECT architecture, Malliaras and coworkers have recently
developed a novel conformable neural interface array that can record local field potentials and
action potentials in vivo from the surface of the brain without the need of penetrating
electrodes.111,112
The same principle of interplay between ionic and electronic transport is used, in a specular manner,
in organic electronic ion pumps (OEIPs), mainly developed in the laboratories of Berggren and his
collaborators.113
These devices operate basically as transistor for ionic species, where their transport
is regulated by an electronic gate signal. They have been employed as platform for the precise
spatio-temporal release to biological preparations of simple ions (like calcium)114
but also signaling
molecules and drugs;115,116
in particular, by delivery of different neurotransmitters, OEIPs can be
used to modulate electrical activity in neural tissues.
Another interesting application is the exploitation of the superficial oxidation state of an organic
semiconductor, in particular conducting polymers, to control the ability of cells to grow and
proliferate on top of it. Indeed, different groups have demonstrated, for different types of cells, that
they preferentially grow on oxidized region of the material surface, probably because of a different
interaction with the substrate of the proteins mediating the adhesion process.107,117
Closely related to
these studies is the possibility to control the outgrowth of neurites of cells grown on conducting
polymers with the application of short electrical pulses to the substrate,118
demonstrating the
importance of bioelectrical signals not only at the level of action potential transmission but also in
modulating the cell fate.
1.4 Photoactive bio-polymer interfaces
Although much of the interest in organic semiconductor is related to their unique optical properties,
the possibility of using them as transducer of light stimuli into bioelectrical signals has only recently
started to emerge.39
The basic idea is to exploit the photovoltaic action in organic materials,119
and
12
in particular in bulk heterojunctions120,121
of conjugated polymers with fullerene-based electron
acceptors, to generate electrical charges upon illumination; these charges should then be able to
modulate the membrane potential of a cell grown on top of the device.
1.4.1 The hybrid solid-liquid organic photovoltaic cell
The first step in this direction was to demonstrate that an organic photovoltaic cell could work in a
liquid environment like that of a cell culture. Indeed, organic solar cells are quite known to suffer
from degradation problems due to oxygen and humidity;122
these issues stem not only from the
intrinsic electrochemical stability properties of conjugated polymer,123
but also from the degradation
of the highly reactive low work function metals usually employed as cathodes in these kinds of
solar cells.124
This problem was solved by realizing that the electrolytic solution of the extracellular
medium is by itself a charge conductor and can be used directly as an electrode, without the need
for a metal one. The device architecture reported by Antognazza et al.125
was thus composed of a
glass substrate covered with a transparent conductive oxide (ITO, indium-tin oxide), on top of
which a thin film of the photoactive layer was deposited via spin-coating. The organic materials
used in this first report where the conjugated polymer poly[2-methoxy-5-(2’-ethylhexyloxy)-p-
phenylene vinylene] (MEH-PPV) as light absorbing material and hole conductor, and the fullerene
derivative C61-butyric acid methyl ester (PCBM) as electron acceptor. The device was then put in
an electrolytic solution resembling the extracellular medium (Krebs-Ringer’s solution, KRH) and
the circuit was closed with a gold wire put as a counter-electrode in the solution. In the work, the
authors were able to demonstrate that such a hybrid solid-liquid photovoltaic device was able to
support the generation of a photocurrent, with an action spectrum similar to the one of a standard
organic solar cell.
In a subsequent work Lanzarini et al.126
showed that also other conjugated polymer could work as
light harvesting material in these hybrid devices and proposed that, under continuous illumination,
hydrogen evolution could occur at the polymer/electrolyte interface. Among the semiconductors
used in this report, poly(3-hexylthiophene-2,5-diyl) (regioregular P3HT) was selected as the choice
material for the applications in biological interfaces. Guerrero et al.127
were also able to fabricate
organic photoelectrochemical cells (OPECs) based on P3HT:PCBM blends with quantitative
photocarrier conversion in which, by proper selection of the redox couple, the organic layer was
able to provide either holes or electrons, expanding the device applicability to the production of
different solar fuels.
13
1.4.2 Poly(3-hexylthiophene)
P3HT is one of the prototypical conjugated polymers used in the field of organic electronics and it
has been by far the most investigated polymer in the understanding of the photophysics behind the
functioning of organic photovoltaic cells.128
It is composed of a backbone of thiophene rings that
determine its optoelectronic properties, functionalized with alkyl side chains to confer solubility in
organic solvents. In its regioregular version (rr-P3HT) the side chains are periodically ordered along
the backbone, allowing interdigitation with adjacent polymeric chains. In the solid state, P3HT
tends to form lamellar structures with interchain stacking of the thiophene rings, leading to an
increased delocalization of the electron density and increase in charge mobility. Depending on the
polymer molecular weight and the deposition process, films usually present a crystalline fraction of
lamellae embedded in an amorphous matrix.129,130
The material presents a broad absorption in the visible peaking in the green (500-550 nm), with a
side shoulder at 605 nm attributed to the formation of the ordered lamellar phase. As in other
organic semiconductor, light absorption leads to the formation of a bound electron-hole couple
(exciton), with a binding energy of about 0.7 eV;131
some driving force, usually given by the
presence of an electron acceptor like PCBM, is thus needed to break the exciton and obtain free
polarons. While P3HT is generally considered a p-type material (i.e. a hole conductor), careful
investigation of its transport properties have revealed similar mobilities for holes and electrons in its
intrinsic state;132
however doping due to exposure to air results in traps for electrons that drastically
reduce electron mobility.133,134
Different studies have investigated P3HT stability upon illumination. As in many conjugated
systems, exposure to UV light in presence of oxygen leads to irreversible degradation of the
material, due to oxidation by radical chain mechanisms that destroy the backbone conjugation with
a deterioration of optoelectronic properties.123
Upon illumination with visible light, instead, only a
reversible effect is observed, attributed to the formation of a charge-transfer complex with
molecular oxygen.135,136
This process occurs, at a much lower rate, also in dark and it should be
favored by the presence of humidity.137
Interestingly for biological application, upon illumination
degradation of films in direct contact with a saline solution is not enhanced with respect to films
kept in air.138
Due to the presence of the alkyl side chains, P3HT surfaces are usually quite hydrophobic. To allow
the attachment of cell cultures, films usually need to be pre-treated with some adhesion layer.
Scarpa et al.139
demonstrated the possibility of growing mouse fibroblasts on P3HT films by using
interlayers of proteins like fibronectin, polylysine or collagen. However, also a mild plasma
treatment, to oxidize the surface and increase hydrophilicity, was seen to be sufficient to promote
14
cell adhesion and proliferation. Biocompatibility of P3HT and P3HT:PCBM films has been studied
up to four weeks in-vitro also for cultures of primary cells like neurons140,141
and astrocytes,142
demonstrating viability rates and electrophysiological properties similar to cells cultured on
standard control substrates.
1.4.3 Photostimulation of primary cells
In 2011 Ghezzi, Antognazza et al.140
published the first report about the photostimulation of
primary neurons via light absorption in a P3HT-based biointerface. The device consisted of a thin
film (≈ 150 nm) of a P3HT:PCBM blend (in relative weight ratio 1:1) deposited on an ITO-coated
glass substrate. Rat hippocampal neurons were cultured on top of the active material, pretreated
with a poly-L-lysine adhesion layer to promote cellular adhesion. Pulsed (20 to 50 ms) light
excitation was obtained with a 532 nm green laser, delivered to the preparation through the
microscope objective (in an upright configuration) with an intensity of about 10 mW/mm2.
Recording performed with standard patch-clamp techniques showed that, upon illumination on the
cell body of the neuron, action potential firing was clearly elicited, with a success rate higher than
85 %. Interestingly, moving the illumination spot outside of the cell body did not produce any
excitation of cell activity, demonstrating an intrinsic spatial selectivity of the stimulation
architecture. The actual coupling mechanism between the device and the neuron was not fully
elucidated, but the author proposed a capacitive charging of the polymer/electrolyte interface upon
charge generation in the active material. In a later paper, it was reported that also in devices with an
active layer composed of only P3HT neuronal excitation could be reliably obtained with similar
power intensities; this result indicated that, contrary to what happens in organic solar cells, charge
generation in the bulk of the semiconducting material is not the main physical phenomenon leading
to cellular stimulation in these hybrid interfaces.
In a subsequent work by Benfenati, Martino et al.142
the same device architecture was used to
investigate the effect of photostimulation on astrocytes (Appendix B). In this case, continuous
illumination was used (λ = 560 nm, intensities up to 13 mW/mm2). A progressive depolarization of
the cell was observed along with a modification of the rectification properties of the membrane.
Pharmacological experiments demonstrated that the photostimulation was causing the opening of
the chlorine channels ClC-2. Based on the known stimuli involved in modulation of ClC-2
conductances, it was proposed that a local acidification was occurring upon illumination; this could
be due either to a capacitive rearrangement of charges at the polymer/electrolyte interface or to the
occurrence of electrochemical reactions.
15
1.4.4 Ex-vivo experiments on blind retinas
The capability of the polymeric interface described above to transduce optical signals into a
bioelectrical stimulation of neuronal cells makes these devices an interesting platform for
neuroscientific research, but has also important implications in the field of vision restoration to
blind people. Diseases that affect the photoreceptor layer in the retina impairing its light sensitivity,
like Retinitis Pigmentosa, age-related macular degenerations and Stargardt’s disease, are the main
cause of legal blindness in the western world. Restoring light sensitivity in a degenerated retina is
thus one of the main focuses of research in developing visual prostheses.
The first report of a successful interfacing of a blind retina with a photoactive polymeric-based
device came in 2013 by Ghezzi et al.141
Photoreceptor degeneration was induced by prolonged
exposure to intense light in albino rats; the retinas were then explanted and put in contact with a
P3HT-based device in a subretinal configuration, i.e. with the degenerated layer of photoreceptors
facing the polymer. Multi-unit activity and local field potentials were recorded with an extracellular
electrode from the ganglion cell layer upon illumination with pulses of light (λ = 532 nm) over a
wide range of intensities (from 10 nW/mm2 to 4 mW/mm
2). While for blind retinas on control glass
substrates only small responses to light stimulation could be measured and with a threshold at quite
high intensities (80 μW/mm2), in retinas placed on photoactive devices the response was
significantly increased and the activation threshold was reduced (0.3 μW/mm2).
Similar results were also obtained in 2014 by other groups. Gautam et al.143
used a blend of P3HT
and a n-type conjugated polymer, P(NDI2OD-T2), to successfully excite blind embryonic chick
retinas. The same experimental model of retinal degeneration was used by Bareket et al.144
to
demonstrate a device architecture based, instead of semiconducting polymers, on a mesh of carbon
nanotubes, used as electron acceptors and transporters, sensitized for visible-light absorption with
inorganic core-shell quantum dots. In both cases, excitation intensities of the same order of
magnitude as the ones used by Ghezzi et al. were reported (tens to hundreds of μW/mm2); again, a
capacitive charging of the device interface was reported as the excitation mechanism.
16
Chapter 2 – The plasma membrane
Eukaryotic cells are complex systems organized in many specialized compartments and organelles
that have evolved to perform different tasks essential for the correct functioning of the cell.145
Many
of these organelles, like the nucleus, the Golgi apparatus and the endoplasmic reticulum, are defined
by a membrane that separates them from the rest of the cell. Moreover, each cell is separated from
the extracellular space by what is called the plasma membrane.146
The basic elements that constitute
all these systems are double layers of amphiphilic lipids that spontaneously tend to form
bidimensional structures to balance hydrophilic and hydrophobic forces. Biomembranes, however,
are not simply partitioning elements, but are usually an active part of the organelles, fundamental in
their functioning. Their functionality is mainly given by the presence of several proteins, embedded
in or bounded to the surface, involved in a variety of biological processes.
One of the fundamental characteristics of biological membranes is their selective permeability, i.e.
the ability to block the diffusion of some molecular species while letting other pass through. The
diffusion of charged species in particular, from simple ions to charged macromolecules, is tightly
regulated by the presence of very selective transporting proteins that span the membrane, called ion
channels.147
This precise control of ion concentrations leads in many cases to the establishing of a
transmembrane potential difference that is fundamental in controlling several of the membrane
functions.3 In particular, the plasma membrane of virtually all eukaryotic cells is characterized by a
potential difference between the inner and outer side; for animal cells in physiological conditions
this membrane potential is usually in the range between - 80 mV and - 40 mV and it has been
shown, for example, to be correlated with the phase of the cell cycle and the cell ability to
proliferate.148,149
The properties of plasma membrane potentials have been mainly studied in the
context of signals transmission in excitable cells and in particular in neural circuit.
Since one of the main goals of the hybrid polymer-based devices introduced in Chapter 1 is the
ability to modulate the electrical potential in cell membrane, a deep understanding on their
composition and functioning is of great importance. In this Chapter, we first introduce (Section 2.1)
the fundamental structure of a typical biomembrane, composed of a lipid bilayer with embedded
proteins. We then discuss a particular class of transmembrane protein, the ion channels, which
regulate the flowing of ion species through the membrane (Section 2.2) and how they are involved
in the arising of the membrane electric potential (Section 2.3). The discussion here is focused on the
17
plasma membrane of the cell, even if several of the elements presented are common also to other
systems.
2.1 The structure of the plasma membrane
A biological membrane is an object that is able to separate two different aqueous compartments by
selectively control the flow of molecules between them. In particular, the plasma membrane defines
the cell boundary, dividing the extracellular space from the interior of the cell. It is thus the main
element that controls the uptake and release of substances, regulating the composition of the
cytoplasm. Membranes are mainly composed of three different kinds of molecules: lipids, proteins
and sugars.146
Lipids are a broad class of hydrophobic or amphiphilic small molecules with different biological
functions, from energy storage to signaling and structural properties. The basic constituents of the
membrane are a particular class of lipids, called phospholipids. They are amphiphilic molecules
composed by a hydrophilic head containing a phosphate group to which a hydrophobic tail is
attached, usually made of two fatty acid chains. The head of phospholipids found in membranes can
be either negatively charged or zwitterionic (i.e. presenting both a negative and a positive charge);
positively charged phospholipids are not found in nature, but can be synthesized in laboratory. To
balance the hydrophobic and hydrophilic forces, phospholipids in aqueous solutions usually tend to
aggregate in structures that screen their hydrophobic tails from interactions with water molecules.
One of the most common structures is the lamellar phase, in which phospholipids arrange
themselves in a bilayer with hydrophobic tails in the middle and hydrophilic heads towards the
solution. This two-dimensional structure is the basic component of a biological membrane. The
presence of a hydrophobic core renders lipid bilayers generally quite impermeable to polar and
especially charged molecules, while apolar molecules like O2, N2, CO2 and fats can usually rapidly
permeate through them. A notable exception is water, which can permeate through membranes
although being polar; the actual mechanism by which this happens is however still debated.
Proteins are large macromolecules formed by one or more chains of amino acids and are the main
functional elements in living systems. In membranes, proteins perform a variety of tasks, among
which is the formation of selective pores for the transport of molecules for which the lipid bilayer is
normally impermeable. Membrane proteins can be classified based on how they are associated with
the lipid bilayer. Integral proteins are molecules that are permanently bound to the membrane, either
spanning the entire lipid bilayer (polytopic or transmembrane proteins), or being attached only to
one side, usually the inner one (monotopyc proteins). Peripheral membrane proteins are instead only
18
temporarily attached, either to an integral protein or to the lipid bilayer, via a combination of non-
covalent interactions.
Sugars in biological membranes can be found in complexes with other molecules, either with lipids
(glycolipids) or with proteins (glycoproteins). Since they can form many different structures in
relatively short chains, sugars are mainly used as distinguishing features that enable recognition
processes between biomolecules. For this reason, in membranes they usually occur on the outer
side, allowing specific cell-cell interactions and targeting of signaling molecules like hormones.
Figure 2.1 | Schematic representation of the composition of the plasma membrane.
The basic model that describes a cell membrane is the fluid mosaic model, introduced in 1972 by
Singer and Nichols.150
It treats the membrane as a two-dimensional fluid composed of a matrix of
lipids in which proteins are embedded. In this model, lipids and proteins can easily diffuse and no
long-range order is present in the membrane, which is seen as a homogeneous system. This model
has been refined in the following years to take into account the observation that both lipids and
proteins may distribute inhomogeneously, forming clusters and domains in the membrane.
Biological membranes actually contain a great variety of lipids that differ for both the length of the
hydrophobic tails and the nature of the hydrophilic head. Also, transmembrane proteins can have
different lengths of the hydrophobic surface embedded in the membrane; if this length does not
match the hydrophobic core of the bilayer, unfavorable interactions can arise. Lipids with longer or
shorter fatty acid tails tend thus to accumulate around proteins with different core length to
compensate this effect. In the mattress model151
of Mouritsen and Bloom (1984) this phenomenon,
called hydrophobic matching, is proposed to explain the accumulation of certain lipids around
different proteins and the attraction between proteins due to capillary forces. Similarly, the
interaction mismatch between different species of lipids can explain the formation of aggregates and
domains.
19
It is thus quite clear that biological membranes are complex systems in which thermodynamics
plays a fundamental role. Lipid bilayers can exist in a variety of phases, from crystalline to gel to
fluids and indeed phase transitions are observed in membranes at physiologically relevant
temperatures.146
The actual properties of a membrane considerably depend on its particular
composition. Different types of cells possess membranes with quite different composition of lipids
and proteins. Interestingly, it has also been shown that the same cells can be characterized by
different compositions of the membrane if grown at different temperatures or pressures. Moreover,
also the two leaflets composing the bilayer have different distribution of lipids, which rarely
spontaneously flip from one side to the other.146,152
Indeed, specific proteins like flippases and
scramblases are present in the membrane to allow the translocation of lipids between the two
monolayers. This asymmetry153–155
is fundamental in determining the electrostatic properties of the
membrane and actually the inner leaflet is usually more negatively charged with respect to the outer
one.
2.2 Ion channels
While lipid bilayers by themselves are impermeable to many molecules, especially polar and
charged ones, transmembrane proteins allow the transport of molecules from one side to the other of
biological membranes. These proteins can be classified in two broad categories depending on
whether they work or not towards thermodynamic equilibrium:156
passive pores (or channels) that
facilitate the diffusion of particular chemical species through the membrane, or active transporters,
which utilize energy (for example in form of ATP) to move substances against the electrochemical
gradients between the intracellular and extracellular compartments.
Among these membrane transport proteins, ion channels are a particular class of passive pores that
mediate the passage of ions (mainly K+, Na
+, Cl
-, Ca
2+) present in the electrolytic solution
composing the physiological media. Ion channels are fundamental in determining the bioelectrical
properties of membranes, promoting the establishment of a transmembrane potential and controlling
its value upon the presence of proper stimuli. Being able to modulate the intracellular ion
concentrations, they can also regulate cell volume by driving osmotic flow of water through the
membrane. A vast variety of ion channels with diverse properties have been discovered;157
their
expression is extremely variable between different cells, and also in the same cell at different phases
of its development, conferring them diverse bioelectrical properties. In particular, ion channels
present two peculiar characteristics that make them extremely flexible tools in controlling the flow
of ions through the membrane:3 (i) they can have a very high selectivity for a particular ionic
20
species, blocking the passage of all the others; (ii) they can be activated or inactivated by the
presence of external stimuli.
2.2.1 Ion channel structure and selectivity
As for all proteins, the basic functioning of ion channels is strictly dependent on their geometry.
While many characteristics of these proteins had been already inferred via indirect methods, the first
actual high-resolution crystallographic structure of an ion channel became available only in 1998.158
The core part of an ion channel is a transmembrane unit with a central pore that spans through the
entire width of the membrane. This region is generally made up by the symmetrical arrangement of
different subunits of the protein around a central axis, forming a channel that can be filled with
water molecules. Along this channel, specific structures made by amino acid residues are present to
form what is called the selectivity filter,159,160
i.e. a region where a particular ionic species may be
recognized and allowed to pass. To allow very fast transport of ions, in many cases the pore is
formed by a long passageway where ions can diffuse freely, interrupted by a quite small selectivity
filter. Indeed, ion channels can transport up to 108 ions per second.
3 Apart from this central part, ion
channels can also have domains in the extracellular or intracellular space to sense the presence of
different stimuli.
The mechanism of channels selectivity is the result of different competing phenomena:160
(i) the
geometrical width of the filter, which can block larger particles from passing; (ii) the hydration state
of the ions that increases their geometrical hindrance; (iii) the electrostatic properties of amino acid
residues present in the selectivity filter. To pass through the selectivity filter, hydrated ions need to
lose their shell of water molecules and temporarily bind to the amino acid residues, from which they
are subsequently released by thermal energy. The channel selectivity between anions and cations is
thus given by the actual charge on the side chains of the amino acid forming the selectivity filter.
The selectivity for different ions with the same charge is instead given by the fact that smaller ions
(like Na+) have a stronger interaction with water molecules than bigger ones (like K
+). When an ion
reaches the selectivity filter, it binds to it only if it is in a more energetically favorable situation with
respect to the hydrated state. A small pore with a high binding energy is thus able to strip the water
molecules from a Na+ ion and let it through, while a K
+ ion is too big to pass from the filter. Instead,
a larger pore with a lower affinity can still provide sufficient energy to dehydrate the K+ ion, while
the Na+ ion retains its water shell and cannot pass.
21
Figure 2.2 | Schematic representation of an ion channel. To pass through the pore, a
hydrated ion (A) needs to lose its water shell to bind to specific residues of the selectivity
filter (B). Conformational changes in the channel structure due to external stimuli may
close the pore, a mechanism called gating. © The Nobel Foundation.161
2.2.2 Gating mechanisms
Another peculiar property of ion channels is that they usually exist in at least two relatively stable
different conformations.3 In particular, channels have always at least an open and a closed state, and
can go from one to the other in the presence of appropriate stimuli. Moreover, closed channels can
be in a resting state, in which they can be opened upon the occurrence of the stimulus, or a
refractory state, in which they are insensible to external signals. This mechanism is called gating,
and it is fundamental in controlling many bioelectrical properties of cells. The stimulus triggering
the gating of a channel may be of different natures, depending on the actual functionality of the
channel itself.159,162
Voltage-gated channels are controlled by a variation in the membrane potential. These
channels posses a subunit (the “voltage sensor”) that can sense the local electric field and
trigger a conformational change in the protein. Although they are usually activated by a
depolarization of the membrane (i.e. a variation of the potential towards more positive values),
examples of voltage-gated channels that open upon hyperpolarization have also been found.
This mechanism is essential in the formation and propagation of action potentials in neurons,
22
based on the interplay between the rapid opening and closing of sodium and potassium
channels.
Ligand-gated channels switch from a conformation to another upon binding of a specific
chemical species in a selective pocket on either the intracellular or extracellular side of the
membrane. There is a variety of substances that can act as gating agents, from simple ions like
Ca2+
to signaling molecules like neurotransmitters.
Mechanically-gated channels respond to a mechanical deformation of the membrane or of the
cytoskeleton of the cell; they are the main transducer of sensory stimuli like touch and hearing,
but are also involved in cardiovascular regulation and osmotic homeostasis.
Light-gated channels contain an isomerizable chromophore (like retinal) that changes
conformation upon light absorption, triggering the opening and closing of the channel. Only one
class of natural light-gated channel, namely channelrhodopsin from unicellular green algae, is
currently known; however a lot of research efforts have been devoted to develop new light-
gated channels for optogenetics.
Temperature-gated channels are found in the transient receptor potential (TRP) group, in
particular in the TRPV subfamily, and are responsible for the sensation of heat and pain, but
also for the regulation of body temperature.
The gating mechanisms allow the cell to change the permeability of its membrane in response to
either external or internal stimuli, exploiting the related variations in membrane potential and ion
concentrations to trigger different biophysical processes.
2.2.3 Channel conductance and temperature dependence
The conductivity properties of ion channels vary greatly from type to type. The introduction at the
end of the ‘70s of the patch-clamp technique by Sakmann and Neher19
allowed the recording of
currents through single channels and opened the way to a precise understanding of their electrical
characteristics. While some channels have linear voltage-current characteristics, others have been
seen to present rectifying behavior because of asymmetries in the structure of the pore and of the
selectivity filter. Typical conductivity values of a single ion channel can vary between 0.1 to 100
pS.147
The membrane conductivity is then given by the sum of the single conductivities of all the
open channels at a certain moment.
Since the transport of ion in the pore of a channel is determined by a trap-and-release mechanism
from the selectivity filter, the actual speed at which this happens is greatly dependent on
temperature, that provide the thermal energy for the releasing of the ion. Indeed, channels
conductivities have been experimentally measured to vary with temperature. A common way to
23
give an estimate of such dependence is via the Q10 temperature coefficient, which measures how the
conductivity changes upon a temperature variation of 10 °C and is expressed by the following
formula:163
where G2 and G1 are the channel conductivities at temperature T2 and T1 respectively. Typical
values for the Q10 coefficient of ion channels are in the range between 1.2 and 1.6, but particular
channels with a coefficient of about 5-6 have been demonstrated.164
It is important to highlight that this temperature dependence is a general mechanism that applies to
all channels modulating their conductance, and it’s given by the fact that the ion transport is a
thermally activated process. The temperature-gating properties of channels like TRPV are a
different mechanism, in which the channel completely changes its conformation when a certain
temperature threshold is reached, passing from an open to a closed state or vice versa.
2.3 The membrane potential
The instauration of a potential across the cell membrane is the result of an electrochemical
equilibrium due to the combination of two essential factors that characterize the plasma membrane:3
An asymmetric distribution of ions between the intracellular and extracellular space. The main
ions that compose physiological media are K+, Na
+, Cl
- and Ca
2+, plus large anions (A
-)
constituted by proteins with charged residues. Of these main cations, the cytosol, i.e. the
electrolytic solution permeating the cytoplasm, is rich in potassium, while the extracellular
medium is rich in sodium. In normal conditions, the concentration of calcium ions is maintained
at very low values in the cytoplasm by active pumping towards the extracellular space or
sequestering in internal stores; the release of Ca2+
is in fact an important signaling event that can
trigger a variety of biological processes in the cell. For the anion, the charged proteins are
mainly confined inside the cell, since they cannot easily cross the membrane, while chloride is
usually found in the extracellular medium. Table 2.1 summarizes typical values for the
concentrations of the different ionic species for mammalian neurons in physiological
conditions.
The selective permeability of the membrane, that allows some ions to pass easily than others.
As described in the previous section, the membrane permeability to ions is mainly given by the
presence of ion channels and many of these channels are very selective only for a particular ion
(2.1)
24
species. The actual permeability properties of a membrane are thus determined by the level at
which each channel is expressed, and this distribution can greatly vary from cell to cell. In
particular, in most cases the plasma membrane in normal conditions is mainly permeable to
potassium ions, while the conductances for other ions are lower. Gating mechanisms are
exploited to change the membrane permeabilities and thus modify the fluxes of different ions.
Table 2.1 | Typical values for ion concentration in the intracellular and extracellular
media for mammalian neurons, reproduced from Ref. 165.
2.3.1 Electrochemical equilibrium in biological membranes
To understand how these two factors determine the formation of the membrane potential, let’s
consider initially a situation in which only potassium conductances are present. The potassium
concentration in the intracellular compartment is much higher than that in the outer region ([K+]i >
[K+]o). Because of this concentration gradient, potassium ions tend to diffuse out of the cell, while
the movement of all other ionic species is hindered by the membrane selectivity. As K+ ions exit the
cell, the outer side of the membrane becomes positively charged, while the opposite happens on the
intracellular side. This charge accumulation at the two sides leads to the formation of a potential
difference across the membrane and thus an electric field that starts to drive back potassium ions
into the cell. A dynamic equilibrium is reached when the diffusional and electrical forces exactly
balance each other; the transmembrane potential at which this happens can be calculated from the
concentration of the ionic species in the intracellular [X]in and extracellular space [X]out with the
Nernst equation:3
Where R is the ideal gas constant, T the absolute temperature, F the Faraday constant and z the
valence of the ionic species (+1 in the case of K+). In the case of potassium ions, for the
concentrations reported in Table 2.1, the Nernst equilibrium potential Veq,K ≈ -89 mV.
If a cell has a plasma membrane only permeable to a single ionic species, the Nernst equation is
sufficient to determine its membrane potential. This is the case for example of several types of glial
Intracellular Extracellular
Potassium (K+) 140 mM 5 mM
Sodium (Na+) 5-15 mM 145 mM
Chloride (Cl-) 4-30 mM 110 mM
Calcium (Ca2+
) 100 nM 1-2 mM
(2.2)
25
cells in resting condition. However, while usually cell membranes are mainly permeable to
potassium, also other conductances are expressed that allow the flow of lower but anyway sizable
currents of different ions. In these cases, the membrane potential assumes a value intermediate
between the Nernst equilibrium for the different ions involved, with the more permeable ones
having a higher weight in determining the final result. The precise value can be calculated in the
case of physiologically most relevant monovalent ions with the Goldman-Hodgkin-Katz (GHK)
equation:166
where the Pi terms refer to the membrane permeabilities for the different ionic species. For neurons
at resting conditions, for example, the typical relative ratios of the different permeabilities are:3
From the values of Table 2.1, sodium and chloride have Nernst equilibrium potential of respectively
Veq,Na ≈ 60 mV and Veq,Cl ≈ -70 mV (for [Na+]in = 15 mM and [Cl
-]in = 8 mM). While the main
contribution to the membrane potential is still given by the potassium ions, the actual equilibrium is
shifted towards more positive values for the presence of these other conductances to Veq ≈ -68 mV.
In these conditions, the single ionic species are not in equilibrium, since the membrane potential is
different from their Nernst equilibrium. In particular, there is a net efflux of K+ ions out of the cell,
since its diffusional driving force is not completely balanced by the electrical gradient, and an influx
of Na+ ions, since both electrical and chemical gradients tend to drive them into the cell. However,
these two currents balance each other to give a zero net current through the membrane. As for
chloride, in many cases its concentrations are such that its electrochemical equilibrium is very close
to the membrane potential, so the driving force acting on it is negligible; the actual Cl- currents
flowing through the membrane are thus usually quite small in resting conditions, even if the
membrane has a substantial permeability to this ion.
The membrane potential of a quiescent cell that is not subject to any particular stimulus is usually
referred as the resting potential. Its value can be different from cell to cell as it is determined by the
distribution of ion channels expressed in the membrane that are actually in the open state in this
resting condition. However, the membrane potential can be modulated by different factors and its
variations are involved in determining the cell response to internal and/or external stimuli. The main
pathway by which a stimulus can influence the membrane potential is through the gating
mechanisms described in the previous section. When an ion channel opens, the membrane
permeability changes producing a variation in its equilibrium potential. In electrophysiology, a
variation of the membrane potential towards more positive values with respect to the resting one is
(2.3)
(2.4)
26
termed depolarization, while a change towards more negative values is referred to as
hyperpolarization. From the GHK equation it’s easy to infer that in normal condition, i.e. a cell at
rest with physiological concentrations of ions, the opening of sodium channels leads to a
depolarization of the cell, since the equilibrium potential is pushed more towards Veq,Na; conversely,
the opening of potassium channel is reflected in a hyperpolarization.
2.3.2 Action potentials in excitable cells
The prototypical example of this response is the generation of action potentials (APs) in neurons.4
These cells posses a significant number of voltage-gated sodium (NaV) channels that are closed in
resting conditions, but open in response to a depolarization of the membrane. Upon an appropriate
stimulus (for example the release of neurotransmitters by an adjacent synapse), specific receptor in
the neuron membrane are activated, producing a depolarization of the membrane. This
depolarization triggers the opening of the NaV channels, increasing the membrane permeability to
sodium, which in turn leads to a further depolarization. If the initial depolarization is high enough,
this mechanism becomes self-sustaining and the membrane permeability to sodium increases
exponentially and becomes 10/20 times higher than the one of K+. In this way, the membrane
potential quickly spikes to values close to Veq,Na. The NaV channels however stay opened for a very
short time and quickly enter a refractory state in which they are not sensitive to the membrane
potential. In the meantime, voltage-gated potassium (KV) channels, which have a slower activation
time, opens in response to the membrane depolarization. The closing of NaV channels and
concomitant opening of KV channels leads to a repolarization of the membrane that goes back
towards negative values and actually hyperpolarize since there is an increase in potassium
permeability. This fast spiking is called action potential and is the manner by which neurons
respond to stimulation and conduct information in neural networks. The firing of an APs is an all-
or-none response, which is triggered if the neuron is subjected to a depolarization exceeding a
certain threshold value. Generally, this threshold is reached if the cell is depolarized to potentials
more positive than -50 mV, from normal resting values of about -70 mV to -60 mV. Once the
threshold is reached, the action potential is fired and it evolves in a manner that is basically
independent on the actual intensity of the initial stimulus. The nature and magnitude of the
stimulation can however influence the frequency at which action potentials are generated. For this
reason information in neural network cannot be generally encoded in the intensity of the
propagating signals, but is mainly transmitted as a modulation in the frequency of APs.
While neurons are considered the prototypical example of systems that support the formation of
action potentials, they can also be found in other cell types, generally termed excitable cells. In
cardiac cells,5 APs are involved in the coordination of the heart contractions (pacemaking); in these
27
cells the action potentials have a different time course with respect to neurons, with a plateau of
several milliseconds in the depolarized states before repolarization, given by the action of slower
voltage-gated calcium channel. In muscle cells6 the propagation of action potentials leads to an
increase of calcium ions concentration in the cytoplasm, which acts as a second messenger for
triggering the contraction of muscle fibers. A conceptually similar mechanism can be observed also
in endocrine cells,7 where the intracellular calcium concentration controls the release of hormones
by driving the fusion of vesicles to the plasma membrane.
In non-excitable cells, the lack of specific voltage-gated channels does not allow the instauration of
action potentials. However, also in these cells the membrane potential can vary in response to
external stimulations or internal processes. In this case, the variation of the potential is not an all-or-
none response, but is proportional to the stimulus intensity. These progressive responses that do not
involve the self-sustained opening of voltage-gated channels are called graded potential.3 Also
stimulations of excitable cells that do not trigger the firing of action potentials fall in this category,
like the postsynaptic potentials (PSP) produced in dendrites of neurons upon reception of
neurotransmitters. These stimuli can travel along the cell membrane until a specific region of the
neuron, the axon hillock, where they are summed up; it is in this region rich of voltage-gated
channels that, if the threshold is reached, the action potentials are fired.
2.3.3 Maintenance of ion concentrations
The correct functioning of a cell is tightly linked to maintenance of correct concentrations of ionic
species in both the intracellular and extracellular space. If only passive transport of ions through
channels in the membrane was present the ionic concentrations would rapidly change since, as
described in the previous section, even at equilibrium there is a net efflux of potassium ions and an
influx of sodium in the cell. Moreover, in excitable cells, the firing of action potentials is associated
with a substantial displacement of ions through the membrane. For this reason, cells have a series of
active transport proteins in the membrane that maintain the ionic concentrations at physiological
levels. These proteins use energy from different sources to move ions against their electrochemical
gradients and can move one or more different ionic species at a time.156
The energy needed can be
supplied as chemical energy by a molecule like ATP or by the movement of one ionic species along
its electrochemical gradients to move another species in an unfavorable direction.
The prototypical example of these systems is the Na+/K
+-ATPase, or sodium-potassium pump,
167 a
protein present in the plasma membranes of all animal cells that is fundamental in keeping the
concentrations of Na+ and K
+ in physiological ranges. Being an ATPase, it works using the energy
obtained from the decomposition of an ATP molecule into ADP. The conformational changes
28
caused by the binding of an ATP molecule and subsequent release of ADP lead to the expulsion of
three Na+ ions out of the cell and the concomitant influx of two K
+ ions. Since for each cycle there
is a net efflux of positive charge, this pump is actually electrogenic, in the sense that it has a net
effect on the cell equilibrium potential, driving it towards more negative values with respect to the
one determined by only the diffusion processes considered in the GHK equation.
Figure 2.3 | Schematic representation of the functioning of the Na+/K
+ pump. (1) Three
sodium ions bind to the channel from the intracellular space. (2) An ATP molecule is
hydrolyzed by the pump, which upon phosphorylation undergoes a conformational change
that exposes the Na+ ions to the extracellular space; the affinity to Na
+ of the pump in this
new conformation is lower and the ions are thus released. (3) The pump binds two K+ ions
from the extracellular space, causing a dephosphorylation of the protein. (4) Upon
releasing of the phosphate, the pump reverts back to its original conformation which has a
low affinity for potassium ions, which are thus released in the intracellular space.
It is interesting to notice that some cells do not have active transporters for chloride ions.3 In this
case, the concentration of Cl- in the cell is only determined by the passive flow through ion
channels; it thus tends to reach a value such that its Nernst potential equals the one of the membrane
equilibrium potential determined by sodium and potassium.
2.3.4 Electrical equivalent of a cell membrane
In the previous sections we have seen that the plasma membrane is not just a barrier that separates
the cytoplasm from the extracellular space, but a complex system that actively regulates the
bioelectrical properties of the cell. The study of its behavior in response to different stimuli is thus
fundamental in understanding how a cell functions. While the GHK equation introduced in the
previous section gives useful information on the equilibrium state of the system, it is not suitable to
29
investigate the dynamic properties of the cell. A convenient way to represent the cell membrane in
order to study how its potential can evolve in time is by developing an equivalent electric circuit of
the system. The different elements that make up the plasma membrane can in fact be represented as
electrical components bundled in a single circuit that reproduces its functioning.
The lipid bilayer is an insulating barrier that separates two conducting media, the intracellular
and extracellular electrolytic solution. It thus behaves as a capacitor, with the lipids being the
dielectric medium between the two conductive plates. The membrane capacitance can be
estimated as that of a parallel-plate capacitor, with the distance between the plates as the bilayer
thickness, which is usually between dlip = 3-4 nm and a relative dielectric constant on the order
of εlip = 2.5. This simple model gives a value for the specific capacitance of Cm ≈ 0.7 μF/cm2.
While this parameter is usually considered a biological constant, actual variations are measured
between different cells and in different regions of the same cell. This variability is given by the
different composition in terms of proteins content, which can modify the effective thickness of
the membrane. Moreover, the ions accumulated on the two sides of the membrane are not
exactly concentrated at the dielectric interfaces, but form diffuse layers extending into the
solution for several nanometers. In any case, experimental values are usually in the order of 1-2
μF/cm2.168
Many of the different molecules that make up the plasma membrane are generally charged in
physiological conditions. As already described in Section 2.1, the two leaflets of the membrane
have thus a surface charge and in particular the inner side is generally more negative than the
outer one. Moreover, the different composition of the extracellular and intracellular
compartments produces a different distribution of the ions in the diffuse layers at the two sides
of the membrane. These asymmetries effectively generate an intrinsic potential difference
across the membrane even if there is no charge accumulated on it or, conversely, there is an
accumulation of charges on the membrane capacitance even if the transmembrane potential is
kept at zero. This intrinsic potential can be modeled as a voltage generator (Vσ) in series with
the membrane capacitance.
Ion channels are conducting pores that let charges flow through the membrane and they can
thus be represented as resistances (RX) in parallel to the membrane capacitance. Since currents
across the membrane are carried by different ionic species, the various channels can be grouped
in different resistors each representing the membrane permeability to a particular ion. These
resistances, however, are not fixed, but can vary in time due to the gating mechanisms triggered
by the different stimuli to which the cell is subjected. Moreover, due to asymmetries in the
channel structure or in the ion concentrations on the two side of the membrane, some channels
do not behave as simple resistors (i.e. do not have a linear voltage-current relationship), but can
30
present rectifying characteristics. Also voltage-gating of the channel conformations can result in
a non-linear dependence on the transmembrane voltage.
The difference in the distributions of ionic species on the two sides of the membrane acts as an
electromotive force that drives charges along their concentration gradient. Each of the resistor
for the different species is thus in series with a voltage generator, whose value is the Nernst
equilibrium potential (VX) for that particular ion.
Combining together all these elements, it is possible to come up with an equivalent circuit like the
one depicted in Figure 2.4a. The upper and lower nodes of the circuit represent the intracellular and
extracellular space respectively and the difference in their electrical potential is the actual
membrane potential, which is conventionally defined as Vm = Vin - Vout.
In the following chapters a simplified version of this electrical model is going to be used (Figure
2.4b). In particular, the different ionic conductances can be grouped into an effective membrane
resistance (Rm), driven by a single voltage source that represent the cell equilibrium potential (Veq)
as determined by the GHK equation.
Figure 2.4 | (a) Equivalent electrical circuit of the cell membrane with the different ionic
conductances explicitly modeled. (b) Simplified membrane circuit with only an equivalent
membrane resistance and equilibrium potential; in the scheme also the series resistance of
a patch pipette for electrophysiological measurement is represented.
It is important to remember that all the parameters in this equivalent representation are not fixed,
but depend strongly on the actual conditions of the cell. Apart from the channel resistances that can
be greatly modified by gating mechanisms, one of the main factors that influence the actual values
of the electrical parameters of the circuit is temperature. It has been previously discussed in Section
2.2 that charge transport in ion channels is a thermally activated process and also in the equation for
the Nernst equilibrium potential there is a direct dependence on the absolute temperature of the
system. Moreover, variations in the capacitance of the plasma membrane have been recently
31
observed upon light-induced heating of cells and have been proposed as a stimulation mechanism
for cellular activity.58
32
Chapter 3 – Hybrid interfaces characterization
Photovoltaic devices based on organic semiconductors have been widely investigated in the past
two decades, with power conversion efficiencies now exceeding 10 %.169
Very few reports are
instead present in literature of hybrid devices where the photoactive layer is interfaced with an
electrolytic solution. Previous works from our group demonstrated that these systems are able to
generate charges upon illumination with visible light and produce a photocurrent with an action
spectrum similar in shape to that of standard photovoltaic devices based on the same materials.125,140
The precise mechanisms behind these effects, however, were not fully elucidated. Narayan and
coworkers170,171
have also carried out extensive electrical characterizations of similar architectures,
but their works focused mainly on devices with active layers significantly thicker than those used by
us; they also employed different semiconducting materials, especially for the electron acceptor. In
any case, different reports have demonstrated the successful interfacing of these devices with
biological tissues;140–143
however, the complete understanding of the mechanisms behind
transduction of light stimuli into bioelectrical signal cannot be achieved without an in-depth
knowledge of the functioning of the polymer/electrolyte interface by itself.
In this chapter, a thorough characterization of hybrid polymer/electrolyte interfaces based on the
conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) as the photoabsorbing material is
carried out. In order to better contextualize the results later presented, a first introduction on the
operation principles of standard organic photovoltaic devices is given in Chapter 3.1. The hybrid
polymer/electrolyte structure is then introduced in Chapter 3.2, with details on the devices realized
and measured in the subsequent experiments. In Chapter 3.3 the photophysics of the active layer is
investigated by means of optical spectroscopies, in order to assess the effect of the electrolyte
presence on the dynamics of photoexcited species. A complete electrical characterization of the
device is then carried out in Chapter 3.4. Finally, the thermal phenomena occurring at the device
surface upon photoexcitation are analyzed in Chapter 3.5.
3.1 Standard organic photovoltaic devices
The standard architecture of an organic photovoltaic (OPV) solar cell,172,173
in the simpler version, is
composed of an active layer, where incident photons are transformed to electron and holes,
sandwiched between two electrodes for charge extraction.
33
The active layer is generally composed of two different p- and n-type organic materials for electron
and hole conduction respectively. The p-type material is usually a conjugated polymer that also acts
as the main photoabsorber. For the n-type phase, a fullerene derivative like phenyl-C61-butyric acid
methyl ester (PCBM) is normally used. Upon light absorption an exciton, i.e. a bound electron-hole
pair, is formed in the polymer. These excitons have binding energies on the order of 0.5 eV131,172
and thus thermal energy is not enough to break them into separated charges (as instead it happens in
inorganic semiconductors). Excitons diffuse in the p-type phase until they recombine; usual
diffusion lengths in polymeric semiconductors are on the scale of 10 nm.174,175
In order to break
down the exciton a driving force is thus needed; in the case of an organic solar cell, this is normally
given by the presence of the n-type material that acts as an electron acceptor. When an exciton
reaches an interface between the p- and n-type phases, the difference in the energetic levels for
electrons in the two materials can be enough to overcome the coulomb attraction between the
electron-hole pair and to obtain free charges.173
The precise photophysical mechanism (or
mechanisms) by which this separation occurs is actually still under debate in the scientific
community.176
In any case, the result is to have a hole in the p-type phase and an electron in the n-
type phase of the active layer; these charges are then free to move towards the electrodes, provided
that a continuous path can be found in the relative phases.
The first devices based on this principle were planar heterojunctions of p- and n-type materials,
similar to standard inorganic devices.177
However, the typical absorption lengths in conjugated
polymers are on the order of 50-100 nm, while exciton diffusion lengths are usually not higher than
10 nm. Only the photons absorbed very close to the interface with the n-type material are thus able
to efficiently be converted into charges, while all the other excitons recombine without contributing
to the final current. To overcome this problem, the bulk heterojunction architecture was
introduced;120
in this case, the p- and n-type materials are mixed together in a single layer composed
of an interpenetrating structure of the two, with phase separation on the order of the exciton
diffusion length.
Once charges have been separated, some driving force is needed in order to promote their migration
to the respective electrodes. Electrodes with different work functions are thus usually employed for
hole and electron collection respectively, producing a built-in electric field in the active layer that
directs the drift of charges. At least one of the two electrodes needs to be transparent, in order for
light to reach the absorbing layer; one of the most used materials is indium tin oxide (ITO), a
conductive oxide that presents a very good trade-off between conductivity and transparency. ITO is
usually used as an anode (i.e. the electrode extracting holes) in standard device architectures, but it
has been shown that it can also act as an electron collector.178
Complete photovoltaic devices
34
usually have also electron and hole selective interlayers between the active material and the
collecting electrodes to help charges being collected at the proper electrode.
3.2 Hybrid devices structure
The devices used in this work are hybrid polymer/electrolyte interfaces, where the metal contact of
a standard organic solar cell has been replaced with an aqueous electrolyte, the typical environment
in which cells are cultured.125
The substrate is a thin glass slab (170 μm) coated with an indium tin oxide (ITO) layer. This
small thickness of the substrate is necessary when the samples have to be analyzed in the
electrophysiology setup, which is based on an inverted microscope with an objective with short
working distance. For specific measurements, also bare glass substrates are employed.
The active material is a thin film of P3HT alone or blended to form a bulk heterojunction with
PCBM. The materials are dissolved in chlorobenzene and the solution is deposited directly on
the substrate via spin-coating. The films obtained, depending on the concentration of the
solution and the spin parameters, have thicknesses that can range from tens to few hundreds of
nanometers. After deposition, all the films are annealed in air at 120 °C for 2 hours; this step
improves crystallinity of P3HT, but it serves also as a sterilization process, mandatory in
experiments with cells. In the following, if not specified otherwise, we refer to the
polymer/electrolyte interface to describe in general a device with an active layer made either
with by pristine P3HT or a P3HT:PCBM blend.
For specific control measurements, another material has been used as the photoabsorbing layer,
namely the photoresist MicroPosit® S1813
®. This material can absorb light in the visible (in
particular in the blue), but has insulating properties and it does not support the generation of
charges when illuminated.
The electrolytes (el) used are different depending on the experiment. Basic electrical
characterization of the interface is generally performed with a solution of sodium chloride at a
concentration of 200 mM, resembling physiological ionic strengths. For experiments with cells,
appropriate solutions are used, like Krebs-Ringer-Henseleit buffer (KRH) or complete cell
growth media.
35
3.3 Spectroscopic characterization
In this section, the characterization of the optical properties of P3HT based films is presented. In
particular, we investigated the dynamics of the photoexcited species in this material on different
timescales by means of pump and probe spectroscopies. While the photophysics of P3HT and
P3HT:PCBM blends has been broadly studied in the past twenty years,179,180
the possible
interactions of these materials with a saline solution are still an open problem. Our main goal was to
assess if the photoexcited species in the active layer could interact with the saline solution to give
photochemical reactions.
3.3.1 Absorption and fluorescence
P3HT is a conjugated polymer that absorbs in the blue-green region of the visible spectrum, while
PCBM absorption band is shifted in the UV, with a small tail in the blue. The fluorescence spectrum
of P3HT is red-shifted with respect to the absorption, peaking between 650 and 700 nm; upon
addition of PCBM, however, singlet excitons are efficiently quenched and luminescence
significantly drops. Typical absorption and fluorescence spectra for thin films (d ≈ 70 nm) of P3HT
and P3HT:PCBM are reported in Figure 3.1.
Figure 3.1 | Absorption and fluorescence (PL) spectra for P3HT and P3HT:PCBM thin
films. The PL spectra are collected for an excitation at 510 nm.
In the absorption spectra, especially for the pristine polymer, the vibronic replicas of the main
electronic transition are visible, consistently with the semicrystalline nature of the polymer. The
shoulder at 605 nm is usually attributed to the formation of a lamellar structure with a π-π stacking
of the thiophene rings.181
The vibronic structure is also visible in the luminescence spectrum of
P3HT, while, as expected, the signal from the blend is significantly quenched. Upon addition of
PCBM, its absorption in the UV below 400 nm becomes clearly visible. Also, the vibronic structure
36
of the P3HT becomes less accentuated, especially the shoulder at 605 nm, because of the presence
of the acceptor molecules that can lower the formation of P3HT crystallites.
In a previous study from our group, the effect of light, oxygen and water on the absorption and
luminescence spectra of P3HT was investigated in depth. In particular, it was demonstrated that
while absorption properties of the polymer are only marginally modified by the presence of oxygen,
the luminescence yield is greatly reduced, especially when the effect of oxygen is combined with
prolonged illumination by visible light.123,133
This effect was attributed to a reversible doping of the
polymer, due to the formation of a charge-transfer complex between P3HT and oxygen molecules.
While this doping mechanism does not break the conjugation of the polymer, conserving its
absorption properties, the charge-transfer complexes act as quenching sites for singlet excitons, thus
diminishing the fluorescence yield of the material. Interestingly, in the same work it was shown that
the presence of water, instead of ambient air, does not introduces significant differences,138
but
actually slightly reduces this doping effect, probably because a lower concentration of oxygen
molecules dissolved in water with respect to air or a different electrostatic coupling with the
environment.
3.3.2 Pump and probe spectroscopies
Optical spectroscopies can be employed to study the evolution in time of excited states in a
material. In particular, transient absorption (TA) techniques have been widely applied for the
investigation of charge generation and recombination processes in organic photovoltaics.182,183
These techniques are based on the pump and probe concept. The basic idea is to photoexcite the
material with a first beam of light resonant with one of its absorption transitions; the generated
photoexcited species modify the electronic transition of the material. A second beam of light is
employed to monitor the evolution in time of these variations, from which the dynamics of the
different excited states can be inferred.
The variations in the absorption spectra due to presence of excited states are quite small (usually no
more than few percents). The relevant signal that is recorded is the normalized differential
transmission (ΔT/T),183,184
i.e. the difference in the probe transmission signal in presence and in
absence of the pump, divided by the ground state transmission. This signal is measured for different
wavelength of the probe beam (TA spectra) and at different delays from the pump excitation (TA
dynamics). Three different kinds of phenomena can contribute to the final TA signal:
Ground-state bleaching (GSB, ΔT/T > 0). Excited species have different electronic transitions
with respect to the ground state. The absorption of the material at the wavelengths resonant with
37
the ground-state transitions thus decreases in the presence of the pump beam, leading to a
positive differential transmission.
Stimulated emission (SE, ΔT/T > 0). When molecules are in singlet excited states, the
impinging probe photons at resonant wavelengths with fluorescence transitions can promote
stimulated emission from the material. Since more photons come out from the material with
respect to the incident ones, the differential transmission is again positive.
Photoinduced absorption (PA, ΔT/T < 0). The presence of excited species in the material
generates new optical transitions not present in the ground-state. At wavelengths resonant with
these new transitions, the differential transmission is thus negative, since absorption is
increasing in the presence of the pump beam.
Once the different bands appearing in the TA spectra have been assigned to the relative excited
species, their populations can be followed in time by looking at the TA dynamics at the relative
wavelengths. However, due spectral congestion, i.e. overlapping of bands arising from different
signals, single-wavelength dynamics can be made up of contributions from more excited species; in
these cases, extracting the temporal evolution of the different populations is not straightforward.
In the spectroscopic measurements that follows, the samples used are thin films of P3HT and
P3HT:PCBM deposited on glass substrates. The films have similar thicknesses of about 70 nm. To
study the effect of the electrolytic solution, we firstly recorded the measurements in air (these data
are referred to as “dry” condition); after that, the sample were put in contact with a saline solution
(NaCl 0.2 M in ultrapure water) and the measurements were performed again (“wet” condition).
3.3.3 Femtosecond transient absorption spectroscopy
Charge generation processes in semiconductors usually occur on the timescale of
femto/picoseconds, but electronic instrumentation is too slow to follow such fast dynamics. To
reconstruct TA dynamics on such ultrafast timescales, mode-locked lasers producing femtosecond
pulses are employed. Both pump and probe are ultrafast pulses, with the probe that is delayed with
respect to the pump varying its optical path by means of a translation stage. The probe transmission
through the sample can thus be measured at different delays to reconstruct the TA dynamics. The
time resolution of the measurement is given by the convolution of the pump and probe durations
and in standard setups is on the order of 100 fs. The maximum delays that can be measured depend
basically on the length of the translation stage and are usually in the range from hundreds of
picoseconds to few nanoseconds. A detailed review of ultrafast transient absorption setups can be
found in Ref. 184.
38
The photophysics of P3HT has been widely investigated in literature.180,185,186
A typical visible TA
spectrum of regioregular P3HT (Figure 3.2a at different probe delays) presents a broad GSB
positive ΔT/T signal between 500 nm and 620 nm and a negative PA2 band peaking at 650 nm. The
PA2 signal has been assigned to interchain polaron pairs (or charge-transfer states), i.e. an electron-
hole couple delocalized between adjacent polymeric chains. These polaron pairs cannot easily
separate into free charges and thus recombine to the ground state in the first 100 ps after
photoexcitation. There is however a very small fraction of photoexcitations that are able to
overcome coulomb attraction and separate into free charges; these are responsible for the two small
PA1 and PA3 bands that become visible at 450 nm and 690 nm especially at longer time delays.
Figure 3.2 | Ultrafast pump-probe spectra on P3HT films in “dry” (a) and “wet” (b)
conditions, taken at different times after photoexcitation (100 fs – 300 ps).
Figure 3.3 | Comparison between the dynamics of the pump-probe signals at 560 nm and
650 nm for the samples in “dry” (red) and “wet” (blue) conditions. The traces of the “wet”
sample have been rescaled to the peak of the “dry” one, in order to have a clearer
comparison of the dynamics.
The presence of saline solution (NaCl, 200 mM) in contact with the polymer does not significantly
change the photophysical picture (Figure 3.2b). Apart from a small variation in the signal intensity,
39
related to smaller pump intensity due to increased losses in the presence of water on the optical
path, the shape of the spectra are the same. Also the dynamics at the different relevant wavelengths
do not show any variation in the decay evolution in time (Figure 3.3).
Figure 3.4 | Ultrafast pump-probe spectra on P3HT:PCBM films in “dry” (a) and “wet”
(b) conditions, taken at different times after photoexcitation (100 fs – 300 ps).
Figure 3.5 | Comparison between the dynamics of the pump-probe signals at 560 nm, 650
nm and 690 nm for the samples in “dry” (red) and “wet” (blue) conditions. The traces of
the “wet” sample have been rescaled to the peak of the “dry” one, in order to have a
clearer comparison of the dynamics.
Upon addition of PCBM, some differences arise in the transient absorption spectra of the films. The
GSB signal is still present for wavelengths below 620 nm, but there is an increase of the signal in
the blue region of the spectrum, consistent with the steady-state absorption spectra reported in
Figure 3.4a. The PA2 band at 650 nm attributed to interchain polarons is still present; more
interestingly, the PA3 signal at 690 nm is here much more visible with respect to the pristine
polymer case (the PA1 band is however hidden by the high GSB signal in the blue). This increase in
the PA3 band is consistent with a much higher generation of free charges in the blend due to the
presence of the electron acceptor. It has also to be highlighted that, in contrast to the pristine case,
40
even after 300 ps there is still a significant fraction of the photoexcited population that has not
recombined back to the ground state. This long-lived population is related to the presence of free
holes and electrons in the material that are spatially separated in the two different phases of the
materials (P3HT and PCBM), thus having a much longer lifetime compared to bound states.
However, also in the case of the P3HT:PCBM blend, no significant differences can be observed
between the “dry” and “wet” conditions, as it can be seen by the shape of the spectra in Figure 3.4b
and the comparison of the dynamics in Figure 3.5.
3.3.4 Nanosecond transient absorption spectroscopy
Next, we analyzed the behavior of the photoexcited species in the ns-μs regime. This is again a
pump-probe experiment, but given the different timescales involved, the setup implementation is
different. Since the operation frequency of electronic measurement instruments is abundantly
beyond the GHz, the temporal dynamics can in this case be followed in real time with an
oscilloscope. For the probe beam is thus sufficient in this case an incoherent lamp. The pump signal
instead, which needs to be shorter than the desired temporal resolution, is given by a Q-switched
laser (pulse duration ≈ 7 ns).
As it has been demonstrated above with ultrafast spectroscopy, only charged species are left in
P3HT after the first few hundreds of ps upon photoexcitation (if intersystem crossing towards triplet
states can be considered negligible).185,186
The dynamics of photobleaching and charge absorption
signals are thus the same and only one of the two needs to be measured in order to have a complete
picture. In Figure 3.6 the normalized temporal traces at 570 nm (in the P3HT bleaching band) are
reported for both P3HT and P3HT:PCBM samples in “dry” and “wet” conditions.
Figure 3.6 | Comparison of the normalized dynamics (presented as the difference in
optical density, ΔOD, in presence and absence of the pump) for the photobleaching band
at 570 nm of P3HT (a) and P3HT:PCBM (b) samples in “dry” (red) and “wet” (blue)
conditions.
41
As it can be expected, the lifetime of charged species is longer in the blend sample with respect to
the pristine polymer. More interestingly, in both cases a slight difference can be observed between
the measurements in “dry” and “wet” conditions. However, this is to be attributed just to an
enhanced thermal conductivity of water with respect to air (kwater = 0.6 W m-1
K-1
, versus kair = 0.024
W m-1
K-1
).187
Indeed, similar variations in the dynamics have already been reported in literature to
occur upon changing the thermal conductivity of the substrate. In particular, faster dynamics were
observed in samples with a more thermally conductive substrate (namely sapphire, with respect to
standard glass).188
In our case, the same explanation can be applied, considering that water has a
higher thermal conductivity with respect to air.
3.3.5 CW photoinduced absorption spectroscopy
Free charges recombine on the nano-microsecond timescale; after that, however, in the material
some trapped species still remain up to the millisecond range. Since their number is usually quite
small, the relative variations in the transmission spectra are on the order of 10-4
. To detect such
small signals, an oscilloscope is not sensible enough and lock-in detection is needed. In this case,
both the pump and the probe are continuous-wave beams; the pump is periodically modulated,
usually with a mechanical chopper, and the corresponding variations in probe transmission are
extracted by the lock-in amplifier. This is not a transient technique and actually only information on
steady-state populations can be detected. However, information on the lifetimes can be obtained
performing a frequency-domain analysis varying the pump modulation.186,189
In pristine films of P3HT, detrapping of the trap states due to thermal energy quickly favors the
recombination of these species. At room temperature it is thus very difficult to detect a CW-PIA
signal.189
To slow down this process, cryogenic measurements are usually performed; however, if
the contribution of direct contact with water is to be assessed, this strategy cannot be applied. For
this reason, we could perform only measurements on P3HT:PCBM blends. In this material, the
higher number of charges generated and the spatial segregation between electrons and holes gives
higher steady-state populations that can be reliably detected.
Visible and near-IR spectra for both the “wet” and “dry” case of a P3HT:PCBM thin film are
reported in Figure 3.7. Apart from the bleaching at wavelengths shorter than 620 nm, a broad
negative signal is visible between 650 nm and 1050 nm. This signal is actually made up by two
different bands, PA1 and PA2 peaking at 690 nm and 980 nm respectively. These bands have been
assigned to two different types of polarons: PA2 band is related to intrachain polarons in the
amorphous fraction of the material; PA1 signal is instead attributed to delocalized polarons in the
42
lamellar structures. In any case, also on the timescales no significant variation can be observed
between the “wet” and “dry” case.
Figure 3.7 | CW-PIA spectra in for P3HT:PCBM films in “dry” (red) and “wet” (blue)
conditions upon excitation at 560 nm.
3.4 Electrical characterization
The spectroscopic measurements of the previous section have shown that charge transfer reactions
from the photoactive material to the ionic conductor are not occurring, at least in a sizable amount.
It is thus very likely that, in contrast to the metallic electrode of standard OPV devices, the liquid
electrolyte does not behave as an ohmic contact for charge extraction. This observation suggests
that the functioning of these hybrid devices can be quite different from a standard solar cell.
Previous measurements from our group have demonstrated that P3HT in presence of oxygen, and
especially upon illumination, gets quickly p-doped via the formation of a charge transfer complex
between the polymer backbone and the O2 molecule.138
In particular, concentrations of free holes in
the range of Np = 1017
-1018
cm-3
have been estimated from capacitance measurements of devices
kept in electrolytic solution under illumination. Given these levels of doping, at the polymer/ITO
interface electrons should be injected into the polymer from the contact, creating an interfacial
dipole. This dipole can screen the internal electric field in the active layer, which in standard OPVs
drives the photogenerated charges towards the electrodes. Upon photoexcitation of the active
material, the absence of a net electric field in the bulk implies that in this region holes and electrons
do not get efficiently separated and should thus tend to recombine, not contributing to the final
photocurrent. Only in the interfacial region with ITO the electric field is able to separate
photogenerated electrons and holes, and to produce a sizable current. In particular, it should be
expected that electrons are collected by the ITO.
43
In order to support this picture and to understand the actual coupling of the electrical signals
generated upon illumination in the active material with the ionic conductor, we have investigated in
more details the electrical functioning of the hybrid polymer/electrolyte systems. To simplify the
analysis, we start by studying the case of the pure P3HT active layer. In particular, we first
investigate the charge generation and separation processes in the material by measuring the
photovoltage produced in the ITO/P3HT/el device; we then study the coupling mechanisms at the
polymer/electrolyte interface with photocurrent and surface potential measurements. Finally, we
briefly compare the results obtained in the case of using the P3HT:PCBM blend as the active
material.
The following study is aimed at understanding the behavior of our hybrid devices upon illumination
with short pulses of light (i.e. few tens of milliseconds), since these are the relevant timescales when
capacitive stimulation of cells is considered as the final goal. The picture we draw here cannot thus
be transposed to the case of prolonged illumination, in which other physico-chemical phenomena
can intervene. Upon continuous illumination electrochemical reactions are indeed activated in these
hybrid interfaces, as it has been shown in previous works of our group;126,127
the description of these
mechanisms is however beyond the scope of this investigation, which is focused on the processes
occurring over short timescales.
3.4.1 Photovoltage measurements
We start the investigation of electrical properties of the hybrid polymer/electrolyte interfaces by
presenting an in-depth analysis of transient photovoltage measurements performed on ITO/P3HT/el
devices. Since we are dealing with an electrochemical system, in which both electronic and ionic
conductions are present, particular attention must be paid in the measurements. In particular, we
used for the recordings a three-electrode configuration. In this scheme, the current flows between
the working electrode (WE), which is the ITO/P3HT device we want to measure, and a counter-
electrode (CE) made of an inert material (in our case a platinum wire); however, the potential of the
WE is measured against a third electrode, the reference electrode (RE), whose potential is well-
known and stable. For our measurements, an Ag/AgCl electrode in a saturated KCl solution was
employed. The photovoltage transient generated at the WE is measured as the potential necessary to
counterbalance the current flowing through the CE, i.e. to keep the system in open circuit condition.
The complete measurement apparatus is described in more details in Appendix A.
The measurements presented in this section were performed by recording the photovoltage of
ITO/P3HT/el samples upon illumination with a 50 ms pulse of light from a collimated white LED.
The samples had approximately an area of 200 mm2 and were entirely illuminated by the spot; the
44
electrolyte was a water solution of NaCl at a concentration of 0.2 M. A typical recorded trace is
reported in Figure 3.8a.
Figure 3.8 | (a) Photovoltage response measured on a P3HT sample upon illumination
with a 50 ms light pulse (represented by the light blue box). (b) Typical photovoltage trace
measured in a standard all-solid ITO/P3HT/Al photovoltaic device.
Before switching on the light, a constant potential Vdark is present. Its value is related to the
electrochemical equilibrium condition for the system with respect to the Ag/AgCl potential and is
subject to variations from sample to sample, with usually positive values ranging up to about 200
mV. This variability is attributed to small differences in the polymeric films, in terms of
morphology, defects and doping due to previous, unavoidable exposure to light and ambient
oxygen. Upon illumination, the potential has a downward dynamic that saturates after few
milliseconds at a plateau value (in this case, about ΔV ≈ -100 mV), consistently with our hypothesis
of electrons being collected at the ITO electrode.190,191
As the light is switched off, the system tends
to return to its original equilibrium condition, but, interestingly, this process is characterized by a
markedly longer time constant with respect to the onset of the excitation. It has also to be noted that,
during the light pulse, the potential does not remain constant at the plateau value and a slight
variation towards positive values can be observed. This process is probably related to the
instauration of electrochemical reactions at the polymer/electrolyte interface;126,127
since however
their effect is minimal over short timescales, we will neglect this component in the following
investigation.
As a comparison, the photovoltage generated in a standard all-solid device (ITO/P3HT/Al), in
which the liquid electrode is replaced with an aluminium contact, is shown in Figure 3.8b. In this
case, the signal is positive, consistently with the internal electric field in the bulk of the device that
drives the electrons towards the aluminium, while the ITO is here collecting the holes.
45
In order to corroborate our hypothesis of an efficient generation of charges only in proximity of the
ITO interfaces, we recorded transient photovoltage measurements on ITO/P3HT/el samples of
different thickness and by changing the side from which light impinge on the device (i.e. from the
ITO or from the electrolyte). All measurements were performed at the same light intensity of 267.5
μW/mm2. From the traces, reported in Figure 3.9, the initial offset in dark condition has been
subtracted, in order to highlight the actual variation in the ITO potential upon illumination between
the different cases.
Figure 3.9 | Photovoltage responses of P3HT films with increasing thicknesses upon
illumination with a 50 ms light pulse (light blue box) from the ITO side (a) and the
electrolyte side (b).
Figure 3.10 | Dependence on thickness of the voltage peak (a) and rise time (b) of the
photopotentials measured upon illumination of the P3HT device from the ITO (blue) or
electorlyte (red) side.
As it can be expected, the peak potentials reached during the light pulse are quite dependent on the
film thickness; however, it is interesting to notice that this dependence is markedly different for the
two cases with different side of illumination (Figure 3.10a). In the case of light coming from the
ITO side, the potentials show a monotonic increase with the thickness of the active layer, reaching
46
saturation at the higher values (d > 150 nm). In the case of illumination from the electrolyte,
instead, the photopotentials have a maximum for an optimal thickness (around ≈ 40 nm), and then
start to decrease as the films get thicker. It can also be noticed that, while the onset of the
photovoltage has a similar time constant in all the traces for the ITO illumination, in the other case
the building up of the signal gets slower for increasing dimensions (Figure 3.10b).
The data here presented are in good agreement with the photophysical picture proposed before.
When light is impinging from the ITO side, it is mainly absorbed close to this interface. The local
electric field can efficiently separate the hole and electron pairs, with the electron being collected at
the ITO and the building up of the photopotential. The higher thicknesses of the devices initially (d
< 50 nm) are reflected in a higher number of absorbed photons. However, upon a further increase of
the dimensions this improvement is less important because of two concomitant factors. Firstly,
according the Lambert-Beer law the highest fraction of light is absorbed in the first few tens of
nanometres (the P3HT absorption length of about 100 nm); increasing further the device thickness
does not lead to a sizable gain in the number photons absorbed. Secondly, the photons absorbed
further from the ITO interface than the region where the electric field is present do not contribute to
the final signal in a significant manner. The situation is different when the illumination comes from
the electrolyte side. In this case, the majority of photons are absorbed close to the P3HT/el interface.
For thin samples (d < 50 nm) this is not an issue, because the absorbed photons are anyway also
close to the ITO/P3HT interface and the photoexcited electrons in the active materials can still be
efficiently collected at the electrode. However, as the films get thicker, the number of photons
absorbed in the region with a sizable electric field decreases and the generation of the photovoltage
accordingly becomes less efficient.
We also performed measurements in dependence of the light intensity impinging on the sample.
Figure 3.11 reports different photovoltage transients recorded with illumination (from the ITO side)
ranging from 4.7 μW/mm2 to 197.5 μW/mm
2. The peak potential, as expected, increases with higher
intensities; in particular, a logarithmic dependence of the voltage can be observed (Figure 3.12a).
Moreover, also in this case an interesting slowing down of the ITO charging is observed when the
number of photogenerated charges is lower (Figure 3.12b), with a time constant for the
photovoltage formation that quickly drops of more than an order of magnitude (from 34 to 2 ms).
The logarithmic increase of the voltage with light intensity is an indication that the system behaves
as a rectifying junction. Indeed, the contact between P3HT and different metals (among which also
ITO) has been studied in details and it has been seen to form a Schottky junction.192–195
The strong
dependence of the lifetimes on the light intensity can be explained by considering that the
photocurrent flowing through the junction is, at least in a first approximation, proportional to the
number of absorbed photons, while the junction potential increases only logarithmically. In
47
particular, to increase the potential is necessary to charge the junction capacitance and thus to
accumulate a charge that is proportional to the photopotential. Also this charge has a logarithmic
dependence on the light intensity, but since the photocurrent increases linearly with it, at higher
intensities a shorter time is needed to charge the junction. The same reasoning can also be applied to
the case of the increasing time constant with device thickness for illumination from the electrolyte
side, since also in this case what is actually happening is that less photon are absorbed in the
junction region.
Figure 3.11 | (a) Time traces of photovoltage measurements performed on a P3HT sample
(d ≈ 65 nm) upon illumination with a 50 ms pulse at increasing light intensities.
Figure 3.12 | Intensity dependence of the photovoltage peak (b) and rise time (c).
The photovoltage measurements reported above give us information mainly on what happens at the
ITO/P3HT junction upon illumination. In the discussion of the data, we have not considered
eventual effects occurring at the P3HT/el interface. This assumption is supported by the following
measurements (Figure 3.13), in which the photovoltage has been measured upon changing the
concentration of the ion species in the electrolyte. In particular, the recordings have been performed
with NaCl at 200 mM, 20 mM and in ultrapure water. The peak potential reached in all cases
remains basically the same, suggesting that the ion species in the electrolyte are not involved in its
48
formation. Interestingly, only at the higher concentration the photovoltage signal is seen to slightly
tend towards positive values as the illumination continues, confirming our initial hypothesis that
this process can be attributed to some phenomena occurring at the P3HT/el interface. However, for
what concerns the initial negative photovoltage peak, its independence from the ionic strength of the
solution is a strong indication that the P3HT/el interface does not give significant contributions to
its formation and it is thus mainly determined by the photophysics of the ITO/P3HT junction.
Figure 3.13 | Dependence of the photovoltage signal on the concentration of the
electrolyte solution, measured on a P3HT sample (d ≈ 65 nm) for illumination from the
ITO side.
3.4.2 Photocurrent measurements
While the photovoltage measurements presented in the previous section give an exhaustive picture
on the functioning of the ITO/P3HT interface, they do not provide direct information on the effects
(at short times) of this photoexcitation on the device surface, which is the region where cell are
eventually cultured. In this section we present photocurrent measurements on the ITO/P3HT/el
devices, in which the ITO contact is short-circuited with a platinum counter-electrode in solution
(details on the experimental setup are given in Appendix A). Figure 3.14 shows the traces recorded
for a ≈ 70 nm P3HT film upon illumination from the ITO side with pulses of light (λ = 530 nm) at
different light intensities, ranging from 15.4 to 384 μW/mm2.
The positive signal upon illumination is consistent with the current flowing from the ITO/P3HT
device to the counter-electrode through the solution, i.e. with electrons from the polymer being
collected at the ITO as demonstrated with photovoltage measurements. As expected, the recorded
signals increase with light intensity but, interestingly, the photocurrent decays back to zero quite
rapidly during the light pulse, with a decay time constant that is on the order of few milliseconds,
decreasing for higher illuminations. In a conventional solar cell the current produced upon
illumination is, at least ideally, constant over time; the fact that in the ITO/P3HT/el device it drops
to zero after few milliseconds is again an indication that this system behaves differently with respect
49
to standard OPVs.196
In particular, this behavior is an indication that there is an element in the
device structure that tends to block the flowing of current at steady state. Consistently with our
previous observations that there are no significant redox reactions taking place, on the investigated
timescales, at the P3HT/el interface, it is thus reasonable to conclude that this interface behaves,
electrically, as a capacitance (Figure 3.15). In electrochemical terms, we can say that the
photocurrent measured in this system is purely capacitive, with no appreciable faradaic
contributions.
Figure 3.14 | Photocurrent measurements on a P3HT film upon illumination with a 50 ms
pulse of light (light blue box) at increasing light intensities.
This finding is actually quite relevant in the context of using such hybrid devices for the
photostimulation of biological systems: eventual redox reactions could in fact lead to the generation
of harmful chemical species for the cell like oxidizing agents. Moreover, irreversible chemical
reactions would compromise the device stability in time. On the downside, this capacitive behavior
of the interface limits the total quantity of charge that the device can displace upon illumination:
once the P3HT/el capacitance has been charged, the current stops flowing through the device even
if the light is still on.
It is thus clear that one of the most important parameters that determine the functioning of the
device is the P3HT/el capacitance. This capacitance is given by the double layer of charges
accumulated on one side in the polymer at its surface and on the other in the diffuse layer of ions in
the electrolytic solution.170,197
The typical capacitance value that we measured, by means of
impedance spectroscopy, for P3HT is around Cint = 2 μF/cm2.197
This value is actually about one
order of magnitude higher than those usually found for standard inorganic devices based on silicon
used for capacitive stimulation. Such an increase is mainly due to the fact the that polymer surface
is in direct contact with the electrolyte in our hybrid devices, while in the case of silicon, an oxide
layer is always present between the semiconductor and the aqueous medium.91,102
This oxide layer
can have a thickness ranging from tens to hundreds of nanometers and has thus a significant effect
50
in reducing the interface capacitance (a 10 nm layer of SiO2 has a geometric capacitance of Cox =
0.34 μF/cm2).
Figure 3.15 | Schematic representation of the ITO/P3HT/el architecture. The green box
represent the illuminated region. Cint: interfacial capacitance; Rbulk: resistance of the
polymer bulk; Iph: photocurrent generated at the Schottky junction between P3HT and
ITO.
3.4.3 Surface potential measurements
The measurements of the previous section show that the ITO/P3HT/el device is indeed able to
generate a photocurrent, even though transient, in short-circuit conditions, i.e. with the ITO contact
connected to a counter-electrode in the electrolyte. However, when they are used as active
substrates for cell culture and stimulation, these devices work in a different configuration:140–142
(i)
the ITO electrode is not directly contacted with an external circuit; (ii) only a small fraction of the
active area is illuminated (ideally, it would be desirable to address single cells, thus using a spot of
about 10-20 μm of diameter).
In order to understand if the capacitive charging of the P3HT/el interface occurs also in these
conditions, we measured the local potential generated at the surface of the device in conditions
similar to those used in experiments with cells. The sample was put in a petri-dish and immersed in
the electrolytic solution (NaCl 0.2 M); the local surface potential was measured with a glass
micropipette electrode positioned in close proximity (≈ 2 μm) of the P3HT interface. The device
was illuminated from the bottom (i.e. the ITO side) through a 40x microscope objective; the light
spot had a diameter of 540 μm. A more detailed description of the experimental method is reported
in Appendix A. The recorded traces for illumination with a 50 ms light pulse (λ = 470 nm) at
different intensities are reported in Figure 3.16a.
These measurements show that, upon photoexcitation, there is a flow of charges in the device even
if the ITO electrode is not contacted. This current produces a variation of the potential at the
51
P3HT/el surface. Also in this case, because of the capacitive nature of the interface, the current
drops to zero quite rapidly, in few tens of milliseconds. In order for this current to flow, the
electrical circuit between the ITO layer and the electrolyte needs to be closed. Since no external
contact is made in these measurements, there has to be a parasitic coupling between the two, as
depicted in Figure 3.17a. This coupling may be due to a parasitic capacitance between the ITO
electrode and the solution or to physical contact between the two at the borders of the device or
cracks and defects in the polymer layer.
Figure 3.16 | (a) Local potentials measured at the P3HT/el interface with a glass
micropipette electrode upon illumination with a 50 ms light pulse (light blue box) focused
on the active surface (A ≈ 0.23 mm2) at increasing light intensities. (b) Surface potential
measurement on a glass/P3HT/el samples without the ITO contact.
In order to confirm the role of the ITO layer in the establishment of this parasitic coupling, we
measured the dependence of the surface potential signal from the lateral dimension of the ITO
contact. We realized ITO patches of increasing areas (from 0.37 to 18 mm2, in any case bigger than
the illumination spot size) on a glass substrate by selective etching with HCl of the conductive
oxide before the deposition of the P3HT active layer. The plot of Figure 3.17b shows that indeed the
peak of the surface potential measured depends on the dimension of the ITO contact underneath,
with a saturation for areas larger than ≈ 4-5 mm2. This result supports the hypothesis of a parasitic
coupling that increases with larger ITO electrodes. For small ITO areas, the low value of this
parasitic capacitance limits the current that can flow in the circuit; when the electrode gets bigger,
however, the current is no more limited by this coupling, but by the actual capacitance of the
polymer/electrolyte interface, and thus the signals does not grow any larger.
It is thus clear that the ITO contact is fundamental for the electrical charging of the device, because
it promotes the formation of a photovoltage at the junction with the ITO but also because in non-
contacted conditions it is fundamental for having the parasitic coupling with the solution that closes
the circuit, allowing the formation of a surface potential at the device interface. Interestingly,
52
surface potential measurements performed on devices in which the active layer is deposited on bare
glass substrates without the ITO layer did not give any signal, confirming that in this situation the
capacitive charging of the interface is hindered (Figure 3.16b).
Figure 3.17 | (a) Electrical scheme of the parasitic coupling between the ITO electrode
and the electrolytic solution in the surface potential measurements. Rel: resistance of the
electrolytic solution; Cpar: parasitic capacitance; RE: reference electrode. (b) Dependence
of the surface potential signal on the dimensions of the ITO electrode; the data represent
the peak voltage measured on the onset of the light pulse (λ = 470 nm, I = 235 μW/mm2).
3.4.4 Measurements on P3HT:PCBM
We conclude the characterization of the electrical properties of the hybrid interfaces by analyzing
their behavior when the active layer is replaced with a P3HT:PCBM blend. In this case, the charge
generation processes in the material are favored due to the presence of the electron acceptor, as also
shown by the spectroscopic data presented in Section 3.3. The photovoltage, photocurrents and
surface potential measurements for P3HT:PCBM films of ≈ 50 nm of thickness are reported in
Figure 3.18.
Figure 3.18 | (a) Photovoltage measurements of a P3HT:PCBM film upon illumination
with a 100 ms light pulse. (b) Photocurrents recordings for a P3HT:PCBM film
illuminated with a 20 ms pulse at increasing light intensities. (c) Surface potential elicited
at the P3HT:PCBM/el interface upon illumination with a 20 ms pulse of light at different
intensities.
53
Qualitatively, the data recorded on the blend are similar to the case of the pristine P3HT active
layer, suggesting a similar functioning mechanism of the hybrid interface. However, it is evident
from the traces that the blend presents higher signals, consistently with the increased charge
generation efficiency. This increase is also accompanied by faster dynamics with respect to the
P3HT case, due to the fact that, with higher currents, the surface P3HT/el capacitance gets charged
more quickly. Also in the case of P3HT:PCBM, devices without the ITO contact did not give any
surface potential signals.
3.5 Thermal characterization
Upon illumination of the active material, the photogenerated excitons can recombine or be
dissociated into charges; these charges, however, are not extracted from the device. After an
equilibrium situation has been reached with the charging of the interface capacitance, all
photogenerated charges will recombine. Given the low fluorescence yield of P3HT, especially in the
doped state,138
recombination of excitons and charges occurs mainly via non-radiatively pathways.
Most of the energy of the photons absorbed by the material is thus transformed into vibrational
energy, i.e. into local heating of the active layer. This thermal energy is then dissipated through both
the substrate and the electrolyte, with a local increase in temperature.
3.5.1 Local temperature measurements
In order to assess the thermal effects of photoexcitation of the active layer, we have measured the
local temperature variations of the bath at the polymer/electrolyte upon light pulses of different
duration and intensity. The measurements were carried out with the technique of the calibrated
pipette.198
This technique exploits the temperature dependence of the resistance of a glass
micropipette filled with an electrolytic solution (Figure 3.19). If this relationship is known, the
temperature evolution in the close proximity of the micropipette tip can be known simply by
measuring the current flowing through the pipette for a fixed applied voltage.
The measurements were carried out in the standard electrophysiology setup described in Appendix
A. Aqueous solutions of sodium chloride at 200 mM were used as both the bath and the pipette
filling media, in order not to have liquid junction potentials arising at the pipette tip. To calibrate the
pipette resistance, we measured the current response flowing through the pipette for a potential step
of ΔV = 5 mV at different values of the bath temperature controlled with an external heater (Figure
3.20a). Plotting the measured currents against the temperature in an Arrhenius plot results in a
straight line relationship (Figure 3.20b), from which a constant activation energy Ea = 11.7 kJ/mol
54
for the process can be extracted. The relationship between current and temperature can thus be
expressed in the following form:198
where I0 is the current flowing through the pipette at a base temperature T0 and R is the ideal gas
constant.
Figure 3.19 | Typical variation of the resistance of a pipette tip placed in proximity of a
photoactive interface upon illumination with a 200 ms pulse of light (light blue box).
Figure 3.20 | (a) Current responses of the pipette upon the application of a 5 mV potential
difference with respect to the reference electrode at different temperatures of the bath. (b)
Arrhenius plot for the temperature dependence of the current, with a linear fit to extract
the activation energy Ea.
We measured the temperature variations at the polymer/electrolyte interface upon illumination for
thin films deposited on glass substrates of different active materials: P3HT, P3HT:PCBM and
Photoresist. The deposition processes for the three samples were carefully controlled in order to
obtain films with similar light absorption coefficient at the selected excitation wavelengths (λ = 475
(3.1)
55
nm for P3HT and P3HT:CBM, λ = 435 nm for Photoresist, see Figure 3.21). The pipette tip was
micromanipulated in close proximity of the interface (≈ 2 μm) and an offset potential was applied
with respect to the reference electrode in order to have a current of about I0 = 4 nA at the base
temperature of T0 = 23 °C. The time evolution of the current was then recorded upon illumination of
the substrate with pulses of 20 ms and 200 ms at different light intensities. The temperature profile
was finally extracted with the formula of Equation (3.1).
Figure 3.21 | Absorption spectra for the P3HT, P3HT:PCBM and Photoresist films used
to measure the local temperature increase upon illumination. The blue and light blue boxes
represent the two wavelengths used to excite Photoresist (λ = 430 nm) and P3HT-based (λ
= 475 nm) samples respectively. Different wavelengths were chosen in order to have
comparable absorption in the three samples.
The recorded traces (Figure 3.22) show similar dynamics for all the investigate samples, indicating
that they posses similar properties in terms of heat generation. When light is switched on,
temperature increases monotonically with a decreasing slope. Upon illumination, heat is generated
in the active material and transferred to the bath in proximity of the interface; as time passes,
however, this thermal energy starts to be dissipated away from the surface and the temperature
increase becomes slower. As the light pulse ends, temperature starts to decrease until the base value
is finally reached.
The measured increases in temperature scale linearly with light intensity; in particular, at the
maximum light intensity used, temperature variations of about 3 °C and 7 °C are measured at the
end of the 20 ms and 200 ms light pulses respectively.
56
Figure 3.22 | Local temperature dynamics in close proximity of the device surfaces for
P3HT (left), P3HT:PCBM (center) and Photoresist (right) upon illumination with light
pulses (represented by the blue and light blue boxes) of 20 ms (top) and 200 ms (bottom)
at different intensities.
3.5.2 Numerical simulations
In order to corroborate the origin of the recorded signal as arising from local temperature variations,
we conducted a numerical simulation of heat generation and diffusion in the system. A classical
heat diffusion model was implemented with the geometry depicted in Figure 3.23, with the
following hypotheses:
1. All energy absorbed by the active layer is transformed into heat, consistently with the fact that
virtually no charges are extracted from the devices and luminescence yield is negligible.
2. The bottom surface of the substrate is thermally isolated from the sample holder.
3. The simulated domain of the bath is large enough that the temperature at its extremes can be
considered constant and equal to the base temperature.
57
Figure 3.23 | Scheme of the geometry used for the numerical solution of the heat diffusion
problem to calculate the temperature dynamics at the polymer/electrolyte interface (the
drawing is not in scale).
Given the circular section of the light spot (radius 270 μm), a cylindrical symmetry was assumed in
solving the problem. Given the similar experimental results obtained for the three different
substrates, the case of P3HT was considered in the simulations. Actually, given the very small
thickness of the active material with respect to the other two domains (glass and water), no
significant effect on the results is expected by small variation of its thermal parameters. The
physical parameters for the different materials (glass, water and P3HT) were taken from literature
and are summarized in Table 3.1.
Table 3.1 | Values of the parameters used for the resolution of the numerical model of
heat diffusion. cp: specific heat at constant pressure; ρ: density; k: thermal conductivity; α:
absorption length; t: thickness.
The temporal traces of temperature variation in the bath in close proximity with the surface of the
device were extracted from the simulation and compared with the experimental results (respectively
open circles and solid lines in Figure 3.24). A very good agreement can be seen between the two,
validating the experimental calibrated pipette method used for measuring the local temperature.
Glass Water P3HT
cp [J kg-1
K-1
] 840 4181.3 1400 [199
]
ρ [kg m-3
] 2500 1000 1100 [200
]
k [W m-1
K-1
] 1 0.6 0.2 [201
]
α [cm-1
] - - 105
t [μm] 170 2000 0.15
58
Figure 3.24 | Comparison between the experimentally measured temperature dynamics on
a P3HT substrates (open circles) and the results of the numerical simulations (solid lines)
upon stimulation with 20 ms (a) and 200 ms (b) light pulses (represented by the light blue
boxes) at different intensities.
The spatial distributions of temperature as obtained from the numerical simulation, at different
times during and after a 200 ms pulse, are reported in Figure 3.25. They show that the temperature
increase is an effect basically localized to the illuminated area of the device and its immediate
surroundings, quickly decaying in few hundreds of micrometers from the interface, with the bulk of
the electrolyte remaining at the base temperature during the light pulse.
Figure 3.25 | Distribution of the local temperature values in space (along the radial and
axial direction) at different times during and after a 200 ms pulse (switched on at t = 0 ms)
of 57 mW/mm2. The vertical light blue lines separate the light by the dark region, while
the horizontal dashed lines is located at the position of the thin-film of active material.
59
Chapter 4 – Coupling hybrid interfaces with cells
In Chapter 3 we have characterized the functioning of the polymer/electrolyte interfaces in terms of
the physical phenomena occurring upon photoexcitation. In particular, we have observed that two
different effects can take place at the surface of these hybrid devices: (i) a capacitive charging of the
interface that leads to a superficial transient potential; (ii) a local heating of the electrolyte following
the thermalization of the photoexcited species in the absorbing layer.
In this chapter we try to understand if and how these two effects can be exploited to modulate the
bioelectrical activity of cells. In particular, we are interested in studying the variations of the
membrane potential of cells grown on the active surface upon illumination. Our previous
publications have demonstrated that such interfaces are actually able to stimulate electrical activity
in neurons,140,141
astrocytes142
and explanted retinas.141
However, they are quite complex biological
systems and it is difficult to assess the direct effects of the polymer-mediated stimulation and
decouple them from the active responses they trigger. We thus decided here to use simpler cellular
systems to understand the actual effects of photostimulation on the membrane properties of the cell.
The cells used for this investigation are introduced in Chapter 4.1, along with a brief introduction on
experimental techniques used for measuring their bioelectrical properties. In Chapter 4.2 we
conduct experiment of photostimulation on these cells and we observe that both thermal and
electrical effects can take place in modulation of their membrane potential. Chapter 4.3 is dedicated
to an in-depth analysis of the thermal mechanisms and their influence on the membrane electrical
properties. Finally, in Chapter 4.4 we discuss about how the capacitive charging of the interface can
affect the membrane potential.
4.1 Human Embryonic Kidney (HEK) 293 cells
The previous works published by our group on the subject of photostimulation of biological systems
with P3HT-based hybrid interfaces were mainly focused on the study of neurons.140,141
These cells
are the main active elements of the nervous system, where they transport and elaborate the electrical
signals that control virtually all the vital functions in the body. Neurons are thus the main target in
the development of brain-machine interfaces to sense and control the functioning of living
organisms.90,91
The response of a neuron to a stimulus is generally represented by an action potential
(see Chapter 2.3); this rapid variation of the membrane potential is however a codified response,
60
which is basically the same independently on the origin or the intensity of the stimulus, as long as a
certain threshold is reached. It is thus difficult to directly correlate the response of a neuron to a
specific cause and usually pharmacological modulation of different electrophysiological properties
of the cell is needed to address the problem.
In order to have a clearer understanding of the effects of polymer-mediated photostimulation on the
electrical properties of cellular membranes we thus decided to employ a simpler biological system.
Although only neurons and few other excitable cells (like myocytes and endocrine cells) are able to
sustain the firing of action potentials, all cells (for simplicity’s sake, we refer here to animal cells)
possess a plasma membrane and maintain a potential difference across it. The cells we selected for
our investigation are Human Embryonic Kidney 293 (HEK-293) cells.202
As their name suggests,
these cells have been isolated from the kidney of a human embryo in 1973 in the laboratories of
prof. Alex van der Eb (Leiden, The Netherlands) and have been subsequently genetically modified
to yield an immortalized cellular line. These cells are widely used in cell biology due to their
easiness in handling and can be grown in adhering cultures on different substrates. In particular,
they have found a lot of applications in electrophysiology for different reasons:202
(i) they are easy
to transfect; (ii) they are non-excitable cells that express quite low intrinsic conductivities and thus
can be used as a platform to study the behavior of exogenously expressed ion channels; (iii) they
have a compact shape with few processes, reducing space-clamp artifacts in electrophysiological
measurements.
Apart for their easy handling and their suitability for electrophysiological measurements, we
decided to use this particular cell type as a simple model of a cellular plasma membrane with a
small intrinsic conductivity.203
Our goal is thus to understand the actual biophysical mechanism that
couples the thermal and capacitive effects occurring at the polymer/electrolyte interface to the
electrical properties of the cell membrane. In principle, other simpler, non-living systems could be
used for this purpose, for example suspended lipid bilayers204
or vesicles.89
However, these artificial
systems lack all the complex machinery of a real cell that mediate the adhesion to the substrate.
With these lipid-based systems we would thus probably have a quite different interaction with the
substrate, both in terms of distance and composition of the cleft (i.e. the space between the surface
of the device and the basal membrane of the cell). HEK-293 cells can be considered as a good
compromise between a complex biological cell like a neuron and a too simplistic artificial system
like a lipid vesicle.
The experiments reported in this chapter on the photostimulation of HEK-293 cells, while
interesting on their own, cannot be directly transposed to more complex systems like neurons and
retinas. Different cells have different compositions of the membrane, both in terms of the lipid
content and of the actual transmembrane proteins expressed (especially ion channels);147
the effects
61
of photostimulation can thus be in principle different on different cellular types. However, the
understanding of the basic biophysical mechanisms occurring in HEK-293 cells can be used as a
foundation upon which building the investigation of more complex systems. We will comment
more on this important issue in Chapter 5.
4.1.1 Cultures of HEK-293 cells on polymeric substrates
Before starting with electrophysiological investigation of the photostimulation of HEK-293 cells, it
is necessary to assess if they can be cultured on our interfaces based on semiconducting polymers.
The biocompatibility of P3HT-based thin films has actually been already investigated in the past on
different cellular types like fibroblasts, neurons and astrocytes (see Chapter 1.4).
Given the hydrophobicity of P3HT films, cells cannot be grown directly on them. An interlayer is
thus usually needed in order to promote cellular adhesion. Among the different molecules that can
be used for this purpose, we opted to use fibronectin, a glycoprotein of the extracellular matrix that
had already been investigated by Scarpa et al.139
for adhesion of cells on P3HT. The devices on
which we cultured HEK-293 cells are the same described in Chapter 3.2 and are basically composed
of a thin glass substrate (170 μm) with or without an ITO coating, on top of which a thin-film of the
active material (P3HT, P3HT:PCBM or photoresist) is spin-coated. The procedure for cell culturing
is reported in the following.
1. The devices are first sterilized in an oven at 120 °C for 2 h. This step is crucial to eliminate
bacterial contaminations that can hamper the growth of animal cells. From this point on, all
handling of the substrates needs to be done in sterile conditions under a biological hood.
2. The devices are put in a petri-dish or a multiwell and a solution of fibronectin in PBS
(Phosphate Buffered Saline) at a concentration of 2 μg/ml is casted on its surface. In order to
keep the solution on the hydrophobic substrates, PDMS (polydimethylsiloxane) wells are used.
The devices are incubated at 37 °C for at least 30 minutes to promote adhesion of the
fibronectin to the surface of the polymeric film. The substrates are then rinsed with PBS.
3. After the deposition of the fibronectin layer, HEK-293 cells are seeded on the surface of the
devices at a desired density (number of cells per cm2) in complete growth medium (see
Appendix A). The cells are then incubated at 37 °C and 5 % CO2.
In normal conditions, HEK-293 cells adhere to the substrate in few hours after seeding. If the
seeding density is not too high, they can be initially found as single cells; however, they quickly
start to replicate until a continuous monolayer covering the entire surface is formed. Depending on
the initial density, this process occurs in few days up to about a week.
62
In order to have more quantitative data on the possibility of culturing HEK-293 cells on P3HT-
based surfaces, we performed the MTT assay for cell viability. This test is based on the reduction of
the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its
formazan form, which is insoluble and has a purple color. This reduction is operated in living cells
by enzymes involved in mitochondrial activity.205
The quantity of the MTT that is reduced to the
formazan form is thus dependent on the number of living cells on the substrates and can be
measured optically by collecting its absorption in at a selected wavelength (we used λ = 570 nm, see
typical absorption spectrum in Figure 4.1a). By repeating the assay at different times after seeding,
growth curves can be obtained to assess if a particular material is better or worse in promoting cell
culturing with respect to a control substrate. The growth curves measured on P3HT and
P3HT:PCBM films are reported in Figure 4.1b for 1, 2, 3, 4 and 7 DIV, compared to standard glass
substrates (also coated with fibronectin). The three curves show comparable growth rates,
confirming previous literature data on the biocompatibility of these materials.
Figure 4.1 | (a) Typical absorption spectrum of MTT in ethanol extracted from cells to
evaluate their vitality; the green bar (λ = 570 nm) represents the wavelength used to
evaluate the absorbance. (b) Growth curve up to 7 DIV for HEK-293 cells grown on
different substrates: glass (control), P3HT and P3HT:PCBM.
4.1.2 Basic electrophysiology of HEK-293 cells
After having assessed the viability of HEK-293 cells grown on the P3HT-based films, we started
with the investigation of their basic electrophysiological properties, to see if they were comparable
to the data present in literature.
As briefly introduced in Chapter 1.1, the gold standard for measuring bioelectrical activity of cells
is the patch-clamp technique, especially in the whole-cell configuration.21
A thorough description of
this technique and the experimental setup is given in Appendix A. Briefly, the substrates with HEK-
293 cells are put under the microscope in a petri dish with a Krebs-Ringer-Henseleit buffer (KRH,
extracellular solution). A glass micropipette filled with a solution resembling the ionic composition
63
of the cytoplasm of the cell (intracellular solution) is then pressed against the plasma membrane. By
forming the so-called gigaseal, the patch of membrane inside the micropipette tip is removed and
the pipette enters in electrical contact with the inside of the cell. Having an Ag/AgCl electrode in
the pipette and a counter-electrode of the same material in the extracellular solution, it is possible to
measure the potential difference across the plasma membrane.
After the formation of the patch, different measurements can be performed on the cell, depending
on which properties need to be investigated. Some of the electrical parameters of the cell are readily
available to the experimenter: the resting potential V0, the membrane capacitance Cm and the patch
series resistance Rs. The resting potential is the potential difference measured across the membrane
at electrochemical equilibrium (Veq), i.e. no net current flowing through the membrane, when the
cell is not perturbed by an external stimulus; the value of Veq is regulated by the Goldmann-
Hodgkin-Katz (GHK) equation, as already introduced in Chapter 2.3:
In the following, we indicate with V0 the cell resting potential, i.e. the membrane equilibrium
potential Veq at the base temperature (T0) before the cell has been excited in any way. The
membrane capacitance and the series resistance can be read from the patch amplifier after the
capacitive transients present upon electrical stimulation have been corrected during the formation of
the patch.
One of the basic characterization that can be done to understand if the cell is behaving correctly,
and thus the patch is good, is to measure the I-V characteristic of the membrane. We performed this
measurement by applying a sequence of increasing voltage steps (with pulse duration of 500 ms)
from -100 mV to +50 mV (in steps of 10 mV) to the cell and measuring the correspondent current
elicited, as depicted in Figure 4.2a for a typical HEK-293 cell. Between the voltage steps, a holding
potential of Vhold = -40 mV was kept, a value close to the ideal resting potential of HEK-293 cells.
The period between each voltage step was 10 s. The I-V curve for the membrane was then
reconstructed by plotting the mean value of the current in the last 100 ms of the pulse against the
respective value of applied potential. Given that the actual current flowing through the membrane is
proportional to the cell area, it is usually convenient to normalize the measured current for the
capacitance of the cell, which is proportional to the plasma membrane area. The intersection of
these curves with the x-axis is the potential at which no current flow through the membrane, and
thus the equilibrium potential of the cell. The slope of the curve in this point is related to the
membrane resistance Rm; more precisely, it is the membrane conductance Gm, or the specific
(4.1)
64
conductance spG if the current has been normalized by the cell capacitance. Figure 4.2b shows
recorded I-V traces on different HEK-293 cells on glass substrates.
Figure 4.2 | (a) recorded current traces on an HEK-293 cell upon the application of a
stepping potential protocol from -100 mV to 50 mV (in 10 mV steps). (b) I-V curves
extracted from the current measurements for 7 different HEK-293 cells cultured on control
glass substrates.
The curves reported above show a generally rectifying behavior of the HEK-293 plasma membrane,
with small inward currents at negative potentials and bigger outward currents at positive potentials.
This behavior is consistent with the existing literature on endogenous currents in HEK-293 cells.203
However, a great variability in the actual values of the currents is observed in the different cases,
implying a substantial variability in the actual ion channel content of the membrane of the different
patched cells. Similar measurements have been performed also on cells grown on P3HT and
P3HT:PCBM devices; while the variability in the membrane properties like specific conductance
and resting potential is still high, no substantial difference can be observed for the different
substrates, as shown in Figure 4.3.
Figure 4.3 | Distributions of the values of membrane potential (a) and resistance (b) of
HEK-293 cells in resting conditions measured on different substrates: P3HT (n = 48),
P3HT:PCBM (n = 14) and glass (n = 12).
65
4.2 Measurements on different substrates
We now turn to the study of the interaction mechanisms between the polymer-based interfaces and
the cell membrane upon illumination. A direct way to investigate the effect of photoexcitation of the
active material on the bioelectrical properties of HEK-293 cells grown on top of it is to measure the
corresponding variation in membrane potential with the patch-clamp. Typical whole-cell recordings
for a cell cultured on an ITO/P3HT:PCBM substrate are shown in Figure 4.4, upon illumination
with 20 ms light pulses (λ = 475 nm) at different intensities ranging from 7.7 to 47 mW/mm2.
Figure 4.4 | Typical traces of membrane potential measurements on an HEK-293 cell
grown on an ITO/P3HT:PCBM sample upon illumination with 20 ms pulses of light
(represented by the light blue box) at increasing intensities (7.7, 15, 35, 47 mW/mm2).
Two different components of the signal (A and B) may be distinguished based on their
characteristic timescales.
Figure 4.5 | Membrane potential measurements on four different HEK-293 cells cultured
on bare glass substrates for light pulses (I = 57 mW/mm2) of 20 ms (a) and 200 ms (b).
Two main components can be detected in the elicited signal during illumination: (i) a fast spiking at
the onset of the light pulse (A); (ii) a slower transient depolarization evolving during the
illumination (B). When the light is switched off, a complementary behavior is observed, with a
66
reversed spike at light offset and a hyperpolarization afterwards. Both components increase with
higher light intensities. In order to univocally correlate the recorded signal to an effect mediated by
the active material, control measurements of cells on bare glass substrates were also taken (Figure
4.5). Accordingly, in this case, no variation in the cell membrane potential could be recorded.
The spiking signals (A) on ITO/P3HT:PCBM substrates are reminiscent of the capacitive charging
of devices described in Chapter 3.4. The slower component (B) may be a consequence of such
electrical coupling, but could also be related to other effects mediated by the photoactive interface.
In order to disentangle the origin of these two signals, the same measurements were conducted also
on ITO/P3HT and ITO/photoresist substrates. In pristine P3HT, charge generation is still occurring,
but with a much lower efficiency with respect to the blend; in the photoresist, instead, no charge is
generated upon illumination. The intensity of the components in membrane potential variations
related to the capacitive charging of the interface should thus be different in the three cases. Indeed,
the intensity of the A-component at light onset and offset is clearly dependent on the substrate
electrical properties, as shown in Figure 4.6. However, the B-component of the recordings is
qualitatively the same in all the devices.
Figure 4.6 | Membrane potential recordings of HEK-203 cells on different photoabsorbing
materials deposited on ITO-coated substrates: (a) P3HT:PCBM , (b) P3HT, (c)
Photoresist. Magenta boxes highlight the presence of fast spikes on devices with charge
generation capabilities. The different traces refer to increasing same light intensities (7.7,
15, 35, 47 mW/mm2).
To demonstrate that the fast spikes are related to the capacitive charging of the interface and not to
other effects due to charges generated in the active layer, measurements on glass/P3HT:PCBM and
glass/P3HT substrates were also performed. As it has been demonstrated in Chapter 3.4, the
presence of the ITO layer is crucial for closing the circuit with the solution. Its absence thus
prevents the instauration of a surface potential at the polymer/electrolyte interface. Accordingly, the
fast spikes in membrane potential recordings are suppressed in this case for both substrates (Figure
4.7). Nonetheless, the slower component is still present with similar intensities with respect to the
previous cases.
67
Figure 4.7 | Membrane potential recordings of HEK-203 cells on different photoabsorbing
materials deposited on glass substrates: (a) P3HT:PCBM , (b) P3HT. The different traces
refer to increasing light intensities (7.7, 15, 35, 47 mW/mm2).
The origin of the B-component seems not to be correlated to an electrical effect mediated by the
active layer upon generation of charges. It has been shown in Chapter 3.5 that, apart from electrical
activity, photoexcitation of the active materials used here leads to a local heating in the bath close to
the surface. Indeed, for substrates with comparable absorption coefficients, similar temperature
transients have been measured when the light intensities used are the same, consistently with the
qualitative similarity observed for the membrane potential signals recorded on the different devices.
Thermal effects can thus be involved in the observed depolarization/hyperpolarization response.
4.3 Analysis of thermal effects
We focus now our attention on the characteristics of the slow (B) component recorded in the
previous section, in order to understand its origin and if it is actually due to thermal effects
mediated by the active layer. Since it has been shown that this component is qualitatively the same
in all the different substrates, we continue the analysis on cells grown on glass/P3HT devices, in
which only the B-component is present. Recordings of the membrane potential variations in four
different cells at the same light intensity (57 mW/mm2) are reported in Figure 4.8 for both 20 ms
and 200 ms pulses.
The recordings for the short pulses are similar to the traces already presented in the previous
section; however, it is evident that there is a great variability on the recorded signals from cell to
cell, both in terms of the intensity of the signals recorded and of their temporal dynamics. Looking
at the traces for the long pulses (200 ms), we can actually identify three different parts: (i) an initial
transient depolarization after the light is switched off; (ii) a gradual hyperpolarization occurring
during the illumination that takes over the initial depolarization signal; (iii) a further
hyperpolarization following the end of the pulse. In order to avoid confusion between the two
68
hyperpolarization signals, we named them hypon and hypoff, respectively. Given that the hypoff signal
seems to be just the reverse processes of the initial depolarization, we focus on the two components
occurring during the light pulse.
Figure 4.8 | Membrane potential measurements on four different HEK-293 cells cultured
on glass/P3HT devices for light pulses (I = 57 mW/mm2) of 20 ms (a) and 200 ms (b).
4.3.1 Transient depolarization
First, we investigate the transient depolarization that occurs in the first milliseconds after the light
onset. As already observed, the traces in Figure 4.8a indicate that there is a great variability of this
depolarization component from cell to cell. This variability is confirmed by the boxplots in Figure
4.9a, where the peak depolarization values (ΔVpeak) for different light intensities are reported for n =
51 cells. However, by carefully observing the traces of Figure 4.8a, it can be seen that the variability
of the peak depolarization values is not casual, but is clearly correlated with the time the signal
takes to reach the peak (time-to-peak, tpeak): the faster the peak is reached, the lower is the value of
the depolarization. If the ΔVpeak values for all the cells (n = 51) are plotted against tpeak, a clear
correlation can be see between the two (Figure 4.9b).
Figure 4.9 | (a) Distribution of peak depolarization values obtained upon 20 ms
illumination at different light intensities for HEK-293 cells cultured on glass/P3HT (n =
69
48) and control glass (CTRL, n = 12) substrates; for the control case, only the maximum
intensity (57 mW/mm2) is reported.. (b) Scatter plot of the peak depolarization values (I =
57 mW/mm2) versus the time in which the peak is reached upon the onset of the light
pulse for the data on glass/P3HT.
There should thus be a parameter of the cell that determines the dynamics of its temporal response.
Since the cell membrane can be schematized as the parallel of a capacitance (Cm, given by the
charges accumulated across the lipid bilayer) and a resistance (Rm, given by the ion channel
conductances), an obvious candidate is the system time constant given by the product of the two (τm
= RmCm). The membrane capacitance of each cell is known from the compensation of the capacitive
transients performed after the gigaseal is established, while the membrane resistance can be
extrapolated from the cell I-V curves (in particular, from the slope of the curve at the crossing with
the x-axis). Figure 4.10 shows the statistical distributions for the measured Cm and Rm, as well as for
their product τm; it is clear that the different cells have quite different values for the membrane time
constant, as already observed in Section 4.1, which could explain the great variability observed in
the measured dynamics.
Figure 4.10 | Distribution of the membrane electrical properties for the HEK-293 cells
cultured on glass/P3HT (n = 48): (a) membrane capacitance, (b) membrane resistance, (c)
membrane time constant, i.e. the product τm = CmRm.
Indeed, plotting the values of peak depolarization ΔVpeak and time-to-peak tpeak versus the time
constant of the corresponding cell, it is evident the influence of τm in determining the behavior of the
cell dynamics (Figure 4.11).
A behavior similar to the depolarization observed here was reported by Shapiro et al.58
in their
investigation of the physical mechanism behind Infrared Neural Stimulation (see Chapter 1.2). INS
is a cell stimulation technique based on illumination of biological tissues with an IR pulse that is
absorbed by water causing a local increase in temperature. In that paper, the authors demonstrated
that the depolarization measured in different experimental systems (oocytes, HEK-293, lipid
bilayers) upon photoexcitation was related to an increase in the membrane capacitance with
temperature. Interestingly, in their model the cell depolarization was indeed dependent on the
70
membrane time constant. As the membrane capacitance increases, the potential difference across it
decreases (ΔV = Q/C), i.e. the cell depolarizes. However, this variation in potential from the resting
value Vr leads the membrane out of electrochemical equilibrium and a net current is established
across the membrane to restore the equilibrium potential. This re-equilibration mechanism explains
why the observed depolarization during the illumination is only transient; moreover, the rapidity
with which the equilibrium is reached depends on how much currents can flow through the
membrane, and thus on its specific conductance (spG), i.e. the conductance of the membrane per
unit area. Since the area of the membrane is proportional to the capacitance, spG is usually
expressed in terms of the ratio Gm/Cm, which is just the inverse of the membrane time constant.
Figure 4.11 | Scatter plots showing the dependence of the properties of the membrane
depolarization signal, the peak depolarization (a) and the time to peak (b), from the
membrane time constant τm.
4.3.2 Gradual hyperpolarization
After the transient depolarization, the membrane potential does not come back to its resting value
during the light pulse, but a hyperpolarization of the cell is instead observed (hypon). This signal
increases in time and, at least qualitatively, seems to follow the local temperature dynamics
measured in Chapter 3.5. The boxplots of Figure 4.12a show the maximum hypon signals recorded
just before the end of the light pulse at different light intensities for the n = 51 cells measured on the
glass/P3HT substrates. Again, also for this signal a great variability can be observed. However, this
signal does not show any significant correlation with the membrane electrical parameter (Figure
4.12b,c), thus indicating that its origin is different from that of the initial depolarization.
71
Figure 4.12 | (a) Distribution of maximum hyperpolarization reached during a 200 ms
illumination at different light intensities in HEK-293 cells cultured on glass/P3HT (n =
48) and control glass (CTRL, n = 12) substrates; for the control case, only the maximum
intensity (57 mW/mm2) is reported. (b,c) Scatter plots showing the independence of the
hyperpolarization signal measured at the end of a 200 ms illumination (I = 57 mW/mm2)
from membrane time constant (b) and membrane capacitance (c).
A mechanism that could explain a hyperpolarization proportional to the local temperature is based
on the variation with heating of the equilibrium potential of the membrane given by the GHK
formula. The equilibrium potential Veq can be extracted from the I-V characteristic of the cell as the
potential at which no current flows through the membrane. To see if the hyperpolarization is indeed
related to a variation of the equilibrium potential, we measured its value at the end of a 200 ms
pulse (57 mW/mm2). To have more precise data, we did not measure the I-V curve in the entire -100
mV / +50 mV range, but in a smaller interval close to the resting condition. In particular, the
membrane potential of the cell was clamped at a holding potential (Vhold) close to its resting
potential; a stepping protocol was applied to the cell with 800 ms pulses of potential from -5 mV to
+ 5 mV (with respect to Vhold) in 1 mV steps. During the voltage pulses, a 200 ms pulse of light was
then delivered to the cell. The equilibrium potential at each instant can be extracted as the x-axis
intercept in the corresponding I-V plot. A typical example of such a measurement is presented in
Figure 4.13.
The recorded I-V characteristics show that indeed a variation in the equilibrium potential towards
more negative values occurs upon photostimulation. Plotting this value against the corresponding
hyperpolarization measured in current-clamp recordings (n = 17 cells) clearly show a good match
between the two (Figure 4.14a), indicating that indeed the hyperpolarization is due to a variation in
the equilibrium potential in time.
72
Figure 4.13 | (a) Current response of an HEK-293 cell to a stepping protocol with 1 mV
steps around the resting membrane potential (from -5 mV to +5 mV); the light blue box
represent the 200 ms pulse of light (I = 57 mW/mm2), while the grey and magenta boxes
identify the regions from which the current data for the dark and light conditions were
respectively extracted. (b) Comparison between the I-V curves extracted from the
measurements in panel (a) in dark and light conditions.
Figure 4.14 | (a) Correlation between the cell hyperpolarisation measured in current-
clamp experiments at the end of a 200 ms pulse of light and the variation in reversal
potential as measured from the protocol of Figure 4.13; the grey dashed line represents the
quadrant bisector. (b) Correlation between the variation in cell membrane resistance in the
dark and at the end of the light stimulus; the grey dashed line represent the quadrant
bisector, while the solid blue line represent the line best fitting the data, with a slope of
0.804 ± 0.017. Points in e,f represent data from individual cells.
This observation is consistent with the GHK equation, where it can be seen that Vr is directly
proportional to the temperature. If V0 the cell resting potential at temperature T0, the equilibrium
potential Veq upon heating can be expressed as:
(4.2)
73
Since the resting potential V0 is negative, an increase in temperature leads to a negative variation in
the membrane potential (ΔV), i.e. a hyperpolarization, as it is observed. Moreover, a bigger
hyperpolarization should be obtained for cell starting from a more negative resting potential.
However, this behavior is not observed experimentally, as it can be seen by the scatter plot in Figure
4.15, where the maximum hyperpolarization values (at 57 mW/mm2) are plotted against the resting
membrane potential for the corresponding cell.
Figure 4.15 | Scatter plot of the hyperpolarization measured in a HEK-293 cell at the end
of a 200 ms light pulse (I = 57 mW/mm2) with respect to the resting potential of the same
cell (n = 48).
This result indicates that in the simple temperature dependence of Equation (4.2) something is
missing. In particular, has it has been discussed in Chapter 2.2, the permeability of ion channels is a
thermally activated process164
and thus the parameters P[x] in the GHK equation are actually
temperature dependent. The actual relationship between the equilibrium potential and temperature is
thus more complex and is dependent on the thermal properties of the single channels and their
density in the cell membrane. In any case, since ion channels permeability increases with heating,
the membrane resistance should decrease at higher temperatures. The membrane resistance can be
estimated from the slope of the I-V curves reported in Figure 4.13b; indeed, upon illumination a
steeper characteristic is obtained, which is consistent with a lower membrane resistance. In
particular, a decrease of about 20 % of the initial value can be estimated by the statistical analysis of
the data for all the n = 17 cells reported in Figure 4.14b.
4.3.3 Time evolution of membrane properties
To sum up, from the analysis of the membrane potential traces we hypothesize that the two
components of the signal during the illumination are given by two different mechanisms, both
triggered by the local heating. In particular, we associate the initial depolarization to an increase in
membrane capacitance that leads to a transient variation in the membrane potential before this effect
74
is balanced by the current flowing through the membrane and the cell goes back to its equilibrium
potential. The subsequent hyperpolarization is instead due to an increase in the conductances of the
membrane ion channels that leads to a variation in the cell equilibrium potential.
In order to prove in a direct way the effect of temperature on the membrane electrical properties, i.e.
resistance and capacitance, we tried to measure their temporal dynamics during illumination. The
measurement was performed by analyzing the cell response to an oscillating voltage input21
during a
pulse of light (200 ms, 57 mW/mm2). The cell was patched in voltage-clamp mode and was hold at
its resting potential; on top of this potential, a double sinusoidal perturbation was applied:
In the following measurements, we used A = 10 mV and f = 195.3125 Hz (which corresponds to a
period of T = 5.12 ms). In complex notation, with ω = 2πf, this stimulus can be expressed as:
As explained in Section 2.3, in a simplified version a patch-clamp experiment can be modeled with
a with the parallel of a capacitance (Cm) and a resistance (Rm), making up the membrane, and a
series resistance (Rs), that takes into account the presence of the patch pipette (Figure 2.4b). In the
following analysis the voltage sources VX and Vσ are considered as DC signals. While in reality
these two parameters can vary in time due to the temperature gradient, their variation is slower
compared to the frequency here considered. The result of this approximation is that they thus do not
give any contribution to the final result in the following AC analysis. The complex impedance of
this system can be calculated as:
Upon the application of the input voltage of Equation (4.3), the theoretical output current is given
by the following relationships:
Figure 4.16 show an example of the output current simulated with this model for a cell with the
following electrical parameters: Rs = 15.5 MΩ, Rm = 715 MΩ, Cm = 13.5 pF. For comparison, an
actual current trace measured from a cell with the same electrical properties is also reported.
(4.3)
(4.4)
(4.5)
(4.6)
75
Figure 4.16 | (a) Simulated current response (in red) of a cell with Cm = 13.5 pF, Rm = 715
MΩ, Rs = 15.5 MΩ upon the stimulation with the oscillating voltage protocol depicted in
blue. (b) Current traces actually recorded on a HEK-293 cell with the same electrical
parameter as panel (a).
In order to extrapolate the values of the electrical parameters in time from the measured currents,
the traces were divided in single periods of 5.12 ms. From each period, the instantaneous values of
Cm, Rm and Rs were estimated by fitting the measured current with the one expected from the
theoretical impedance of Equation (4.5). Repeating this process for each period in the measurement,
we reconstructed the temporal dynamics of the three parameters. The measurements were
performed on n = 39 cells; while different cells have different initial values of capacitance and
resistance (see Figure 4.10), the relative variations of these parameters were seen to be quite
reproducible as it can be seen from the statistical distributions of the signals reported in Figure
4.17.
Figure 4.17 | Variation in time of membrane electrical properties upon illumination with a
200 ms pulse (I = 57 mW/mm2): (a) membrane capacitance, (b) membrane resistance, (c)
series resistance.
The results here presented show that indeed all three parameters vary during the illumination pulse.
Interestingly, the traces have similar dynamics to the temperature variations measured in Chapter
3.5, supporting the influence of temperature on the membrane electrical parameters.
76
The series resistance Rs is seen to vary of about 10 % during the 200 ms pulse. This resistance does
not describe a parameter of the cell itself, but it’s a necessary element to perform the measurement.
Since the series resistance is mainly determined by the access resistance of the pipette, it is not
surprising that it varies with temperature. Indeed, the temperature dependence of the resistance of a
micropipette was used in Chapter 3.5 to estimate the local heating at the interface. Also in that case,
a variation of about 10 % of the pipette resistance was measured upon illumination with a 200 ms
pulse at 57 mW/mm2.
The membrane capacitance Cm has a maximum variation of about 2 % at the end of the 200 ms
pulse. This increase in capacitance is consistent with the proposed explanation for the transient
depolarization signal. However, since this phenomenon is transient, only the increase of temperature
occurring in the first milliseconds is actually important in determining the depolarization value
reached. At the same time, however, the current flowing through the membrane is balancing the
depolarizing effect. If we consider a cell with a very small specific conductance spG, the effects of
this second phenomenon can be considered negligible in the first 20 ms; in this case we can
approximate the charge on the membrane as constant. The depolarization reached is thus given by
the following relationship:
In which V0 – Vσ is the potential difference across the membrane capacitance (see equivalent circuit
in Figure 2.4). Considering typical values as V0 = -30 mV and Vσ = 120 mV58
and a relative
variation in the capacitance of about ΔCm/Cm = 1 % in the first 20 ms, a depolarization of ΔV ≈ 1.5
mV is expected. This value is consistent with the maximum depolarization reached in the
experiments from Figure 4.9b, since a cell with a small specific conductance corresponds to the data
for high time constants τ. Moreover, comparing the temperature variations recorded in Chapter 3.5
with the data here, we can estimate a variation in membrane capacitance of about 0.3 % for each
degree of temperature increase. This value is consistent with the experiments reported for HEK-293
cells by Shapiro et al.58
upon stimulation with IR light.
The membrane resistance Rm traces show a decrease at the end of the 200 ms pulse of about 18 %.
This value is consistent with the one extrapolated in the previous section from the I-V curves of the
cells. The variation in membrane resistance has been attributed to the increase in ion channel
conductances with local heating. From the measured decrease of about 20 % upon a local heating of
7 °C, the temperature coefficient Q10 can be estimated as:
(4.7)
77
Actually, it is reported in literature that different ion channels have different temperature
coefficients.164
It is thus difficult to correlate the value calculated here with literature data, not
knowing the exact types and distribution of ion channel in the membrane. This is especially true if
we consider the great variability of membrane resistances in the measured HEK-293 cells, which
means that each cell has a different composition in terms of ion channels expressed. However, a
rough comparison with literature date shows that typical values for the Q10 coefficient in different
families of ion channels usually range from 1.2 to 1.6,164
in accordance with the value calculated
here.
4.3.4 Numerical modeling
The measured evolution in time of the membrane properties (i.e. resistance and capacitance) seems
to follow quite closely the dynamics of the local temperature reported in Chapter 3.5. Here we try to
extrapolate a numerical relationship between the membrane electrical parameters and the
temperature, in order to directly link the membrane potential variations measured in HEK-293 cells
with the local heating. Applying Kirchhoff’s law to the equivalent circuit introduced in Chapter 2.3
(see Figure 2.4), the following differential equation is obtained:
Here, Vm is the membrane potential to be determined (which is equal to the potential inside the cell
if the outside is fixed for convention at zero), while Ip is the current flowing through the pipette.
This term is in principle dependent on the series resistance Rs, but since the measurements of the
membrane potential are taken in current-clamp mode, Ip is in this case it is equal to zero. The
equation can thus be readily solved numerically once the time evolutions of the membrane
parameters (Cm, Rm, Vr and Vσ) are known.
Based on the collected measurements and on literature models, for the membrane capacitance Cm,
we hypothesize a linear relationship of this parameter with the local temperature T, in the form of:
where T0 is the base temperature and αC the proportionality constant. As for the membrane
resistance Rm, it can be readily obtained from the Q10 formulation as:
(4.8)
(4.9)
(4.10)
78
Starting from the temperature transients measured in Chapter 3.5 and setting the parameters Q10 =
1.32 and αc = 0.0032 K-1
, these two formulas can be used to closely reproduce the traces
experimentally found in the previous section, as reported by the solid lines in Figure 4.18.
Figure 4.18 | Simulated traces (blue solid lines) for the variation upon illumination of the
membrane resistance (a) and membrane capacitance (b), compared to the experimental
data from Figure 4.17 (grey shaded regions).
The modeling of the equilibrium potential Veq is a more complex problem. Theoretically, it is given
by the GHK equation. As reported above, however, this relationship has a non-trivial dependence
on temperature that involves the knowledge of the ion channels distribution and their specific
thermal responses. Since we cannot reach such level of insight with our measurements, we need to
use an empirical relationship to simplify the GHK equation:
This formula is a generalization of Equation (4.2), with the parameter αV used to model the complex
temperature dependence of the logarithm part of the GHK equation. The case αV = 1 corresponds to
an “ideal” situation in which all the ion channels in the cell membrane have the same Q10 coefficient
and thus their relative contributions to the total membrane current remains constant; in this case the
logarithm part does not vary during the illumination and the only temperature dependence is in the
prefactor, leading to the formulation of Equation (4.2). Values of αV > 1 are indicative of a tendency
of the cell to hyperpolarize more than the ideal case, i.e., on average, the increase in temperature
favors more the conduction of channels mediating outward currents. The opposite holds true for αV
< 1, and in the case of negative values the cell actually tends to depolarize with increasing
temperature. The last parameter of the model, Vσ, is necessary to take into account the charge
asymmetry on the two sides of the plasma membrane, which is due to the differences in ion
(4.11)
(4.12)
79
concentrations but also to different charging of the inner and outer surfaces of the membrane, as
explained in Chapter 2.3. As a first approximation, we consider this parameter constant in the
temperature range here investigated.
Based on the relationships just introduced and the measured time traces of local temperature
(Section 3.5), Equation (4.9) can be solved to reproduce the experimental traces of the membrane
potential upon illumination. In particular, with this model we fitted the signals from the n = 51 cells
measured on P3HT/glass, both for 20 ms and 200 ms pulses. For each cell, we considered αc and αV
as free parameters in the fitting, while the values of Cm,0, Rm,0 and V0 were available from the
experimental data. For the remaining parameters, we chose common values for the all the cells
which gave the best fit, paying attention that they were compatible with literature data; in particular,
we used Q10 = 1.32164
and Vσ = 160 mV.58
The distributions of the obtained values for αc and αV are
reported in Figure 4.19.
Figure 4.19 | Distribution of the values for the αC (a) and αV (b) parameters obtained by
fitting the numerical model of the cell behavior to the experimental curves.
The results for αc show a quite narrow Gaussian distribution for this parameter, with a mean value
of 0.0031 K-1
and a standard deviation of 0.0004 K-1
. This value is consistent with the observed 0.3
% variation in capacitance for each degree of temperature and the narrow distribution implies that
the capacitance variation upon heating is basically the same in all the cells measured; the variability
of the actual depolarization peaks reached upon illumination is thus given by the differences in the
membrane time constants, as already explained in Section 4.3. The values for αV are instead much
more scattered, from close to 0 up to about 2; this variability is reflected in a great difference in
hyperpolarization dynamics between cells, consistently with what reported in Section 4.3.
The actual dynamics of the simulated membrane potential dynamics for a cell are compared to the
experimental data in Figure 4.20, both for 20 ms and 200 ms light pulses. The dashed lines in the
graphs represent the contributions to the final signal coming from the variation in capacitance (blue
80
lines) and from the variation in the membrane equilibrium potential (green lines). As it was
proposed at the beginning, the first component shows that the transient depolarization upon
illumination and the corresponding hyperpolarization after the end of the pulse are given by the
variation in membrane capacitance with temperature. The second component instead confirms that
the gradual hyperpolarization occurring during the light pulse is related to the shift in the
equilibrium potential.
Figure 4.20 | Results of the numerical fitting (pink solid line) for an HEK-293 cell upon
illumination with a 20 ms (a) and 200 ms (b) light pulse (I = 57 mW/mm2), compared to
the experimental traces. In the two panels are reported also the single contribution to the
total signals from the variations in membrane capacitance (blue dashed lines) and
equilibrium potential (green dashed lines).
4.4 Considerations on capacitive charging
In the previous section we have characterized in depth the effects of local heating on the electrical
properties of the cell membrane in HEK-293 cells and the relative variations in membrane potential.
However, the measurements in Figure 4.6 showed that also an effect related to charge generation in
the active material can be present in samples deposited on ITO-coated substrates. In particular, we
observed very fast spikes in the recordings of membrane potential upon the onset and offset of the
light pulse. These signals were quite similar to the traces of the surface electrical potential due to
the capacitive charging of the polymer/electrolyte interface investigated in Chapter 3.4. For direct
comparison, we measured the surface potentials on the different samples used for HEK-293
experiments at the same light intensity used for cell stimulation (57 mW/mm2).
81
Figure 4.21 | Surface potentials measured on the devices used for cell stimulation with a
light intensity of 57 mW/mm2: (a) P3HT, (b) P3HT:PCBM, (c) Photoresist. Both
architectures with (blue) and without (grey) the ITO electrode were tested.
As already seen in Chapter 3.4, devices without the ITO layer did not give any signals. In the
presence of the conductive contact, instead, surface potentials were visible both on P3HT and
P3HT:PCBM, but not on Photoresist, since it does not support the generation of charges. As
expected, the signal on the blend was significantly higher than the one measured on the pristine
polymer. These signals are perfectly comparable to those measured in the membrane potential of
HEK-293 cells upon photostimulation; however, it is not clear if such signals are an actual variation
in the membrane potential, if they are just an artifact due to the coupling of the surface signal to the
recording electrode, or a combination of both.
Let’s call Vcell the potential inside the cell and Vsurf the potential at the polymer/electrolyte interface
and Vm the transmembrane potential. If we consider the cell as an isolated system in proximity of
the device interface, as depicted in the electrical scheme of Figure 4.22, the potential on the outer
side of the membrane is always equal to Vsurf (we are considering the resistance of the electrolytic
solution negligible) and thus:
A variation in the surface potential does not modify the actual membrane potential. Instead, the
potential inside the cell changes in the same way as the outside potential changes, and thus the
potential difference across the membrane remains constant. This happens because, since every
portion of the membrane feels the same outer potential, no current can flow through the membrane
resistances (i.e. the ion channels) and thus the transmembrane potential cannot change. However, in
a patch-clamp experiment in current-clamp configuration what is measured is not the actual
transmembrane potential Vm (which is the biophysically relevant parameter), but the cell potential
Vcell (with respect to the counter-electrode one Vref, that can be considered equal to zero as a
convention). In this case, a variation in the measured potential is to be considered an artifact due to
the superposition of the Vsurf signal to the actual Vm in the recordings.
(4.13)
82
Figure 4.22 | Simplistic modeling of the interface between a cell and the hybrid polymer-
based interface.
This schematization, however, is not complete. Capacitive charging as a tool for stimulating cells
has been largely investigate with devices based on inorganic technology. In particular, Fromherz
and coworkers91
have studied in depth the interfacing of cells with silicon-based devices (transistors
and capacitors). In these systems it is important to consider that the adhesion to the substrate brings
the basal membrane of the cell at a very small distance from the device surface, which is usually
less than 100 nm. Conduction in such a confined space, called cleft,32,33
is to some extent reduced.
To model this effect we can, at least as a first approximation, divide conceptually the cell membrane
in two different parts: the basal membrane and the lateral membrane. The basal membrane feels, on
its outer side, the potential in the thin cleft (Vcleft); the lateral membrane, i.e. the remaining part of
the plasma membrane in contact with the extracellular medium, feels on the outside the surface
potential Vsurf. The cleft region and the extracellular space are connected via a cleft resistance Rcleft,
as schematized in Figure 4.23. Upon charging of the device interface, the cleft and surface potential
can be quite different if Rcleft is high enough and thus the two portion of the membrane can have
different potentials, in principle different from the resting one; in this case, a stimulation of the cell
can be possible. If Rcleft is too small, instead, the model becomes equivalent to the isolated cell one
and no actual stimulation of the cell can be obtained with the capacitive charging of the surface.
This model for the plasma membrane is a first approximation of the real coupling of the cell with
the device surface. More precise models have been developed, taking into account for example the
actual space distribution of the potential in the cleft region or the different conductivity of the cleft
for the different ion species. In any case, the two-compartment model is sufficient to give at least a
qualitative idea of the effects of capacitive stimulation on the cell membrane potential.
Understanding if the fast spiking signals we measured in HEK-293 cells upon photostimulation of
the semiconducting polymer are just an artifact or are actually correlated with a variation in the
83
membrane potential of the cell is not straightforward. Fromherz and coworkers have studied HEK-
293 cells transfected with genes for the expression of voltage-activated channels206
(both for K+ and
Na+) and have demonstrated that capacitive stimulation (with inorganic technologies) is able to
trigger the opening of these channels. However, in our work we only had at our disposal non-
transfected cells and observing the effects of capacitive charging of the interface in these systems is
not so direct. We thus performed some simulations to understand if, with reasonable values for the
system parameters in our case, capacitive stimulation of the cell membrane can be expected or not.
Starting from the electrical model of the photoactive interface proposed in Chapter 3.4, we used the
cell and cleft modeling presented in literature to obtain the equivalent circuit of Figure 4.23. In
particular, we made the following hypothesis:
The cell membrane is modeled as a two-component system like in the Fromherz model, with the
basal and lateral sections. The cell basal area has been taken from typical values for HEK-293
cells (Acleft = 300 μm2), while the total area has been estimated more or less three times the basal
area (Acell = 1000 μm2).
The cell membrane capacitance (Cm = 15 pF) and resistance (Rm = 500 MΩ) have been taken
from the electrophysiology data (Section 4.1) and divided between the basal (Cm,B e Rm,B) and
lateral (Cm,L e Rm,L) sections proportionally to their area.
The cleft is modeled as a compartment separated from the extracellular space by a cleft
resistance Rc. A typical value for this parameter taken from literature32,33
is 1 MΩ.
The cytoplasm is considered sufficiently conductive that the internal potential Vcell is the same
in every point of the cell. This hypothesis is reasonable for HEK-293 cells, given their compact
morphology without significant processes. However in other cells, like neurons that posses thin
dendrites protruding from the cell body, a more sophisticated model may be necessary to take
into account spatial variations of the internal potential.
The specific capacitance of the polymer/electrolyte interface has been considered the same for
both the cleft and the extracellular space regions.
Table 4.1 summarizes the base values used for the different parameters of the electric circuit. The
values for the description of the active interface have been selected in order to give a surface
potential similar in intensity and dynamics to that measured in P3HT:PCBM devices in Chapter 3.4
(I = 784 μW/mm2). The effect of photostimulation on the transmembrane potentials has been
calculated for both the basal (ΔVB = Vcell – Vcleft) and the lateral membrane (ΔVL = Vcell – Vsurf) upon
a light pulse of 20 ms; their dynamics at the onset of the light pulse are reported in Figure 4.24,
compared to the trace for the elicited surface potential (Vsurf) and the internal cell potential (Vcell, the
signal actually measured in a patch-clamp experiment). The simulations were performed with the
OrCAD® PSpice circuit simulator suite.
84
Figure 4.23 | Electrical schematization of the two-compartment model applied to the case
of cell stimulation by the hybrid interface characterized in Chapter 3.4. The green box
represents the area of the device illuminated during stimulation.
The simulation shows that indeed there is an actual variation in the membrane potential. In
particular, the basal and lateral sections are subjected to different transients: the basal membrane is
hyperpolarized, while the lateral section is depolarized. This double behavior is common for
capacitive stimulation and is the main reason why with this kind of architecture it is difficult to
obtain suppression of activity in excitable cells.207
The two sections of the membrane are always
subject to signals with opposite signs; in the section that is depolarized the voltage-gated ion
channels tend to open, depolarizing the interior of the cell even more and overcoming the
hyperpolarization of the other section of the membrane.
Figure 4.24 | Simulation of the transmembrane potentials elicited by capacitive charging
of the interface in the later (green) and basal (orange) compartments of the cell, compared
to the surface potential (blue) and the potential inside the cell (magenta) measured with
respect to the reference electrode. Given the fast dynamics, the graph reports for clarity
only the first milliseconds after the light onset.
85
Table 4.1 | Base values of the parameters used to simulate the electrical circuit of Figure
4.23.
In our case, it is interesting to observe that both the hyperpolarization and the depolarization
obtained are in absolute values higher than the surface potential elicited by the photoactive
interface. The drawback is that the elicited signals are very fast and, in the case of excitable cells
they could be too short to efficiently trigger the opening of voltage-gated channels. As already
discussed in Chapter 3.4, this issue is related to the value of the surface capacitances of the device
and could be addressed by proper engineering of the interface.
To have a better understanding of how the relevant parameters of the cell can influence the ability to
obtain an efficient stimulation, we performed different simulations by varying their values within
reasonable ranges. The relative variations in the peak signals and stimulus duration (estimated as the
pulse area divided by its peak value) are reported in Figure 4.25.
The cleft resistance Rcleft is the main parameters that determine the coupling of the surface
potential to actual variations in the cell membrane potentials. The membrane signals are linearly
proportional to this parameter, except for a slight saturation at very high values. It is interesting
to notice that the membrane potentials obtained can be much higher than the actual surface
potential elicited (in this case it is about ≈ 500 μV). The duration of the stimulus instead is
initially quite independent of Rcleft, increasing only at high resistances.
By changing the cleft area Acleft keeping the total cell area constant it is possible to simulate the
effect of the cell adhesion to the substrate. Increasing Acleft (i.e. the cell is well spread on the
interface) has a positive effect on both the lateral and basal signals, but while the increase is
more than linear on ΔVL, it saturates for ΔVB. As for the stimulus duration, it remains virtually
unchanged in all cases.
The cell area Acell was modified keeping constant the ratio with the basal membrane (Acleft =
Acell/3.33) in order to simulate cells with different dimensions. Also the membrane resistance
and capacitance have been modified proportionally. Both the lateral and basal signal increase
Description Value Description Value
Is Diode saturation current 17 nA Acleft Cleft area 300 μm2
N Diode ideality coefficient 2 Acell Cell total surface 1000 μm2
CJ Diode junct. capacitance 690 pF Alight Illuminated area 0.23 mm2
Iph Photogenerated current 13.8 μA cint Interf. specific capacitance 2 μF/cm2
Rbulk Bulk resistance 60 kΩ Clight Illuminat. area capacitance cint*(Alight - Acleft)
Rel Electrolyte resistance 100 Ω Ccleft Cleft interf. capacitance cint*Acleft
Cm Cell membrane capacitance 15 pF Rm Cell membrane resistance 500 MΩ
Rcleft Cleft resistance 1 MΩ
86
with increasing cell dimension in a linear fashion, while the duration of the pulses are only
slightly lengthened.
Changing the membrane resistance Rm or the membrane capacitance Cm has no sizable
effects on the stimulation dynamics of the cell, both in terms of peak signals and pulse
durations.
This analysis shows that, to have efficient cell stimulation, the main factor to be considered is to
have a good adhesion of the cell to the substrate, with a highly resistive access to the cleft. Bigger
cells are excited better because of an increased cleft area, while the specific capacitance and
conductance of the membrane do not have significant effects.
Figure 4.25 | Dependence of the elicited variations in membrane potential in terms of
peak signal (orange: basal, green: lateral) and decay time (blue: basal, red: lateral) from
the main electrical parameters describing the cell and its adhesion to the substrate. For the
basal and lateral potential, the absolute value of the variation is reported for clarity; in any
case, it should be reminded that, for the electrical parameters used, ΔVB is always negative
(i.e. hyperpolarizing) and ΔVL is always positive (i.e. depolarizing). (a) cleft resistance;
(b) percentage of the cleft area with respect to the total area; (c) cell total surface, scaling
the cleft area accordingly; (d) membrane resistance; (e) membrane specific capacitance,
i.e. the ratio between the area and the total cell surface.
87
Chapter 5 – Discussion and perspectives
In Chapter 3 and Chapter 4 we investigated the functioning of the hybrid polymer/electrolyte
interfaces in terms of their electrical and thermal properties, as well as the effects that these
phenomena have on the membrane potential of cells grown on their surface.
In this chapter we discuss how the results obtained compare with existing technologies for cellular
stimulation and in particular how they relate to our previous work of photoexcitation on neurons
and retinas. We then give some perspectives by identifying the main parameters of these systems
that need to be optimized and propose some possible future developments.
5.1 Discussion
From the investigation of hybrid polymer/electrolyte interfaces carried out in – Hybrid interfaces
characterization we have found that two main phenomena occur upon photoexcitation of the device
with short pulses of light:
In devices with an ITO electrode, it is possible to elicit a capacitive photocurrent upon
illumination. This signal is driven by the charge separation at the ITO/polymer junction and last
until the capacitance of the polymer/electrolyte interface has been charged. In devices based on
P3HT:PCBM active materials, in which the charge separation process is more efficient, peak
currents of several hundreds of μA/cm2 can be reached at the maximum high intensities used (jpc
≈ 900 μA/cm2 at Ilight ≈ 200 μW/mm
2), in a configuration with all the device active area (A ≈ 1
cm2) illuminated and the ITO contact short-circuited to the reference electrode. This current
however decays very rapidly to zero, with time constants shorter than 1 ms. We have also
shown that, even if in the device configuration used for cell stimulation the ITO electrode is not
contacted, the presence of a parasitic coupling with the electrolytic solution effectively close the
circuit and allow the charging of the polymer/electrolyte interface.
All devices, irrespectively of the presence of the ITO electrode, dissipate the great majority of
the energy of the photons absorbed via non-radiative decay and ultimately by heat transfer to
the surrounding environment. In particular, upon localized illumination of the active devices
(Alight = 0.23 mm2, intensities up to Ilight ≈ 57 mW/mm
2), temperature increases at the
88
polymer/electrolyte interfaces on the order of few degrees Celsius have been measured (ΔT1 ≈ 3
°C and ΔT2 ≈ 7 °C with pulse duration of respectively t1 = 20 ms and t2 = 200 ms).
5.1.1 Capacitive stimulation
Capacitive stimulation of biological preparations has been widely investigated in the past with
architectures mainly based on inorganic semiconductors, in particular silicon chips or insulated
metal electrodes. In the great majority of reports, electrical stimulation, i.e. a voltage pulse, was
employed to elicit a capacitive current at the semiconductor/oxide/electrolyte interface. However,
some reports have also used a mixed electro-optical architecture in which the light was used to
trigger the charging of the device by increasing the semiconductor conductivity.
The capacitive current density elicited by a voltage pulse (Vs) applied to the device is basically
dependent on the temporal derivative of the voltage profile, following the relationship:207
where cint is the specific capacitance of the semiconductor/oxide/electrolyte interface. To improve
the efficiency of the device, i.e. the possibility of obtaining reliable stimulation at a lower applied
potential, a high value of the capacitance is thus desirable.
The standard silicon-based device used for capacitive stimulation is formed by a silicon chip
covered by a layer of silicon oxide that acts as the dielectric with thicknesses ranging from tens to
hundreds of nanometers.89,208
Typical values of specific interface capacitance are mainly determined
by the geometric capacitance of the silicon dioxide layer and are on the order of few hundreds of
nF/cm2 (the geometric capacitance of a 10 nm layer of SiO2 is CSiO2 = 0.34 μF/cm
2). In order to
improve this value, dielectrics with higher dielectric constant and with lower thicknesses have been
developed. For example, with hafnium oxide (HfO2),209
devices with a interface capacitance of
CHfO2 ≈ 1-3 μF/cm2 have been developed,
207,210 thanks to its very high dielectric constant (nHfO2 = 25,
compared to silicon oxide nSiO2 = 3.9).
Typical values of capacitive currents used for stimulation with inorganic devices are in the order of
hundreds of μA/cm2 to few mA/cm
2.210,211
Since the current depends on the derivative of the
potential pulse Vs, ramp of voltages are usually employed, in order to have constant current values.
Typical pulse durations in these stimulation protocols are in the order from few hundreds of
microseconds to few milliseconds, depending on the specific experiment. In order to obtain these
capacitive currents, the potential variations applied to the silicon capacitor are generally on the
order of few volts.
(5.1)
89
In the case of our hybrid polymer based devices, the semiconductor is in direct contact with the
electrolyte, without a dielectric layer in between. In this case the actual identification of a dielectric
with a precise geometrical thickness and dielectric constant is not straightforward. In particular, the
capacitance derives from a combination of different factors, like the average extension of the
diffusion layer of ions in the electrolyte and the dimension of the region where charges are
accumulated in the semiconductor.170
These elements are actually present also in inorganic devices,
but usually give only secondary contributions with respect to the geometrical properties of the
dielectric layer. Another factor that can significantly influence the interface capacitance in polymer-
based devices is the conformation of the polymer at the surface and in particular the nature and
orientation of the side chains.212
Moreover, also the doping state of the material and the eventual
penetration of water molecules into it can effectively vary this parameter.213
From impedance
spectroscopy measurements, we have extracted for our P3HT-based devices capacitance values of
about cint ≈ 2 μF/cm2.197
However, examples reported in literature for devices based on thiophene
derivatives can reach specific interfacial capacitances on the order of cint = 10 μF/cm2.212
It has to be
noted that these values of capacitance have usually a significant dependence on frequency and bias
point of the device. However, in the range of voltages we are interested in, i.e. few hundreds of
millivolts (from photovoltages measurements), the device capacitance is basically constant.170,212
As
for the frequency, the fastest signals we measure have dynamics on the order of hundreds of
microseconds, which corresponds to frequencies of few kilohertz, comparable to the cut-off
frequency measured experimentally for the device capacitance.
The currents we are able to elicit with our hybrid interfaces upon photostimulation reach values
close to 1 mA/cm2 in the case of P3HT:PCBM based devices. While this value is comparable to
typical currents obtained for electrical stimulation, the elicited currents quickly drop to zero with
time constants shorter than 1 ms for the highest illumination intensities, i.e. the currents has a high
value only for few hundreds of microseconds. This spiking behavior can be related to the step
profile of the light pulse, which is reflected in a basically rectangular photovoltage profile at high
light intensities. Moreover, there is a limitation in the photopotentials achievable at the ITO
electrode. Their value is given by the polarization of the ITO/polymer Schottky junction due to the
current generated upon light absorption.192–195
Since this junction behaves like a diode, the increase
in the ITO potential is only logarithmic with the photogenerated current, and thus with the light
intensity.
In this work we focused the study on non-excitable cells in order to understand how the basic
properties of the cell membrane are influenced by the photoexcitation mediated by the hybrid
polymeric interface. As a further step, it would be now interesting to transfer the information
collected to excitable cells, where an active response can be elicited by a variation in the membrane
90
potential due to the capacitive charging of the interface. It is indeed critical to understand to which
extent the capacitive charging of the interface determines the action potential generation by
triggering, for example, the opening of voltage gated channels, and to identify excitations thresholds
and critical conditions.
5.1.2 Thermal stimulation
The measurements of local heating at the polymer/electrolyte interface performed Chapter 3.5
demonstrate that basically all the energy of the absorbed light is released as vibrational energy
following non-radiative decay pathways of the photoexcited species in the active material. In
Chapter 4.3 we have then demonstrated that such increase in temperature is able to modulate the
membrane potential with different mechanisms depending on the considered timescale. In
particular, we have observed an initial transient depolarization related to a change in the membrane
capacitance with temperature. This depolarization reaches values of about 1.5 mV for the highest
power intensity used (57 mW/mm2), but it has been shown to be quite dependent on the membrane
electrical properties and the mechanism becomes less effective for more conductive cells. On longer
timescales, a sustained hyperpolarization is instead observed during illumination, with values again
of the order of 1-2 mV. This second process has been attributed to a variation in the membrane
equilibrium potential due to the temperature dependence of ion channels conductances.
A photothermal stimulation process in biological systems has been proposed to explain the
excitation of neurons observed upon illumination with pulses of infrared light (INS, infrared neural
stimulation).56,60
In 2012, Shapiro et al.58
demonstrated that a local increase in temperature is related
to a variation in the membrane capacitance and that this variation can indeed lead to a transient
membrane depolarization. The data we measured on HEK-293 cells with visible light illumination
of the photoactive devices are in accordance with the INS experiments. In particular, we found in
our system an increase in membrane capacitance of about 0.3 % for each degree Celsius of heating,
quite consistent with the measurements of Shapiro et al.58
on the same cell line, who reported a ΔC
≈ 1.7 % for a ΔT ≈ 10 °C. The main difference with respect INS is the phototransducing element: in
INS, the light is absorbed by water, while in our case the interaction is mediated by the active
polymer layer. INS is a very flexible tool for in-vivo experimentation,65,214–216
since it does not
require the use of an exogenous absorber. The main absorption peak of water usually used for INS
is the one at about 1.9 μm, where water has an absorption coefficient217
on the order of α ≈ 100 cm-
1. The light is usually delivered to the preparation via an optical fiber micromanipulated in close
proximity of the cells. Our polymer-based devices, however, have peculiar advantages that are
interesting for in-vitro experimentation. In particular, since the absorption is in the visible, the light
can be delivered to the preparation directly through the optical path of common microscopes, which
91
usually do not transmit IR light above 1 μm. Desired pattern of illumination can be also easily
obtained with commercially available spatial light modulators or holographic systems. Moreover,
given the very high absorption length of semiconducting polymers like P3HT (α ≈ 105 cm
-1), thin
films of about 100 nm are already sufficient to absorb a large fraction of the impinging light.
The measurements presented in Chapter 4.3 were performed with pulses of light of 20 ms and 200
ms in order to investigate in better details the temporal evolution of the membrane potential.
However, to obtain an efficient thermally-mediated depolarization, it is better concentrate the
luminous energy in shorter pulses, for the following reasons:
Because of diffusion of thermal energy outside of the illuminated spot, increase of temperature
with time is not linear, but tends to saturate at an equilibrium value. The same amount of energy
concentrated in a shorter pulse is thus able to give a higher increase in temperature with respect
to a longer illumination.
The depolarization mechanism related to the change in the membrane capacitance is
counterbalanced by the flow of currents through the ion channels, which evolve on a timescale
dictated by the membrane time constant τmem. Using pulses of light with durations shorter than
τmem, this second mechanism has a lower impact on the actual depolarization reached for a
certain value of membrane capacitance variation.
For the hyperpolarizing effects observed on longer timescales, the measurements here performed
are again consistent with recent literature on photothermal stimulation. In particular, light-induced
heating, either mediated by water absorption or by exogenous transducers, has been shown to be
able to inhibit neural excitability and block transmission of action potentials along nerves.65
In
particular, using gold nanorods are used as near-IR absorbers, S. Yoo et al.87
reported that with
temperature variations up to 8 °C it is possible to reliably inhibit spiking activity in neurons. These
values are perfectly consistent with the heating measured in Chapter 3.5 upon prolonged
illumination in our devices and indeed preliminary experiments have shown that the polymer-based
hybrid interfaces are able to inhibit both stimulated and spontaneous activity in cultured neurons
and brain slices.
5.1.3 Comparison with previous works
In the previous works from our group, we have shown that the photoexcitation mediated by the
hybrid polymer/electrolyte interfaces was able to elicit neural activity both in-vitro with primary
cultures of neurons140,141
and ex-vivo with blind retinas from albino rats.141
In these works, an
electrical excitation due to charge generation in the active material was proposed. However, while
preliminary measurements were presented that already indicated the importance of the ITO
92
electrode in the charge separation processes, no definitive coupling mechanism at the
polymer/electrolyte/cell interface was actually demonstrated. Despite the fact that the measurements
performed here on HEK-293 cells cannot be directly transposed to the case of neurons, given the
significant biophysical differences between the two systems, they can in any case provide important
information on the role that different stimulation mechanisms can play. However, given the
differences in the power intensities used, it is necessary to distinguish between the case of primary
neurons and explanted retinas.
In the measurements with primary neurons, excitation intensities of about 10-15 mW/mm2 were
used, while here we employed intensities up to 57 mW/mm2 for measurements on HEK-293 cells.
In those cases, however, a slightly different experimental configuration was used, with an upright
microscope and the illumination impinging directly on the sample surface. In the measurements
presented in Chapter 4, instead, an inverted microscope is used, with the light that has to travel
through the sample holder and the substrate before reaching the active layer. Taking into account
these losses and the fact that here we used a different wavelength (λ = 475 nm), which is less
efficiently absorbed by the P3HT layer than the one used in the previous works (λ = 530 nm), the
optical excitations in the two cases can be considered comparable. The observation in HEK-293
cells of a thermally-mediated transient depolarization upon illumination is qualitatively consistent
with the measured firing of action potentials in neurons and with literature on infrared neural
stimulation. However, the depolarization levels on the order of 1 mV found in Chapter 4.3 are too
small to explain by themselves a reliable triggering of action potential firing, for which
depolarizations on the order of 10 mV are usually needed. There are different possibilities that
could explain this difference:
The cell depolarization upon heating depends, other than the temperature gradient, also on the
biophysical properties of the cell membrane, in particular the actual coefficient of membrane
capacitance variation with temperature and the value of the Vσ potential related to membrane
asymmetries. The first parameter depends on the actual composition of the membrane in terms
of lipids and proteins and, for example, in the work of Shapiro et al.58
it is shown that a lipid
bilayer has a capacitance variation that is about two-fold with respect to that of an HEK-293
cell at the same light pulse energy. The second one is related to the difference in superficial
charge on the two leaflets of the membrane, which again can vary from cell to cell. A more
detailed analysis of these factors in neural cells is thus necessary to understand what could be
the actual effect of temperature variations on their membrane capacitance.
While Shapiro et al.58
attributed neural stimulation in INS to the transient depolarization given
by the effects on membrane capacitance, other groups proposed that this effect is mediated by
the opening of temperature-gated TRPV channels,57
which are expressed in different neural
93
preparations (but are not endogenously present in HEK-293 cells). For example, TRPV4
channels have been demonstrated to open for temperatures higher than 25 °C,218
which are
consistent with the heating levels reached in our devices.
In the measurement with HEK-293 cells, we mainly characterized the effects of local heating.
However, also the capacitive charging of the interface, as proposed in our previous works,140,141
can take part into cell stimulation. The currents measured in our devices are quite fast compared
to standard protocols used with silicon-based devices; however, Fromherz and coworkers206
have actually shown that upon capacitive stimulation it is possible to elicit significant voltage-
gated sodium currents (mediated by NaV channels) even after few hundreds of microseconds.
For what concerns instead the measurements on explanted retinas,141
in that case the light intensities
used were between two and three orders of magnitude lower than those reported here for thermal
stimulation (significant responses were obtained with Ilight = 10-100 μW/mm2). Since the
temperature increase is linear with the absorbed intensity, the local heating in the experiments with
blind retinas should be on the order of few tens of mK. Such low values of temperature are not
expected to induce significant depolarizing effect or to gate the opening of TRPV channels. In this
case, we thus tentatively attributed the photostimulation effect to the capacitive charging of the
interface. Interestingly, also other groups have been able to elicit activity in blind retinas upon
optical stimulation using devices with architectures similar to ours. In particular, Narayan and
coworkers143
used organic devices based on bulk heterojunctions, while the group of Y. Hanein144
developed a systems based on inorganic nanorods absorbers dispersed on a mesh of carbon
nanotubes. In these works, illumination intensities on the order of tens to hundreds of μW/mm2
were employed and in both cases a capacitive coupling mechanism was proposed.
5.2 Perspectives
From the discussion of the experimental results presented in this work it certainly emerges that
indeed hybrid polymer/electrolyte interfaces harvest a huge interest for the modulation of
bioelectrical activity in biological tissues. These devices give the possibility to interact with cells
through stimuli of different nature, both electrical and thermal. The accepted models for electrical
capacitive stimulation, based on the concept of attached and free compartments of the membrane
described in Chapter 4.4, predict that both anodic and cathodic currents results in an excitation of
neural activity, while inhibition cannot be effectively obtained. In the case of our hybrid interfaces,
the possibility of having also thermal effects, that on long timescales have been demonstrated to
block the generation and propagation of action potentials, opens the way to the realization of
multifunctional platforms for controlling neural activity.
94
However, there are still some issues that need to be addressed to reach a clear understanding of the
functioning of these devices.
The current densities elicited upon photoexcitation of the active material are comparable to the
value usually found in literature for electrical capacitive stimulation, but they rapidly fall to
zero on the millisecond timescale. An increase of the temporal duration of this current pulse is
thus desirable to obtain an efficient and reliable stimulation of neural cells. This goal can be
reached by increasing the interface capacitance and thus the total charge that can be
accumulated in two different ways. The first is to choose an active material with a higher
specific capacitance of the polymer/electrolyte interface; for example, thiophene-based
polymers with capacitance values in excess of 10 μF/cm2 (at f = 1 kHz) have been recently
demonstrated.212
The other way is to increase the active interfacial area between the polymer
and the electrolyte by using nano- or micro-structured electrodes. Also, porous materials like
hydrogels,219
which have been demonstrated to be ideal substrates for cell cultures, may be used
for this goal, upon functionalization with conjugated molecules.
The measurements performed on the HEK-293 cells revealed interesting information about the
effect of polymer-mediated photostimulation on general membrane properties like its
capacitance and overall conductivity; however, to understand the actual mechanisms that can
contribute to excitation in more complex systems like neurons, the effects on specific families
of membrane ion channels need to be taken into account.
o The eventual role of temperature-gated channels, like those of the TRPV family.57,218
These
channels, which are not endogenously expressed in HEK-293 cells, are found in different
kinds of neurons, like retinal and vestibular ganglion cells,220
dorsal root ganglion cells,63
CA1 neurons,62
and also in other cells like hyppocampal astrocytes.61
Interestingly, it is
possible to express TRPV channels in HEK-293 cells via transfection protocols and
photothermal stimulation of HEK-293 cells expressing different channels of this family198
has been already demonstrated with water-mediated IR light absorption.
o The effect of the light-evoked capacitive currents on voltage-gated ion channels. Voltage-
gated sodium (NaV) channels are fundamental in the generation of action potentials in
neurons. These channels can be expressed in HEK-293 cells and it has been shown that they
can be triggered via capacitive stimulation with inorganic silicon-based devices.206
Similar
measurements have also been performed on voltage-gated potassium (KV) channels.211
Moreover, it would be interesting to understand how the actual composition of the cell
membrane, in terms of the lipid and protein content but also of the ionic diffuse layer presents
on the two sides, affects the variation in capacitance and resistance upon local heating.58,146
95
Given the complex nature of the plasma membrane, simplified systems like suspended artificial
lipid bilayers204
and vesicles89
may be used to decouple the effects of the various components.
In the meantime, other directions for a further development of these polymer-based hybrid
interfaces can be envisioned:
The devices presented in this work are based on P3HT as the active material, which absorb in
the blue/green region of the spectrum. However, a great variety of semiconducting polymers is
commercially available, with different absorption properties. A clear possible application of this
tunability is the development of a patterned interface with different organic materials absorbing
in different regions of the visible spectrum,221
in order to try to reproduce the color sensitivity of
the retina. Moreover, polymers absorbing in the near-IR222
could be used for applications
requiring in-vivo implants, since visible light has a low penetration depth in biological tissues.
Organic semiconductors have the great advantage that their molecular structure can be
chemically tuned to add different functionalities to the material without losing in conduction
properties. It is thus possible to produce conjugated polymers that are functionalized with
specific biochemical groups that can recognize particular cell types and allow their efficient
adhesion.118
In this way, it is possible to carefully control the cleft properties, which are
fundamental in determining an efficient electrical coupling between the cell and the device
interface.32,33
The hybrid interfaces discussed in this work have been mainly treated in terms of their
capability to modulate the membrane potential of cells for controlling neural activity. However,
electrical signals have been demonstrated to be involved in many other biological processes. A
fascinating example of an alternative use of our interface can be in the control of cellular
growth and differentiation.106,107,117
It has been widely reported in literature that extracellular
electrical stimulation of neural and neuronal-like cells can greatly affect their growth. Recently,
B. Zhu et al.118
have realized a functional substrate based on a conductive polymer and have
demonstrated that applying short pulses of voltage it is possible to enhance the neurite
outgrowth of PC12 cells grown on its surface. It would be thus interesting to understand if the
same effect could be obtained in our hybrid interfaces; if that was the case, new possibilities
could arise for controlling with precise spatio-temporal resolution the differentiation of
biological tissues. Moreover, it has been recently shown that the ability to modulate the cell
membrane potential can have important implications in the control of the life cycle of cells and
in cancer progression.148,149
The devices presented in this work are based on planar interfaces on which cells are grown.
While this configuration is perfectly suitable for in-vitro measurements, it has several
drawbacks for in-vivo experimentation. For these applications, it would thus be desirable to
96
develop new architectures. One example could be the realization of polymer-coated optical
fiber,223
able to bring the active polymer deeper in tissues and at the same time deliver the
luminous stimuli. Another way could be that of realizing photoactive polymeric micro- and
nano-particle224,225
to be delivered in-vivo via injections; however, care should be taken in this
case on how the electrical and physico-chemical properties of the polymer/electrolyte interfaces
can vary with respect to planar devices.
97
Appendix A
A.1 Optical Measurements
In this section are described the experimental setups used for the pump and probe measurements on
the different timescales presented in Chapter 3.3.
A.1.1 Femtosecond spectroscopy
Femtosecond dynamics of the primary photoexcitations in pristine P3HT and P3HT:PCBM blends
were measured with an ultrafast pump-and-probe setup with time resolution of ≈ 150 fs. A
schematic representation of the setup is depicted in Figure A.1.
Figure A.1 | Typical configuration of a femtosecond pump-and-probe experimental setup.
OPA: optical parametric amplifier.
Both the pump and the probe beam comes from an regeneratively amplified mode-locked Ti:Sapph
laser (Clark-MXR model CPA-1), with a fundamental emission wavelength at 780 nm at a
repetition rate of 1 kHz. The laser output is separated in two branches. The pump beam at 530 nm is
obtained from the fundamental via non-linear processes in an optical parametric amplifier (OPA).
The visible probe beam is achieved via supercontinuum generation focusing the laser through a 2
mm sapphire crystal. The pump beam is mechanically chopped at 500 Hz and it’s delayed with
respect to the probe by means of a motorized translation stage that increases its optical path. The
98
two beams are then focused onto the sample on a spot with ≈ 50 μm diameter, taking care that they
have a good overlapping. The transmitted pump beam is blocked, while the probe is collected and
sent to an optical multichannel spectral analyzer. The acquisition of the probe spectra is
synchronized to the chopper modulation, in order to collect the probe transmission in presence (Ton)
and absence (Toff) of the pump. The final signal is then given by the normalized differential
transmission:
The energy of the pump pulses is kept low (≈ 20 nJ/pulse) in order to avoid bimolecular processes
upon photoexcitation, while the total energy of the probe beam is always at least an order of
magnitude lower.
A.1.2 Nanosecond spectroscopy
Transient absorption measurements on the nanoseconds/microseconds timescale were performed
with a commercial laser flash spectrometer (Edinburgh Instruments LP920). The pump pulses are
provided nanosecond tunable source (Opotek Opolett 355II) based on a Q-switched Nd:YAG laser
with a 10 Hz repetition rate. The probe light comes from a pulsed xenon arc lamp. The two beams
arrive at the sample position with a 90° angle, with the sample kept at a 45° angle respect to them.
The beams are properly focused onto the sample in order to ensure spatial overlap. The transmitted
probe light is spectrally filtered with a monochromator and then detected at a single wavelength by
a photomultiplier with a detection window in the visible.
For the traces reported in Chapter 3.3, an excitation wavelength of 530 nm was used, with an energy
density per pulse of about 20 μJ/cm2. The photobleaching signal was collected at 570 nm with a
spectral bandwidth of the monochromator of 3.5 nm. In order to get a good signal-to-noise ratio,
each of the traces reported is the results of averaging of 200 individual recordings.
(A.1)
99
Figure A.2 | Experimental setup of the laser flash spectrometer used for ns-μs transient
absorption measurements.
A.1.3 CW Photoinduced Absorption
The measurements of CW absorption were performed in a home-made setup, whose schematic
structure is depicted in Figure A.3.
The probe beam is given by a 100 W tungsten halogen lamp that is focused on the sample by two
spherical mirrors; metal mirror are preferred to lenses to avoid chromatic aberrations and thus spots
of different size at different wavelength on the sample. The spot obtained on the sample plane has a
diameter of about 0.5 cm. The light transmitted through the sample is then recollected by two other
spherical mirrors that focus the beam on the entrance slit of a diffraction-grating based
monochromator (Spectral Products DK240). The pump beam is provided by a solid-state laser
source (Oxxius 561-100-COL-PP) with an optical power of about 100 mW, modualated by a
mechanical chopper at a frequency of f = 234 Hz. In order to obtain a good overlap between the
pump and the probe, the laser beam is enlarged by placing a defocusing lens in its path.
After being monochromated, the probe light is collected by a silicon photodetector (Thorlabs FDS
100) and the signal is analyzed with a lock-in amplifier (Stanford Research Systems SR830), locked
in frequency to the modulation of the chopper, with an integration time of 3 s. The measured signal
is thus the modulation in the transmitted light (ΔT) at a certain wavelength due to the presence of
the pump. The complete spectrum is reconstructed by scanning the monochromator through the
desired range of wavelengths, in this case 590 – 1100 nm. This signal is then normalized by the
sample transmission (T), measured in a second moment by modulating with the chopper the probe
beam in the absence of the pump.
100
Figure A.3 | Scheme of the custom-built CW Photoinduced Absorption experimental
setup. PD: photodiode.
A.2 Electrical and thermal characterization
A.2.1 Photovoltage measurements
Photovoltage measurements have been performed in a three-electrodes configuration with a
potentiostat/galvanostat station (Metrohm Autolab PGSTAT). In particular, the station is used in
galvanostatic mode, i.e. controlling the value of the current flowing through the working electrode
(WE) and the counter-electrode (CE) and simultaneously monitoring the potential difference
between the WE and the reference electrode (RE). In the case of the measurements performed in
Chapter 3.4, the current value was kept to zero, i.e. the cell was in open circuit condition. The
samples measured are P3HT-based thin-films deposited on ITO-covered glass substrates (thickness
1 mm) with an active area of about 2 cm2. After the spin-coating of the films (as described in
Chapter 3.2), a strip of the absorbing layer is removed with acetone to allow the electrical contact to
the ITO. The device, which is the actual working electrode, is put in a custom electrochemical cell
in which the distance between the WE, the CE and the RE are fixed, in order to have repeatable
measurements. For the counter-electrode, a platinum wire is used, while the reference electrode is
an Ag/AgCl couple in a saturated KCl solution. The cell is filled with an electrolytic solution of
NaCl 0.2 M (if not otherwise specified) in ultrapure water.
Illumination to the sample is provided by a white LED (Thorlabs MCWHL5-C4), controlled in
intensity and pulse duration via a command signal provided by a function generator (Keithley
101
3390). The light enters the cell through a planar quartz window and illuminates the entire sample
active area.
A.2.2 Photocurrent measurements
Photocurrent measurements were performed in a home-made setup with a two-electrodes
configuration. The samples for these measurements were deposited on ITO-covered glass substrates
(thickness 170 μm) with lateral dimensions of 9x18 mm2. On these substrates, the active material
was deposited via spin-coating methods, leaving a 9x5 mm2 strip of free ITO on the top for making
the electrical contact. The sample was then attached to the inner wall of a transparent cuvette with
square cross-section (1x1 cm2). The cuvette was filled with the electrolytic solution (NaCl 0.2 M)
until the active layer of the device was almost completely immersed, but leaving the free ITO strip
dry. As a counter-electrode, a platinum wire was immersed in the solution at the opposite side of the
cuvette. The current flowing from the ITO to the counter-electrode was measured by connecting the
two terminals to a transimpedance amplifier (Femto DHPCA-100). The amplified signal was then
collected with a digital oscilloscope (Tektronix MSO4054).
The illumination of the device was provided by a LED system (Lumencor Spectra X) with a
collimated output. In particular, a LED in the green was used (central wavelength λ = 530 nm) to
match the absorption peak of the P3HT. The LED intensity was controlled both via software and
with a set of neutral density filters. Light pulses (50 ms at 1 Hz repetition rate) were generated by
controlling the LED driver with a TTL signal provided by a function generator (Keithley 3390), that
also triggered the oscilloscope for acquisition.
A.2.3 Surface potential and temperature measurements
Measurements of surface potential a local temperature at the polymer/electrolyte interface were
performed with a similar experimental configuration using the patch-clamp setup (see Section
A.3.1).
The device to be analyzed was put in a plastic petri-dish and immersed in an electrolytic solution
(NaCl 0.2 M, if not specified otherwise) with Ag/AgCl counter-electrode. A glass micropipette
filled with the same electrolytic solution of the bath was micromanipulated in proximity of the
device surface. The approach was controlled visually by bringing the micropipette tip on the same
focal plane as the device surface. Both measurements were performed using the patch-clamp
amplifier in the voltage-clamp configuration and recording the signals upon illumination of the
active layer through the microscope objective, with a spot size dependent on the magnification used.
102
The following scheme summarizes the electrical configuration in which the measurements were
performed.
Figure A.4 | Schematic representation of the electrical equivalent circuit for the
measurements of the surface potentials and temperatures.
In dark conditions, the current flowing through the pipette resistance (which is the actual parameter
measured in the experiment, Ipip) is determined by the potential different between Vsurf, the potential
at the device surface, and Vpip, which is kept by the amplifier at the offset value (Vset) set by the user:
where Rpip is the pipette resistance, determined by the solution concentration and the dimensions of
the pipette tip, and ΔVR the potential difference at its ends.
The main difference between the two experiments is in the offset applied to the recording pipette:
To measure the surface potentials, the offset was set in order not to have current flowing in the
pipette in dark conditions (ΔVR = 0). In this case, Vset and Vpip are thus equal to the value of Vsurf
in dark conditions, which is in turn equal to the reference potential (Vref = 0). Upon illumination,
Vsurf can change because of the capacitive charging of the interface, while Vpip is kept to the
reference value. The surface potential can thus be easily calculated as:
To measure the local temperature, the offset was set at a value that gave a constant current of
about Ioff = 4 nA. In this case, since the measurements were performed only on samples without
the ITO electrode, there is no capacitive charging of the interface upon illumination and Vsurf is
thus constant throughout the experiment, and thus ΔVR is also constant and it’s equal to the
product IoffR0, where R0 is the pipette resistance in dark conditions. Upon illumination, the value
(A.2)
(A.3)
103
of the pipette resistance changes because of the local temperature variation, and can be
determined from the measured current from the following relationship:
The local temperature dynamics can then be recovered from the values of the pipette resistance
after proper calibration, as described in Chapter 3.5.
It should be noted that, also in the case of surface potential measurements the pipette resistance
maybe changing with time due to local heating. In principle, this variation should be taken into
account when calculating the surface potential value from the measured current. However, this
effect becomes important at longer times during the light pulse, while the surface potential signal is
falling to zero quite rapidly, especially in the blend, within the first few milliseconds. As a first
approximation we thus considered the pipette resistance constant in the surface potential
measurements.
A.3 Electrophysiology measurements
A.3.1 Electrophysiology setup
Measurements on HEK-293 cells, but also surface electrical and thermal characterizations of the
hybrid interface, were performed on a standard electrophysiology setup. This setup can be basically
divided in two parts:
An optical system for imaging the cells and guide the patch, with the possibility of shining light
pulses on the sample. It is composed of an inverted microscope (Nikon Eclipse Ti-S) coupled to
a CCD camera (Photometrics CoolSNAP MYO) for video acquisition. The light from the
microscope illuminator is filtered in order to use only wavelength longer than 750 nm for
imaging. In this way, the imaging light does not photoexcite the active material, which absorbs
in the visible. The excitation beam is provided by a collimated LED system (Lumencor Spectra
X) coupled to the microscope port for the fluorescence excitation source. The LED system is
equipped with six different LEDs, of which we use the blue (λ = 430 nm), the cyan (λ = 475
nm) and the green (λ = 530 nm). Power of the light impinging on the sample can be controlled
both via software and with neutral density filters in the optical path of the excitation light.
Measurements were performed with a 40x air objective (Nikon S Plan Fluor ELWD), giving an
(A.4)
104
excitation spot on the sample with a diameter of 540 μm (that correspond to an active area of
about 0.23 mm2).
An electronic system for signal acquisition and amplification. The recording electrode is a
chlorinated silver wire enclosed in a glass pipette filled with an electrolytic solution, while the
counter-electrode is a pellet of Ag/AgCl. The acquisition is performed via a dedicated amplifier,
specifically designed for patch-clamp measurements (Molecular Devices Axopatch 200B). The
recording electrode is mounted on a headstage, which hold the pipette but also performs a first
amplification of the signal before sending it to the main unit of the amplifier. To allow precise
positioning of the pipette, the headstage/pipette system is mounted on a 3-axes
micromanipulator (Sutter Instruments MP-225). The amplifier is interfaced to a PC via a data
acquisition system (Molecular Devices Digidata 1550), which also commands the LED system
for triggering the light pulses.
For all the experiments, pipettes were freshly pulled just before each measurement with a
Flaming/Brown puller (Sutter Instruments P1000) from fire-polished glass capillaries (WPI
1B150F-4).
A.3.2 Electrolytic solutions and cell growth medium
HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM - high glucose, Sigma
D5796), supplemented with 10 % fetal bovine serum (Euroclone ECS 0180L), 100 U/ml penicillin
and 100 μg/ml streptomycin.
For patch-clamp experiments, the composition of the intracellular and extracellular solutions used
are reported in the tables below.
Intracellular solution
KCl K-Gluconate MgCl2 CaCl2 HEPES EGTA ATP-Na2
12 mM 125 mM 1 mM 0.1 mM 10 mM 10 mM 10 mM
Extracellular solution
NaCl KCl MgCl2 CaCl2 Glucose HEPES
135 mM 5.4 mM 1 mM 1.8 mM 10 mM 5 mM
In measurements not involving the presence of cells we normally employed, if not specified
otherwise, a solution of NaCl in ultrapure water at a concentration of 0.2 M. Sodium chloride was
105
selected because Na+ and Cl
- are the two main ions in the extracellular solutions and the
concentration was chosen in order to roughly mimic the typical ionic strength of physiological
media.
106
Appendix B
The analysis performed in this thesis has focused only on the behaviour of the polymer/electrolyte
hybrid interface, both on its own and coupled with cells, under illumination with short light pulses
up to few hundreds of milliseconds. The device, in its complete architecture with an ITO contact,
has been shown to sustain the generation of a capacitive photocurrent that charges the
polymer/electrolyte interface in few milliseconds. However, it has also been proven in other works
that under prolonged illumination electrochemical reactions can be promoted. We have thus also
performed a study on the effect of continuous photoexcitation of the active material on cells grown
on the device. In particular, we have performed experiments using primary neocortical astrocytes.
Astrocytes are cells that belong to the family of glial cells, which comprises the various types of
non-excitable cells present in the nervous system. Until the end of the last century, the function of
glial cells was thought to be only of support and protection for neurons. However, increasing
evidence in the recent years indicates that glial cells, and especially astrocytes, may have a more
active role in the functioning of the nervous system than previously believed. In particular,
astrocytes are found in the central nervous system, where they are the most numerous macroglial
cells. They have a fundamental role in regulating the concentrations of ions in the extracellular
space and in the recycling of neurotransmitters released by the synaptic terminals of neurons during
the propagation of the nervous signals. Moreover, astrocytes have a major role in the response of
the neural tissue to pathological situations like traumas, infections or neurodegenerative diseases
and also in the reaction to medical implants.
It is thus clear the importance of understanding the effects of photoexcitation on glial cells if the
polymeric interfaces here developed are to be applied for in vitro experimentations on models of the
nervous system or for in vivo implants.
Since the work that follows (which has been published in Ref. 142) is based on the use of prolonged
illumination protocols, the effects observed here cannot be directly compared to what observed in
the main part of this thesis. However, they are reported in this Appendix because they give in any
case interesting complementary information on the possible applications of the photoactive
polymeric interfaces.
107
B.1 Astrocyte cultures and electrophysiological properties
In this work, we used mainly P3HT:PCBM films (with a thickness of about 50 nm) as the active
layer of the devices. We started by characterized the possibility of growing astrocytes on these
materials assessing the viability of cell cultures at several days in vitro. In order to promote
adhesion of the cells to the substrates, they were pre-coated with a layer of poly-d-lysine (PDL), a
commonly used molecule for cultures of brain cells.
We firstly performed a fluorescence assay based on the detection of fluorescein diacetate (FDA)
emission. This molecule, which is initially non-emissive, is hydrolyzed inside the cells by non-
specific esterases, transforming it into a fluorescent probe. Since this process can happen only in
live cells, the intensity of fluorescence can be related to the viability of cells. Figure B.1a,b shows
typical fluorescence images taken on control substrates (ITO+PDL) and on the active devices
(ITO/P3HT:PCBM+PDL) at 1 day in vitro (DIV) after re-plating. In the histograms of Figure B.1c
are reported the viability of the cells on the two different substrates at 1DIV and 4DIV, showing
that indeed astrocytes can be cultured on P3HT:PCBM thin films without significant effects on their
vitality.
Figure B.1 | (a,b) Fluorescence confocal images of astrocytes stained with fluorescein
diacetate grown on control ITO substrates (a) and on ITO/P3HT:PCBM devices (b); the
images are taken 1 day after plating. (c) Viability of astrocytes cultured on the two
different substrates at 1 and 4 DIV.
We then characterized the basic electrophysiological properties of the astrocytes cultured on the
active devices and compared them to those grown on control substrates. The cell membrane
properties were measured via patch-clamp techniques in whole-cells recordings with control
extracellular and intracellular solutions (see Section B.3 for details). A voltage ramp from -120 mV
to + 60 mV (duration 600 ms) was applied to the astrocytes and the relative membrane current was
measured in a voltage-clamp configuration. The complete protocol is depicted in the inset of Figure
B.2. The current traces recorded during the voltage ramp are reported in Figure B.2a,b for the case
of the cells grown on the control substrates and on the active material respectively. The membranes
108
show a strong rectifying behavior for negative potentials, with negligible currents at values more
hyperpolarized than -40 mV.
Figure B.2 | Membrane currents recorded on ITO (a) and ITO/P3HT:PCBM (b) substrates
upon the application of the protocol reported in the inset. Only the current trace relative to
the ramp part of the stimulus is reported.
The electrophysiological parameters extracted during the measurements on both substrates are
reported in Table B.1. No significant difference can be observed between the values recorded on the
two cases (control: n = 9; blend: n = 10).
Table B.1 | Electrophysiological properties recorded on ITO (control) and
ITO/P3HT:PCBM (blend) substrates. Vm: membrane resting potential; Cp: membrane
capacitance; Rin: patch input resistance; spG: membrane specific conductance; I-120mV and
I60mV: current density (i.e. normalized by the cell capacitance) measured at -120 mV and
60 mV respectively.
The biophysical properties of the currents here measured are perfectly consistent with those already
characterized in immature glial cells grown on standard plastic Petri dishes for tissue culturing, that
have been mainly attributed to the presence of the delayed rectifier potassium channel (KDR).226,227
These data confirms that conjugated polymers are suitable substrates for culturing primary cells like
astrocytes and that they do not modify their basic electrophysiological property with respect to
control substrates normally employed in studies in vitro.
Vm
[mV]
Cp
[pF]
Rin
[MΩ]
spG
[pS/pF]
I-120mV
[pA/pF]
I60mV
[pA/pF]
Control −47±3 42.1 ± 5.9 443 ± 105 0.1 ± 0.02 −6.3 ± 1.2 52.3 ± 7.6
Blend −48±7 38.3 ± 9.8 599 ± 138 0.07 ± 0.03 −4.7 ± 2 44.3 ± 16.5
109
B.2 Photostimulation of astrocytes membrane conductances
After having assessed the characteristics of membrane conduction in astrocytes in dark, we
investigated the effects on those properties of photostimulation of the active material. The
membrane potential was monitored during continuous illumination of the active layer with light
intensity of 13 mW/mm2 (λ = 561 nm) in a spot with a diameter of about 100 μm around the
patched astrocyte. Upon the onset of the light stimulus, the membrane potential of the cell starts to
depolarize, passing from -49 ± 4 mV to -19 ± 2 mV (n = 5) during 50 s photoexcitation, while no
effect can be observed for measurements on control substrates (Figure B.3). This behavior is
apparently in contrast with the measurements presented in Chapter 4 on HEK-293 cells, in which a
hyperpolarization was measured at longer times. However, it has to be reminded that the timescales
investigated here (several seconds) are completely different the pulses used in the HEK-293
recordings, which arrived up to 200 ms at maximum. Indeed, preliminary measurements (data not
shown) on astrocytes with short pulses confirm the presence of a behavior comparable to that of
HEK-293 cells on those timescales.
Figure B.3| Recording of the variations in membrane potential of an astrocyte upon
illumination with continuous light (13 mW/mm2) for P3HT:PCBM (red trace) and control
(grey trace) substrates.
To understand what biophysical phenomenon could be responsible of the depolarization observed in
Figure B.3, we repeated the membrane characterization with a voltage ramp protocol of Figure B.2
at different times during the illumination increasing the light intensity. In particular, the protocol
was applied every 10 s to measure how the membrane currents were affected by the
photostimulation. It can be seen in Figure B.4a that indeed the conductance properties of the
membrane vary during the illumination; in particular a significant increase of inward currents at
negative potentials could be observed, an effect that was augmented by using higher light intensities
(Figure B.4c). At the same time, a shift of the zero current potential (which correspond to the
equilibrium potential) towards more positive values was observed, consistently with the
110
depolarization measured in Figure B.3. Again, no significant effect was recorded on control
substrates (Figure Figure B.4b,d); indeed, eventual small variations in the measured currents are
clearly independent of the presence of the light stimulus.
Figure B.4 | (a,b) Membrane currents recorded in dark and at different light intensities
during illumination with the voltage protocol of Figure B.2 for P3HT:PCBM (a) and
control (b) substrates. (c,d) Variation in time of the current measured at -120 mV (i.e. at
the start of the voltage ramp) during continuous illumination at different light intensities
(represented by the colored boxes) for P3HT:PCBM (c) and control (d) substrates.
The inward current observed upon photostimulation remained also after switching off the light and
we attributed the effect to the activation of a membrane conductance. Considering the simultaneous
shift of the zero-current potential toward more positive values, we could hypothesize the
involvement of a chloride conductance or of a nonspecific cation channel. We thus exposed the
cells after the light-mediated stimulation to an extracellular solution in which all the monovalent
cations were replaced equimolarly by the non-permeable ion NMDG+. The ramp currents elicited in
the membrane with control saline and with this modified solutions (NMDG-Cl) were however the
same (Figure B.5a), indicating that the photoactivated current was not mediated by cations and was
thus most likely due to Cl- ions. We then investigated the biophysical properties of this current by
analyzing its time dynamics. In order to isolate this current from the other conductances of the cell,
we replaced equimolarly the cations in the extracellular solutions with NMDG+ and the potassium
in the intracellular solution with Cs+. We measured the time traces of the evoked currents before
(Figure B.5b) and after (Figure B.5c) photostimulation of the astrocytes upon the application of a
111
voltage step protocol from -120 mV to + 60 mV (see inset in Figure B.5b). While in resting
conditions virtually no conductances could be measured at every potential, after the light excitation
hyperpolarizing stimuli were able to elicit a time-dependent current. This current displayed a first,
fast component and a second slowly activated, non-deactivating one that grew larger for higher
hyperpolarizations. This behavior is characteristic of the chloride conductance mediated by the ClC-
2 channel, which has been previously characterized in cortical astrocytes both in vitro and in
situ.228,229
Interestingly, this channel is normally not activated in astrocytes cultured in vitro in
resting condition, unless a long-term pharmacological treatment is used.
Figure B.5 | (a) Ramp currents recorded on astrocytes after 50 s photostimulation (13
mW/mm2) with control extracellular saline (gray trace) and the NMDG
+-Cl solution
(black trace). (b,c) Current traces evoked in astrocytes upon a step voltage protocol (from
-120 mV to 60 mV in 20 mV steps) with intracellular CsCl and extracellular NMDG-Cl
before (b) and after (c) photostimulation for 50 s at 13 mW/mm2.
To corroborate the hypothesis of the activation of the ClC-2 conductance, we exposed the cells after
photostimulation to a 200 μM extracellular concentration of Cd2+
ions, which have been
demonstrated to inhibit these ion channels.229
Indeed, the currents evoked in the astrocytes upon the
application of the Cd2+
-containing solution were clearly reduced with respect to the ones measured
with the control NMDG-Cl saline (Figure B.6).
The evidence collected demonstrates that long-term photostimulation of astrocytes with visible light
mediated by the hybrid polymer-based interfaces results in the activation of specific chloride
currents mediated by the ClC-2 ion channel. This channel is expressed in different tissues, like the
brain, intestine, stomach, kidney, salivary glands, and heart.230
While a clear understanding of the
physiological functions of ClC-2 still has to be determined, there is a growing body of evidence that
this channel can play different roles, in the different tissues in which it is expressed. Results
obtained from mice in which its expression was inhibited revealed that ClC-2 disruption resulted in
leukoencephalopathy, blindness and male infertility.231,232
In this context, the possibility to stimulate
112
ClC-2 currents by polymer photoexcitation may represent an interesting tool to help clarifying the
roles of this ion channel in central nervous system.
Figure B.6 | Current traces evoked with a voltage step protocol (inset) after
photostimulation (50 s light at 13 mW/mm2) with NMDG-Cl saline (a) and upon the
addition of sub-millimolar concentrations of Cd2+
ions (b).
Regarding the activation process of the channel upon photostimulation, the actual mechanism is still
unclear. It is known that ClC-2 conductances are modulated by different stimuli, like membrane
hyperpolarization, hypotonicity-induced cell swelling, moderate acidification of the extracellular
medium and disruption of F-actin filaments.226,228,233,234
During the measurements, we did not
observe any visible morphological change of the cell upon photostimulation. Membrane
hyperpolarization was indeed measured in HEK-293 cells upon prolonged illumination. However,
also electrochemical reactions at the polymer/electrolyte interface have been demonstrated to occur
on these long timescales and these reactions may be accompanied by a variation in the local pH felt
by the cell. More in-depth characterizations of these processes are thus necessary to clearly
understand the biophysical mechanisms leading the specific activation of ClC-2 channels.
B.3 Experimental methods
Organic conducting polymers preparation
Rr-P3HT has a regio-regularity of 99.5 % and molecular weight of 17500 g/mol. An accurate
cleaning of the substrate was required: the substrate was rinsed in an ultrasonic bath with,
sequentially, a specific tension-active agent in water solution (HELLMANEX® II, 3 %), deionised
water, pure acetone and isopropyl alcohol. 1,2-Chlorobenzene solutions of P3HT and PCBM were
prepared separately. P3HT was diluted to a final concentration of 15g/l. PCBM was prepared at a
concentration of 15g/l and then mixed (1:1 volume ratio) with P3HT using a magnetic stirrer.
Solutions were then heated at 50 °C, stirred and finally deposited on ITO-covered and ITO-less
glass substrates, previously heated, by spin-coating. Spinning parameters (first step: 800 rpm,
113
angular acceleration 1500 rad/s, rotation duration 2 s; second step: 1500 rpm, angular acceleration
4000 rad/s, rotation duration 30 s) were carefully selected in order to obtain suitable optical quality
and film thickness. After deposition, organic layers were annealed and properly sterilized by heating
at 120 °C for 2 hrs. Control substrates were sterilized in the same way.
Cell culture preparation and maintenance
Briefly, cerebral cortices devoid of meninges were triturated and placed in cell culture flasks
containing Dulbecco’s modified Eagle’s medium (DMEM)–glutamax medium with 15% fetal
bovine serum (FBS) and penicillin–streptomycin (100 U/mL and 100 lg/mL respectively) (Gibco-
Invitrogen, Milan, Italy). Culture flasks were maintained in a humidified incubator with 5% CO2
for 2–5 weeks. Immunostaining for glial fibrillary acidic protein (GFAP) and the flat, polygonal
morphological phenotype of the cultured cells indicated that more than 95% were type 1 cortical
astrocytes. At confluence, astroglial cells were enzymatically dispersed using trypsin–EDTA in
P3HT-PCBM/ITO or ITO substrates treated for 30 min with poly-D-lysine (0,01 mg/ml in PBS) at a
concentration of 1x104 per substrate and maintained in culture medium containing 10% FBS.
Cell Viability Assay and Counting
Astrocytes plated on the different substrates were mounted in a custom-made perfusion chamber
and incubated for 5 min with FDA (Sigma Aldrich). After rinsing with physiological saline
solution, a sequence of confocal images (10 to15 different fields of 0.6 mm × 0.6 mm for each
sample) was taken using a Nikon TE 2000 inverted confocal microscope (20× objective). Living
cells were counted and number of cells per mm 2 was calculated and compared at each time point
analyzed.
Electrophysiology and photostimulation
Current recordings were obtained with the whole-cell configuration of the patch-clamp technique.
Light excitation was provided by a CW laser diode (OXXIUS) peaking at 561 nm, with an intensity
was properly varied by a neutral density filter with variable optical density, in the range 0.7–13
mW/mm2. Laser light was coupled to the microscope, impinging on the sample from the ITO side.
Patch pipettes were prepared from thin-walled borosilicate glass capillaries to have a tip resistance
of 2-4 MW when filled with the standard internal solution. Membrane currents were amplified (List
EPC-7) and stored on a computer for off-line analysis (pClamp 6, Axon Instrument and Origin 6.0,
MicroCal). Because of the large current amplitude, the access resistance (below 10 MW) was
corrected 70-90%. Experiments were carried out at room temperature (20-24°C). The reference
electrode was an agar bridge filled with either 150 mM NaCl or 1 M KCl saline for experiments in
which substitution of extracellular Cl– was required. Experiments were carried out at room
114
temperature (20–24°C). Current densities were calculated by dividing the current values measured
at each membrane potential by the cell capacitance derived from the correction of the capacitive
transients of the recorded cells by means of the analogue circuit of the patch-clamp amplifier.
Solutions and chemicals
For electrophysiological experiments the standard bath saline was (mM): 140 NaCl, 4 KCl, 2
MgCl2, 2 CaCl2, 10 HEPES, 5 glucose, pH 7.4 with NaOH and osmolarity adjusted to ~315 mOsm
with mannitol. The intracellular (pipette) solution was composed of (mM): 144 KCl, 2 MgCl2, 5
EGTA, 10 HEPES, pH 7.2 with KOH and osmolarity ~300 mOsm. When using external solutions
with different ionic compositions, salts were replaced equimolarly. The different salines containing
pharmacological agents were applied with a gravity-driven, local perfusion system at a flow rate of
~200 ml/min positioned within ~100 mm of the recorded cell. In order to isolate Cl– current, the
external bath perfusion, termed control saline, was (in mM): 140 NMDG-Cl, 4 NaCl, 2 MgCl2, 2
CaCl2, 10 TES, 5 glucose. The intracellular (pipette) solution was composed of (in mM): 126 CsCl,
2 MgCl2, 1 EGTA, 10 TES, pH 7.2 with CsOH, and osmolarity adjusted to approximately 290
mOsm with mannitol.
115
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Acknowledgements
“No man is an island entire of itself; every man
is a piece of the continent, a part of the main”
John Donne (1624)
It was the spring of 2008 when I first entered a lab for my bachelor degree. It was in the Physics
Department and it was called “Cappa-Chimico”, from the fumehood in a corner behind the big
optical table. I witnessed its moving to the infamous garage of the Administrative Building, where it
became the first ARCO-lab, not knowing that I would later spend an entire year in its cold and dark
rooms working on the first version of the cw-confocal for my master thesis. Nor did I knew that
ARCO was meant to be just the original nucleus of what is now the Center for NanoScience and
Technology (CNST for friends), which I witnessed being transformed from a series of empty (and
wet) rooms into a thriving center at the forefront of international research, and which pretty much
became my second home during the four years of my PhD. It has been seven long years since this
adventure started, and it has now arrived the time to thank all the people that have accompanied me
during this period, also because I’m still in debt of a proper acknowledgment to many of them since
my master thesis.
First of all, I have to thank my supervisor, Dr. Maria Rosa Antognazza for supporting me during
these five years, even if I sometimes made it difficult with my chronic lateness, and for always
taking into high consideration my personal and professional interests and needs. Thanks also to
prof. Guglielmo Lanzani, for all his ideas and for allowing me to work in such a scientifically
exciting and socially pleasant environment as the CNST. Thanks to both of them for having given
me the opportunity to work on extremely fascinating projects and to meet very interesting people.
I would like to thank the colleagues from my research group that were essential in providing both
experimental help and theoretical discussions fundamental for the outcomes of this work:
Sebastiano, for the electrochemical measurements; Matteo, for all the numerical calculations; Katia,
for teaching me how to grow cells. Moreover, I have to thank Erica, Elena, Susana, Gabriele,
Andrea, Giovanni, Daniele and Lucia.
I also have to thank our collaborators that guided me from the reductionist world of physics into the
holistic realm of biology and neuroscience. In particular, I would like to thank Dr. Valentina
Benfenati and Paul Feyen for introducing me to this strange discipline called electrophysiology and
131
for teaching me the art of the patch-clamp, but also for all the invaluable discussions we had. I also
have to thank Prof. Fabio Benfenati, Dr. Diego Ghezzi, Dr. Fabrizia Cesca, Dr. Elisabetta Colombo
from the NBT Department in Genova, as well as Prof. Michele Muccini, Prof. Stefano Ferroni and
Dr. Stefano Toffanin at the CNR and Univeristy in Bologna.
I need to thank Dr. Giulia Grancini for being my first guide into ultrafast and confocal
spectroscopy, Dr. Mario Caironi and Dr. Calogero Sciascia for the fruitful collaboration on Charge
Modulation Microscopy and Dr. Alessandro Luzio for making me state-of-the-art transistors. I also
wish to thank all the people I have collaborated with in the disparate projects I was involved in
during these five years: Dr. Annamaria Petrozza, Dr. Daniele Fazzi, Dr. Ajay Ram Srimath
Kandada, Dr. Margherita Zavelani-Rossi, Dr. Valeria Russo, Dr. Massimiliano Bianchi, Dr. Assunta
Pistone, Dr. Grazia Pertile, Dr. Maurizio Mete and Dr. Silvia Bisti.
I wish also to thank all my friends and colleagues from CNST that have made (or still do) working
in this institute not only an invaluable opportunity for my scientific formation, but also a unique
experience at the personal level: Ross, for always seeing the best in people; Vale (Vinz), for being
around since the old times of the garage-ARCO; Vitto, for always knowing the right thing to say;
Ale L, not only for making transistors; Marco (and wife) and Mary for the Friday sushis and the
good wines; Giorgio, Andrea and Joy, for making CNST not so lonely on weekends (and for the
darts matches); Sadir and Pupi, the steady couple since the university years; Michele DB, for always
having the latest news; Vale N, for always having a good word for everyone; Piva, for not letting
me be the only Nicola in the center; Luca P, for his half-veals and cassoeulas; Marcelo, for his
perfect English; Ale Mez, for always being the spirit of the parties. Thanks to all the others, past and
present: Sara, James, Alex, Isis, Berri, GEB, Andrea DV, Erika and Simone, Nava, Giacomo,
Giusy, FM, Michele G, Antonio, Nicolas and all those I have for sure forgotten. Thanks to Rob for
the nice time we had during his stay in Italy. A big thanks also to the administrative and technical
offices, Silvia, Tessa, Elena, Alessandra, Stefano, Luca and Enrico.
A long-awaited and sincere thanks to Marghe, who has always been next to me through the nine
years of highs and lows from the first lessons of calculus to the hard life of the researcher, and with
whom I look forward to have an equally exciting time as a new chapter of our life starts in the U.S.
An equally long-awaited thanks to Lalla (and husband) for always being present and supportive,
even if she betrayed science for the dark side of a real job. Not less important, a special thanks is
due to the parents of both of them, who were always concerned with feeding me in a proper way. I
wish to also to thank everyone else from the years of University, especially Ale, Paul, Mirco, Fra,
Nico and Vale, and all my other friends here in Milan, Sica, Macchia, Gigio, Dani, Tobia, Ieva,
Luz, Cora, Giulia and Tommy.
132
A special thanks to Vicky for our 14-years long friendship that has been a constant and essential
element in my life from Alassio to Milano and that grows stronger every year that passes. A second
special thanks to Fede, for being the best friend (and roommate) anyone could hope for. Thanks to
Ivan for always giving me a place in the sun and to Marco for not letting me be the only nerd
engineer at the beach. Thanks also to Silvia and Jessica for being there since I can remember.
Last, but not least, thanks to my mother, for the endless support and love she has always given me
throughout all my life and for never letting me lack anything, to my father, my grandmother, my
cousin, my aunt, my brothers and sister.
133
List of publications
1. N. Martino, P. Feyen, M. Porro, C. Bossio, E. Zucchetti, D. Ghezzi, F. Benfenati, G. Lanzani,
M.R. Antognazza, Phototermal cellular stimulation in functional bio-polymer interfaces. Sci.
Rep. 5, 8911 (2015).
2. M.R. Antognazza, N. Martino, D. Ghezzi, P. Feyen, E. Colombo, D. Endeman, F. Benfenati, G.
Lanzani, Shedding Light on Living Cells. Adv. Mater. (2014) DOI: 10.1002/adma.201403513
3. N. Martino, D. Fazzi, C. Sciascia, A. Luzio, M.R. Antognazza, M. Caironi, Mapping
Orientational Order of Charge-Probed Domains in a Semiconducting Polymer. ACS Nano 8,
5968 (2014)
4. V. Benfenati, N. Martino, M.R. Antognazza, A. Pistone, S. Toffanin, S. Ferroni, G. Lanzani, M.
Muccini, Photostimulation of Whole‐Cell Conductance in Primary Rat Neocortical Astrocytes
Mediated by Organic Semiconducting Thin Films. Adv. Health. Mater. 3, 392 (2014)
5. D. Endeman, P. Feyen, D. Ghezzi, M.R. Antognazza, N. Martino, E. Colombo, G. Lanzani, F.
Benfenati, The Use of Light-Sensitive Organic Semiconductors to Manipulate Neuronal Activity
in “Novel Approaches for Single Molecule Activation and Detection”. Springer Berlin
Heidelberg (2014).
6. G. Grancini, M. De Bastiani, N. Martino, D. Fazzi, H.-J. Egelhaaf, T. Sauermann, M.R.
Antognazza, G. Lanzani, M. Caironi, L. Franco, A. Petrozza, The critical role of interfacial
dynamics in the stability of organic photovoltaic devices. Phys. Chem. Chem. Phys. 16, 8294
(2014).
7. D. Ghezzi, M.R. Antognazza, R. Maccarone, S. Bellani, E. Lanzarini, N. Martino, M. Mete, G.
Pertile, S. Bisti, G. Lanzani, F. Benfenati, A polymer optoelectronic interface restores light
sensitivity in blind rat retinas. Nature Photon. 7, 400 (2013).
8. N. Martino, D. Ghezzi, F. Benfenati, G. Lanzani, M.R. Antognazza, Organic Semiconductors
for Artificial Vision. J. Mater. Chem. B 1, 3768 (2013).
9. G. Grancini, N. Martino, M. Bianchi, L.G. Rizzi, V. Russo, A. Li Bassi, C.S. Casari, A.
Petrozza, R. Sordan, G. Lanzani, Ultrafast spectroscopic imaging of exfoliated graphene. Phys.
Status Solidi B 249, 2497 (2012).
10. G. Grancini, N. Martino, M.R. Antognazza, M. Celebrano, H.-J. Egelhaaf, G. Lanzani,
Influence of blend composition on ultrafast charge generation and recombination dynamics in
low band gap polymer-based organic photovoltaics. J. Phys. Chem. C 116, 9838 (2012).
11. C. Sciascia, N. Martino, T. Schuettfort, B. Watts, G. Grancini, M.R. Antognazza, M.
Zavelani‐Rossi, C.R. McNeill, M. Caironi, Sub‐Micrometer Charge Modulation Microscopy of
a High Mobility Polymeric n‐Channel Field‐Effect Transistor. Adv. Mater. 23, 5086 (2011).
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