Electroactive properties of electrospun silk fibroin for energy harvesting

applications

Vitor Sencadas1,2,3*, Christopher J. Garvey4, Stephen Mudie5, Jacob J. K. Kirkensgaard6,

Gwenaël Gouadec7, Samuel Hauser1

*Corresponding author: Vitor Sencadas (victors@uow.edu.au)

1 School of Mechanical, Materials, Mechatronic and Biomedical Engineering, University of

Wollongong, Wollongong, NSW 2522, Australia2 ARC Center of Excellence for Electromaterials Science, University of Wollongong, 2522

NSW, Australia3 Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522,

Australia.4 Australian Nuclear Science and Technology Organization (ANSTO), Locked Bag 2001,

Kirrawee DC NSW 2232, Australia5 Australian Synchrotron, ANSTO, 800 Blackburn Rd, Clayton 3168, Australia6 Department of Food Science, University of Copenhagen, Denmark7 Sorbonne Université, CNRS, Laboratoire MONARIS, c49, F75252, Paris, France

1

Experimental Section

Purification of the silk fibroin: Silk fibroin was extracted from Bombyx mori cocoons,

following the method described by Rockwood et al. [1]. Briefly, the cocoons were shredded

in small pieces and boiled for 20 minutes in a solution of 0.02 M of sodium carbonate

(Na2CO3, Sigma Aldrich) to remove the sericin protein. Afterward, the extracted fibroin was

rinsed with deionized water and dried overnight at room temperature. Dry fibroin was

dissolved in a 9.3 M lithium bromide (LiBr, Sigma Aldrich) solution, at 60 °C for 4 h. Next,

the solution was dialyzed for 48 h against deionised water, using a dialysis membrane with a

MWCO of 3.5 k (Spectrum labs) and changing the water regularly. The insoluble fraction was

removed by centrifugation and the clear supernatant filtered using a 0.45 m filter, and

lyophilized.

Membrane processing: The desired amount of silk fibroin was dissolved under magnetic

stirring in hexafluoroisopropanol (HFIP, Sigma Aldrich), , until a clear solution was obtained.

The concentration of protein in the solvent ranged from 40 to 70 g.mL-1. After complete

dissolution, the solution was placed into a glass syringe, fitted with a metallic needle (∅=0.5

mm). A syringe pump (KDScientific) allowed controlling the feed rate (between 0.2 and 1

mL.h-1). Samples were processed by applying an electric field (E) between 0.75 and 1.25

kV.cm-1 (Gamma High Voltage). All samples were collected in flat aluminum foil placed 20

cm away from the needle tip. Sample processing was performed at room temperature (21 ± 1

ºC) and a relative humidity of 43 ± 5 %.

Electrospinning parameters were optimized by using an orthogonal design table L9

(Orthogonal Design Assistant II, Sharetop Software studio). The experimental variables used

were the applied electrical field (A), the polymer feed rate (B), and the polymer concentration

(C, table S1).

Table S1: Factors and levels used in the orthogonal experimental design for the optimization

of silk fibroin electrospinning conditions.

Factors

Levels E(kV.cm-1)

Feed rate(mL.h-1)

Polymer concentration(g.mL-1)

1 0.75 0.2 40

2 1 0.5 50

3 1.25 1 70

2

After processing, the membranes were placed in a desiccator under vacuum for 48 h to allow

any traces of remaining solvent to evaporate. The cross-linking reaction was performed in a

chamber saturated with methanol (Anhydrous, 99.8% Sigma Aldrich) in the vapor phase, for

3 h, and at 25 ± 0.5 ºC. Finally, the membranes were air dried at room temperature for at least

48 h before use.

Materials characterization: Electrospun mats were gold coated with ~15 nm of gold layer

using a sputter coater (Smart Coater, JEOL) and their morphology was assessed by scanning

electron microscopy (SEM, JSM-7500FA from JEOL), with an accelerating voltage of 15 kV.

Fiber average diameter and their distribution was obtained by measuring 300 individual fibers

from different regions of the membrane, using Image J [2].

All Raman spectra were recorded in backscattering configuration on a LabRam HR 800

microspectrometer (Horiba Jobin Yvon) using the 514.5 nm line of a Innova 90C-6UV Ar+

ion laser (Coherent), a 600 lines/mm grating and a 10 microscope objective (Olympus

MPlanN, "UIS 2" series, NA 0.25). The estimated spot diameter was ~2.5 µm (as obtained

from the 1.22 λ/ NA diffraction limit). The incident laser power was set at 30 mW (as

measured on the sample) and the sample integrity was controlled optically after each

exposure.

Differential scanning calorimetry (DSC) measurements were performed with a Polyma 214

apparatus (Netzsch). Samples weighing around 5 mg were placed inside 40 L aluminum

pans with pierced lids, heated between 0 and 310 °C at a rate of 10 °C.min-1. All experiments

were performed under nitrogen purge.

Laboratory wide angle X-ray diffraction (WAXD) measurements were performed at the

SAXSLab instrument (JJ-Xray, Denmark) installed at the University of Copenhagen, and

equipped with a 100XL+ micro-focus sealed X-ray tube (Rigaku, Tokyo, Japan) and a 2D

300K Pilatus detector (Dectris, Baden, Switzerland). Measurements were performed with a

pin-hole collimated beam with the detector positioned asymmetrically in two settings to yield

a combined measurement q-range of 0.5 to 4 Å-1 with the magnitude of the scattering vector

defined by q¿4 πsin(θ)

λ , where λ=1.54 Å is the X-ray wavelength and θ is half of the

scattering angle. The samples were loaded in sample holders sealed with 5-7 micrometer thick

mica windows. The scattering from the mica is a low flat background with occasional sharp

reflections which are masked out from the individual 2D data sets.

Small angle neutron scattering (SANS) was carried out at the QUOKKA beamline [3] at the

Australian Centre for Neutron Scattering (Lucas Heights, ANSTO, Australia) to investigate

the nanostructures, and their length-scales, which resist intimate hydration in the methanol 3

treated material. Three instrument configurations (wavelength, sample to detector distance: 2

m, 5 Å; 14 m, 5 Å; and 20 m. 8 Å) were used to give a continuous q-range, 6.8 x 10 -4 < q <

0.4 Å-1. After subtraction of the instrument and sample holder (quartz windows)

contributions, the isotropic 2-dimensional scattering backgrounds were normalised for sample

thickness and incident flux of neutrons and radially averaged to give the familiar I(q)

representation, using IgorPro (Asylum Research) macros based on those of Kline [4].

Topography images were obtained in tapping mode with an MFP3D atomic force microscope

(AFM, Asylum Research) working at 0.5 Hz scanning rate. The piezoresponse of individual

fibers was collected with a Dual AC Resonance Tracking Mode piezoresponse force

microscopy (PFM). Briefly, individual fibers were deposited onto a gold-coated surface

(bottom electrode), and a cantilever (of 2.8 N/m spring constant) with a Pt/Ir-coated silicon tip

was used as the top electrode. The contact frequency varied from 320 to 340 kHz due to slight

changes in sample surface from point to point. A small AC voltage (200 mV) was applied to

oscillate the tip during the measurement. A sweeping DC bias (Frequency = 0.5 Hz) in the

range of ± 25 V was applied to the tip. Five cycles of the sweeping waves were applied to the

tip. The butterfly loops were obtained by using the Igor Pro 6.36 Software (Asylum

Research).

2.4. Piezoelectric bio-e-skin fabrication and sensor performance: The bio-e-skin energy

harvester was manufactured by cutting a piece of the membrane with an area of 3 x 3 cm 2 and

a thickness of around 100 m. Afterward, a conductive silver tape was used as electrodes on

both sides of the electrospun mat, with an effective area of 2 x 2.5 cm 2. Finally, the entire

structure was encapsulated between two layers (~50 m) of PDMS (Sylgard 184, Dow

Corning), which was prepared according to the manufacturer instructions. The sensor was

attached to a flat surface and a dynamic pressure was applied to the top electrode using a

vibrator generator (JZK-5 Modal Shakers from Sinocera), fitted with a force transducer (CL-

YD-303, Sinocera) to record the applied force. The output voltage and current generated by

the bio-e-skin under several dynamic conditions was recorded using a National Instruments

DAQ board (USB 6229) using a sampling rate of 1000 samples per second, interfaced with a

computer with Labview software, and the current measurements were recorded with a

Keithley 6485 picoammeter.

For synchrotron WAXD the distance between the sample and the Pilatus 1M detector

(Dectris, Switzerland) was 568 mm and X-rays of 20 keV (0.6200 Å) were used to

accumulate WAXD patterns in transmission geometry at the Australian Synchrotron’s

SAXS/WAXS beamline.[5] The sample to detector distance was calibrated with the 010

4

reflection of silk II [6]. A unique data acquisition scheme was used to investigate the transient

restructuring of the electro-spun silk mats during the piezo-electric response: Because

obtaining a good signal to noise ratio in the timescale of a single loading cycle (66.66 ms at

15 Hz) would have been impossible, 3.33 ms WAXD measurements were accumulated over

150 cycles at the same loading stage, giving a total exposure time of 500 ms. Assuming the

material adopts the same steady perturbed state in each cycle, this provided an improved

signal to noise ratio compared with a single shot experiment. A LeCroy BNB signal generator

(Teledyne LeCroy, Chestnut Ridge, USA) was used to provide synchronization between the

framing signal for the WAXD acquisitions and the mechanical stimulus for the piezoelectric

measurements (applied force of 10 N at a frequency of 15 HZ). The synchronization of the

mechanically stimulated current and the data acquisition was checked with a video of a LED

which gave a visual representation of the flow current (see supplementary information, video

1).

Optimization of membrane processing parameters

Orthogonal experimental design can provide a valuable assistance during the study of the

electrospinning processing parameters that affect the average fiber diameter of silk fibroin

(SF), saving experimental time and raw materials, on the pursuit of the optimal processing

conditions. In this work the processing parameters that were studied to optimize the SF

average diameter were the applied electric field ( A), the polymer feed rate (B), and the

polymer concentration (C, table S1).

The impact of the different factors and levels on the SF electrospun fibre diameter is

presented in table S2. Overall, depending on the electrospinning conditions used, we could

obtain thin fibers with an average diameter going from 77.92 ±75.6 nm (experiment H), up to

413.6 ±161.9 nm (experiment G).

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Table S2: Factors (A, B and C), levels (1, 2 and 3) and experimental conditions of

Orthogonal experimental design L9(3)4 for producing silk fibroin fibers through

electrospinning.

Factors Results

Run E

(kV.cm-1)

Feed rate

(ml.h-1)

Polymer Concentration

(μg.mL-1)

Diameter

A 0.75 0.2 40 173.0 ±44.7

B 0.75 0.5 50 248.0 ±82.7

C 0.75 1 70 407.7 ±98.8

D 1 0.2 50 232.1 ±56.6

E 1 0.5 70 403.0 ±147.2

F 1 1 40 164.1 ±60.1

G 1.25 0.2 70 413.6 ±161.9

H 1.25 0.5 40 77.9 ±75.6

I 1.25 1 50 255.4 ±133.5

In Table S3, the difference between the maximum and minimum K values are represented by

R, and reflects the useful or detrimental effects on the average fibre diameter. Accordingly,

the maximum value of R corresponds to the most influential factor.

6

Table S3: Factors and levels used in the orthogonal experimental design for the optimization

of silk fibroin electrospinning conditions.

Orthogonal indices Factors

E

(kV.cm-1)

Feed rate

(ml.h-1)

Polymer Concentration (μ

g.mL-1)K 1

a) 276.3 272.9 138.4

K2a) 266.4 243.0 245.2

K3a) 248.97 275.7 408.1

Rb) 27.2 97.4 136.0

Order of

importance

Polymer Concentration ¿ Feed rate ¿ Electrical Field

Optimal level 3 2 1a) K l

F=∑ evaluation indicesat same level of each factor /3

b) RF=max {K lF }−min {K l

F }, where K and R represents the mean and range values,

respectively, for a factor F at the level l

7

Raman spectroscopy characterization

The full collection of Raman spectra measured in this work is presented in Figure S1.

Figure S1: Raman analysis of electrospun silk membranes with laser = 514.5nm, 600

lines/mm grating, a 10 objective (NA 0.25) and 30 mW power. Each spectrum was acquired

in 300s after 120s of photobleaching. a) "as-spun" membrane (18 spectra; the inset shows the

integrated area of consecutive 5s spectra recorded from initial laser exposition); b) membrane

exposed to MeOH vapor (18 spectra); c) membrane after immersion for 3 hours in liquid

MeOH (110 spectra). The black spectra from each series were averaged and the resulting red

spectra are those shown in Figure 3.

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Thermal analysis

Differential scanning calorimetry (DSC) measurements show water evaporation at

temperatures below 120º C, followed by a glass transition (T g), a cold crystallization (T cc)

process and melting of the crystalline regions (T m) present in the polymer fibres (Figure S2).

The as-electrospun samples present a lower glass transiting temperature and the cold

crystallization process shows a stronger exotherm and occurs over a wider temperature range

when compared to the samples submitted to the MeOH treatment, revealing that when the

solvent evaporated during the electrospinning process, the amorphous polymer chains were

frozen, and didn’t have enough time to reorganize in a more ordered fashion. When the

samples were submitted to the MeOH treatment, the solvent could penetrate through the bulk

of the fibers and segmental diffusion was possible, inducing crystallization of the amorphous

regions of the silk fibroin, leading to an increase of the protein T g (table S4).

Figure S2: Evolution of the thermal properties for the silk fibroin membranes before and after

treatment with methanol in vapor or liquid phase.

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Table S4: Thermal properties of electrospun silk fibroin after different treatments with

methanol.

Sample T g

(ºC)T cc

(ºC)T m

(ºC)As-spun 164 225 284

MeOH vapor 180 208 284

Liquid MeOH * * 286

*: not measurable

Mechanical properties

The mechanical properties of the SF electrospun membranes before and after treatment with

methanol in vapor phase was assessed by quasi-static measurements in a universal testing

machine. The representative stress-strain data given in Figure S3 show that the as-spun

membranes have a brittle behavior, with a ultimate tensile strength (UTS) of approximately 6

MPa and a strain at break ε break of 7%, while the samples submitted to the MeOH in vapor

phase had an improvement in the overall mechanical properties, with an increase of the UTS

and ε break values up to 11.5 MPa and 25%. The Young modulus increased from 116 MPa for

the as-spun sample up to 149 MPa for the sample treated with methanol. The enhancement of

the mechanical properties after the MeOH vapor treatment is due to the increase in sample

crystallinity and fiber packing inside the sample.

Figure S3: Mechanical properties of the silk fibroin membranes before and after treatment

with methanol in vapor phase.

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Electromechanical performance

The PDMS layers with the silver electrodes only (the samples had been removed) didn’t show

any electrical activity after pressing (Figure S4a). After placing the samples between the

electrodes and submitting the energy harvester to a periodic oscillation, an output voltage was

observed (Figure S4b), even after reversing the polarization electrode connections (Figure

S4c), which reflects in the nature of the piezoelectric output signals (Figure S4). Furthermore,

an increase in the SF dielectric constant (ε ') was observed after the membrane treatment with

MeOH (Figure S4f).

Figure S4: a) Electrical output voltage recorded for the empty PDMS layers, b) Forward

electrical output voltage for the as-spun membrane, c) Reverse electrical output voltage for

the as-spun membrane, d) Forward electrical output voltage for the membrane submitted to 3

h of MeOH vapor, e) Reverse electrical output voltage for the membrane submitted to 3 h of

MeOH vapor and f) dielectric constant recorded for the as-spun sample and the membrane

submitted to 3 h of MeOH vapor.

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A weight of 20 N was dropped on top of the nano-harvesters from a 10 cm height, and a time

response (τ ) of 3 milliseconds was obtained for the SF electrospun membranes, despite the

treatment performed to stabilize the sample against aqueous environment (Figure S5).

Figure S5: Response time recorded for the silk membranes: a) as-spun and b) sample

submitted to 3 h of MeOH vapor.

Figure S6: Applied voltage dependence displacement for the as-spun and MeOH treated silk

fibroin electrospun fibers.

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Energy efficiency calculation

The piezoelectric energy conversion efficiency of the SF nanogenerators can be estimated as

the η=Eout

E¿ ratio of the generated output electrical energy to the applied mechanical energy:

E¿=F∗∆l=F σlE , where F (10 N) is the force applied to the nanogenerator, ∆ l is the

deformation under applied stress , and E is the Young modulus for a single fiber in the as-

spun or MeOH-treated membranes.

The output energy generated by the nano-harvester can be determined by Eout=12

C ¿V 2,

where C is the sample capacitance.

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References

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