Assembly of photo-bioelectrochemical cells using photosystem I … · 2016-05-19 · concentration of 0.6% and the solution was stirred at room temperature for 20 minutes. Following

SUPPLEMENTARY INFORMATIONARTICLE NUMBER: 15021 | DOI: 10.1038/NENERGY.2015.21

NATURE ENERGY | www.nature.com/natureenergy 1

Supplementary Information

Supplementary Figure 1: Detailed stepwise assembly of the (PQQ)/PSI/Os2+/3+

polyvinylimidazole redox polymer/glucose oxidase, GOx electrodes.

Assembly of photo-bioelectrochemical cells using photosystem I-functionalized electrodes

Ariel Efrati, Chun-Hua Lu, Dorit Michaeli, Rachel Nechushtai, Sabine Alsoub, Wolfgang Schuhmann and Itamar Willner

http://dx.doi.org/10.1038/nenergy.2015.21

2 NATURE ENERGY | www.nature.com/natureenergy

SUPPLEMENTARY INFORMATION DOI: 10.1038/NENERGY.2015.21

Supplementary Figure 2: A set of complementary control photocurrent action spectra

experiments for the (PQQ)/PSI/Os2+/3+ polyvinylimidazole redox

polymer/glucose oxidase (GOx) assembly: (a) Substitution of PSI by a light-

insensitive protein-cholesterol oxidase, 20 mM glucose, (b) Elimination of the

polymeric Os2+/3+-complex from the array and the direct covalent linkage of

GOx to PSI, 20 mM glucose (c) Elimination of the glucose oxidase from

the system using EDC and NHS, 20 mM glucose. (d,e) Exclusion of PQQ from

the array and the direct tethering of the PSI to the aminated electrode using

succinic anhydride, 0 mM and 20 mM glucose for d and e curves respectively.

(f) Full (PQQ)/PSI/Os2+/3+ polyvinylimidazole redox polymer/glucose oxidase

(GOx) assembly, 20 mM glucose.



SUPPLEMENTARY INFORMATIONDOI: 10.1038/NENERGY.2015.21

Supplementary Figure 3: An organised photonically wired pyrroloquinoline quinone

(PQQ)/PSI/Os2+/3+ polyvinylimidazole redox polymer/FAD-dependent

glucose dehydrogenase (GDH) (from Aspergillus sp., EC 1.1.99.10)

assembly on an ITO electrode acting as a photoelectrochemical enzymatic

electrode. The modified electrode results in the photochemical oxidation of

glucose and the generation of photocurrent by a cascade of photo-triggered

electron transfer reactions.




Supplementary Figure 4: Cyclic voltammograms of the pyrroloquinoline quinone

(PQQ)/PSI/Os2+/3+ polyvinylimidazole redox polymer/GDH modified ITO

electrode, scan-rate 10mV/s. Electrocatalytic anodic currents generated in the

presence of variable concentrations of glucose: (a) 0 mM (b) 10 mM (c) 20 mM

(d) 30 mM.




Supplementary Figure 5: Absorption spectra recorded upon the build-up of the layered

array: (a) The PSI linked to the PQQ monolayer. (b) The Os2+/3+-

polyvinylimidazole redox polymer associated with the PSI layer (c) The FAD-

dependent glucose dehydrogenase (GDH) associated with the PSI/Os2+/3+ -

polyvinylimidazole redox polymer interface.




Supplementary Figure 6: Photocurrent action spectra upon subjecting the

PQQ/PSI/Os2+/3+-polyvinylimidazole redox polymer/GDH modified electrode to

variable concentrations of glucose: (a) 0 mM (b) 5 mM (c) 10 mM (d) 15 mM

(e) 20 mM.




Supplementary Figure 7: “ON-OFF” switchable photocurrents upon irradiation of the

PQQ/PSI/Os2+/3+ -polyvinylimidazole redox polymer/GDH modified electrode

with white light (intensity 0.4 mW) in the presence of variable concentrations of

glucose (a) 0 mM (b) 10 mM (c) 20 mM (d) 30 mM. Electrode biased at 0.0 V

vs. Ag QRE.




Supplementary Figure 8: I-V curve corresponding to the photocurrent generated by the

PQQ/PSI/Os2+/3+ -polyvinylimidazole redox polymer/GDH

photobioelectochemical electrode at different bias potentials and under white-

light irradiation and 30 mM of glucose. The short –circuit current at ca. -

0.05V vs. Ag QRE corresponds to ca. 165 nA and the open-circuit voltage

of the system corresponds to -0.14V vs. Ag QRE. The fill-factor derived

from the I-V curve corresponds to 0.52. Error bars derived from N=3

experiments.




Supple

mentary F

gluco

molec

igure 9:

ose dehydro

cular visual

Illustration

genase (GD

lisation prog

of the act

DH) (from A

gram

tive site of

Aspergillus

f the prote

sp. , EC 1.1

in FAD-de

1.99.10), YA

ependent

ASARA




Supplementary Figure 10: Absorption spectra recorded upon the build-up of the layered

array: (a) The Pt-nanocluster/PSI bound to the mercapto siloxane modified

electrode (b) The Os2+/3+-polyvinylimidazole redox polymer associated with the

Pt-nanocluster/PSI monolayer (c) The GOx associated with the Pt-

nanocluster/PSI/Os2+/3+ -polyvinylimidazole redox polymer interface.




Supplementary Figure 11: Cyclic voltammograms of the Pt-nanocluster/PSI/Os2+/3+

polyvinylimidazole redox polymer/GOx modified ITO electrode, scan-rate

10mV/s. Electrocatalytic anodic currents generated in the presence of variable

concentrations of glucose: (a) 0 mM (b) 10 mM (c) 20 mM (d) 30 mM.




Supplementary Figure 12: I-V curve corresponding to the photocurrent generated by the

Pt-nanocluster/PSI/Os2+/3+ polyvinylimidazole redox polymer/GOx modified

ITO electrode, at different bias potentials and under white-light irradiation and

30 mM of glucose. Error bars derived from N=3 experiments.




Supplementary Figure 13: Efficient and Rapid Purification of Mastigocladus laminosus

photosystem I. (A) Separation of phycobiliproteins (PBP), photosystem I (PSI)

and photosystem II (PSII) extracted from Mastigocladus laminosus thylakoids

on TSK Toyo Pearl DEAE 650 anion exchange FPLC column (2.5X35cm) of

the AKTA explorer (Amersham Pharmacia Biotech) . (B) Native Deriphat

indicates that the purified PSI is a trimer (t). (C) SDS-PAGE indicative of the

highly pure nature of PSI.




Supplementary Methods

1. Preparation of the integrated PQQ/PSI/Os2+/3+-copolymer/ FAD-dependent glucose

dehydrogenase, GDH photobioelectro-chemically-active electrode

ITO slides (Delta Technologies, USA) were cleaned by sonication in ethanol at 60C for 30

minutes. In the first step the ITO slides were reacted with a solution consisting of 3-

aminopropyl triethoxysilane in acetone, 20 mM, for a time-interval of two hours. The

resulting slides were rinsed with acetone and water to yield the amine-function electrode

surfaces. The resulting amine-modified surface was immersed in a 3 ml phosphate buffer

solution (50 mM, pH = 7.2) that included 1 mg of methoxatin, PQQ (pre-dissolved in 50 µL

of DMSO), 5 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, EDC and 5 mM N-

hydroxysulfo-succinimide, sodium salt, NHS. After a reaction time-interval of two hours, the

electrode was rinsed with the PB aqueous solution to yield the PQQ-functionalised electrode.

The resulting PQQ-modified surface was subsequently reacted with a PB solution that

includes 5 mM EDC and 5 mM NHS, for a time interval of 15 minutes, to activate the

surface, and subsequently the surface was subjected to a solution of PSI, 1.5 mg Chl/ml in PB

(50 mM, pH = 7.2) for a time interval of two hours to yield, after rinsing with phosphate

buffer, the PQQ/PSI modified surface. Subsequently, the PQQ/PSI-modified electrode was

interacted with a phosphate buffer solution, 100 µl, that included the polyvinylimidazole

Os2+/3+ redox polymer, 0.17%, for a time interval of 20 minutes. The resulting electrode was

rinsed with the PB solution and interacted with a FAD-dependent glucose dehydrogenase

(GDH) solution that included 2 mg GDH in 100 µl of PB solution for a time interval of two

hours. The resulting electrode was reacted with 100 µl of glutaric dialdehyde in the PB




solution, 1% (v/v) for a time interval of 10 minutes, to yield the surface crosslinked GDH

layer.

2. Protocol for the extraction and purification of PSI

The thermophilic cyanobacterium Mastigocladus laminsus was grown and harvested as

described before (1). Thirty (30) gr of cells were suspended in 50mM MES-NaOH pH 6.5,

20% Glycerol, 5mM CaCl2 and 5mM MgCl2 and 1mM of a mixture of protease inhibitors

containing Amino Caproic Acid, Benzamidine and PMSF) and disrupted at 40C with glass

beads (0.1-1mm). Five cycles of 20 seconds blend with 5 minutes pause were performed in

BEAD BEATER (Biospec Products). Following the separation of the broken cells from the

beads, intact cells and cell debris were removed from the green solution by centrifugation for

2 minutes at 2,000 rpm in an SLA 3,000 Sorvall rotor. From the obtained supernatant the

thylakoid membranes were precipitated by ultra centrifugation at 35,000 rpm in a Ti 50.2

rotor for 20 minutes. The green pellet was washed twice in buffer containing an additional

5mM CaCl2. The washed thylakoids were obtained by re-centrifugation for each wash at

35000rpm as described above.

The extraction and separation of the photosynthetic complexes was performed by a minor

modification of the Keren et al. procedure (2). Washed thylakoids were homogenised in

20mM MES-NaOH pH 6.5; 25% Glycerol, 20mM CaCl2 and 10mM MgCl2 to chlorophyll

concentration of 1.6 mg/ml. n-Dodecyl β-D-maltoside (DDM) was added to a final

concentration of 0.6% and the solution was stirred at room temperature for 20 minutes.

Following ultra-centrifugation at 50,000 rpm (in Ti-70 rotor) for 100 minutes in 40C, the

resulting supernatant was filtrated through a 0.45 µm filter and loaded onto a TSK Toyo

Pearl DEAE 650 anion exchange FPLC column (2.5X35cm) of the AKTA explorer




(Amersham Pharmacia Biotech). The column was prewashed with a buffer containing 40 mM

MES-NaOH pH 6.0, 5% Glycerol, 20 mM CaCl2 and 0.02% DDM. The extracted

photosynthetic solution was loaded (10 ml/minute) onto the prewashed column in the buffer

above containing 12.5% MgSO4. The loaded column was washed with 2 column volumes

(CV) with 12.5% MgSO4 buffer. The PSI was eluted from the column at ~ 50% MgSO4

concentration.

Our modification allowed purifying a highly purified PSI in less than 20 hours. The obtained

PSI was subjected to three analytical analyses; absorption spectra ; Native Deriphat-PAGE

(3) and SDS-PAGE (3) in which the different complexes were fully denatured. The PSI

spectra showed a 680nm absorbance peak and the native Deriphat gel indicate that the PSI

isolated was the PSI-trimer. The ultimate proof for the purity of the PSI was obtained with

denatured PAGE using 12.5% acrylamide.

Supplementary References

1. Nechushtai, R., Muster, P., Binder, A., Liveanu, V. & Nelson, N. Photosystem I reaction

center from the thermophilic cyanobacterium Mastigocladus laminosus. Proc. Natl. Acad.

Sci. U.S.A. 80, 1179-1183 (1983).

2. Kern, J. et al. Purification, characterisation and crystallisation of photosystem II from

Thermosynechococcus elongatus cultivated in a new type of photobioreactor. BBA-

Bioenergetics 1706, 147-157 (2005).

3. Peter, G. F., Takeuchi, T. & Thornber, J. P. Solubilization and two-dimensional

electrophoretic procedures for studying the organization and composition of photosynthetic

membrane polypeptides. Methods 3, 115-124 (1991).


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Assembly of photo-bioelectrochemical cells using photosystem I … · 2016-05-19 · concentration of 0.6% and the solution was stirred at room temperature for 20 minutes. Following