International Journal of Biological Macromolecules 176 (2021) 498–509

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Development of robust, ultra-smooth, flexible and transparent

regenerated silk composite films for bio-integrated electronic
device applications
D.V. Santhosh Kumar Gunapu a, Y. Bhavani Prasad a, V. Sushma Mudigunda b, Palguna Yasam c,
Aravind Kumar Rengan b, Rajesh Korla c, Siva Rama Krishna Vanjari a,⁎
a
Department of Electrical Engineering, Indian Institute of Technology (IIT) Hyderabad, Kandi, Sangareddy, Telangana, India
b
Department of Biomedical Engineering, Indian Institute of Technology (IIT) Hyderabad, Kandi, Sangareddy, Telangana, India
c
Department of Materials Science and Metallurgical Engineering, Indian Institute of Technology (IIT) Hyderabad, Kandi, Sangareddy, Telangana, India

a r t i c l e i n f o a b s t r a c t

Article history: Regenerated Silk Fibroin (RSF) films are considered promising substrate candidates primarily in the field of bio-
Received 27 September 2020 integrated electronic device applications. The key issues that ought to be addressed to exploit the inherent advan-
Received in revised form 5 February 2021 tages of silk thin films include enhancing their flexibility and chemical durability. Such films find a plethora of ap-
Accepted 6 February 2021
plications, the significant one being conformal, transparent microelectrode arrays. Elevated temperatures that
Available online 9 February 2021
are regularly used in lithographic processes tend to dehydrate RSF films, making them brittle. Furthermore, the
Keywords:
solvents/etchants used in typical device fabrication results in the formation of micro-cracks. This paper addressed
Chemical durability both these issues by developing composite films and studying the effect of biodegradable additives in enhancing
Conformal flexibility and chemical durability without compromising on optical transparency and surface smoothness.
Flexibility Through our rigorous experimentation, regenerated silk blended with Polyvinyl Alcohol (Silk/PVA) is identified
Regenerated silk fibroin (RSF) as the composite for achieving the objectives. Furthermore, the Cyto-compatibility studies suggest that Silk/PVA,
Silk/PVA along with all other silk composites, have shown above 80% cell viability, as verified using L929 fibroblast cell
Transparent microelectrode array (MEA) lines. Going a step further, we demonstrated the successful patterning of 32 channel optically transparent micro-
electrode array (MEA) pattern, with a minimum feature size of 5 μm above the free-standing and optically trans-
parent Silk/PVA composite film.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
Flexible bio-integrated electronic devices play a significant role in
applications involving soft and curved biological systems. These appli-
cations include basic measurements of electrophysiological signals
[1,2], drug delivery for advanced therapy [3,4], human-machine inter-
faces [5,6], to name a few. Traditional Silicon or Gallium Arsenide
based devices are fundamentally rigid and planar. In contrast, the bio-
logical tissue of the human body is soft and curvilinear. This mechanical
mismatch at the biotic–abiotic interface hampers the development of
seamless, non-invasive, and robust interfaces during natural move-
ments and associated biological processes. The successful development
of flexible and stretchable bio-integrated wearable systems requires ut-
most care and attention during the materials design of both active
Abbreviations: RSF, regenerated silk fibroin; PVA, polyvinyl alcohol; PVP, polyvinyl
pyrrolidone; Chi, chitosan; Gly, glycerol; MEA, microelectrode array.
⁎ Corresponding author at: Department of Electrical Engineering, Indian Institute of
Technology Hyderabad, Telangana 502285, India.
E-mail address: svanjari@iith.ac.in (S.R.K. Vanjari).
devices and supporting substrate [7]. In this regard, researchers ex-
plored a wide range of synthetic and natural polymers towards the de-
velopment of flexible bio-integrated electronic devices [8]. G. Zheng
et al. used nanostructured Cellulose paper as the basic building block
for the applications of advanced energy storage and optoelectronic de-
vices [9]. Several groups have explored Collagen [10], Gelatin [11,12],
human hair keratin [13], deoxyribonucleic acid (DNA) [14], and various
other biomaterials [15] as the gate dielectric in the bio-organic thin-film
transistors. Among natural biomaterials, Bombyx mori silk fibroin gar-
nered special attention as a potential candidate for bio-integrated elec-
tronic devices such as sensor skins [16], brain-machine interfaces [17],
and biomedical diagnosis & therapy [18] owing to their appealing prop-
erties such as natural abundance, superior mechanical properties, bio-
compatibility, biodegradability, bio-resorbability in conjunction with
their lightweight [19]. Another added advantage is its solution-based
processability, making it easy to develop films of various thicknesses
using simple spin-coating/doctor blade coating methodologies. The re-
generated silk fibroin (RSF) film extracted from native Bombyx mori
silk consists of non-crystalline α-helix chains and crystalline β-sheet
chains connected by disulfide bonds [20]. The structural formation of

https://doi.org/10.1016/j.ijbiomac.2021.02.051
0141-8130/© 2021 Elsevier B.V. All rights reserved.
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al.
silk fibroin depends on the method of extraction for RSF film. The most
prominent crystal structures of Bombyx mori silk fibroin are water-
soluble Silk-I and water-insoluble Silk-II [21]. Several methods were re-
ported in the literature to extract regenerated silk fibroin using aqueous
[22] or organic solution [23] based processing techniques. The aqueous
salt-based method generally produces non-crystalline Silk-I structure,
and the transition from Silk-I (random coil or α helix) to crystalline
dominant Silk-II (β sheet) requires further treatment of the extracted
film with alcohols such as methanol/ethanol [24,25] or water annealing
[26]. While RSF extracted using organic solvents such as formic acid [23]
produces a predominantly Silk-II structure that is relatively stable, less
complicated, and transparent than the former method. However,
these Silk-II structures become too stiff and brittle after dehydration of
RSF, which is the major bottleneck for silk fibroin based flexible bio-
integrated electronic devices. Moreover, these films are prone to
forming micro-cracks after exposure to standard solvents/etchants
commonly used in the micro/nano device fabrication processes [27].
So far, existing methodologies employed various techniques such as
micro-contact printing [28], nanoimprinting [29], plasma-based dry
etching [30–32] to create 2D micron/nano resolution patterns of silk
films. Dickerson et al. [33] have utilized multiphoton lithography print-
ing to create 3D silk structures. Several groups have modified silk fi-
broin/sericin structure to act as a photoresist in creating a variety of
high-resolution patterns [34–36]. Murphy et al. [37] have introduced a
photolithographic masking method to pattern silk film surfaces chemi-
cally. However, direct patterning of metal layers with high accuracy
above regenerated silk fibroin while addressing the chemical robustness
issue is essential for deploying fibroin-based bio-integrated devices.
Earlier, many researchers studied flexibility enhancement mecha-
nisms of RSF by combining RSF with several plasticizers such as (3-
glycidyloxypropyl) trimethoxysilane (GPTMS) [38], glycerol [39],
genipin/glycerol [40], dextrose [41], chitosan [42], glucose [43] mainly
targeted for wound dressing and bio-photonic applications. F. Zhang
et al. [44] studied the effect of CaCl2 concentration and stretching on
the directly dissolved RSF to enhance the flexibility of silk films. D.
Kaplan et al. investigated the possibility of flexibility enhancement in
the RSF by stretching Silk-I film in the wet state followed by dehydration
[45]. Nevertheless, all those reports have addressed only the qualitative
improvement of the flexibility of RSF blends. The chemical durability
and transparency retention have not received the desired attention, es-
sential aspects for its applicability in conformal and flexible bio-
integrated electronic devices.
The present work focuses on overcoming the two critical bottlenecks
of RSF, i.e., brittleness in the dry state and low chemical durability for its
potential use in deploying flexible bio-integrated devices, without ad-
verse effects on optical transparency, surface smoothness, and biocom-
patibility. We assessed the enhancement of flexibility & chemical
durability while retaining the optical transparency and surface smooth-
ness of RSF by combining different biocompatible & biodegradable poly-
mers widely reported in the literature, such as Glycerol, Chitosan,
Polyvinyl alcohol (PVA), and Polyvinyl pyrrolidone (PVP). Silk/PVA
blend film suffices all the criteria mentioned above, that was substanti-
ated by standard structural, optical, and material characterizations such
as RAMAN spectroscopy (RAMAN), Fourier transform infrared spectros-
copy (FTIR), X-ray diffraction study (XRD), Optical microscopy, Atomic
force microscopy (AFM), and UV visible spectroscopy (UV–Vis). We es-
timated the flexibility from the tensile properties plotted by Digital
image correlation (DIC) method using the Universal testing machine
(UTM). The role of water absorption of silk composites was assessed
using Thermogravimetric analysis (TGA). The biocompatibility aspects
were assessed using MTT assay and Live-dead assay with mouse fibro-
blasts (L929 cell lines). Finally, we verified the improvement in the crit-
ical criteria for its potential use in conformal bio-integrated electronics
by fabricating 32 channel MEA having electrodes with a minimum fea-
ture size of width 5 μm and thickness 10 nm onto an optically transpar-
ent free-standing Silk/PVA film.
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International Journal of Biological Macromolecules 176 (2021) 498–509
2. Materials and experimental methods
2.1. Materials
Bombyx Mori silk yarns produced in non-dyed form were purchased
from M/S Bombyx mori Silks & Textiles, Srinagar, Jammu & Kashmir,
India. Formic acid of purity (98%) was purchased from Rankem chemicals.
PVA, PVP, and Chitosan chemicals were procured from Sigma Aldrich.
Glycerol anhydrous (99.5%) was purchased from Sisco research laborato-
ries (SRL) Pvt. Ltd. Calcium chloride dihydrate was purchased from Merck,
Germany. Microposit S1813 positive photoresist was purchased from
Shipley. Oxidized p-type 〈100〉 SiO2 substrates were used as the base sub-
strates. Deionized (DI) water with 18.2 MΩcm−1 resistivity, obtained
from the Merck Millipore system, was used in all the experiments. L929
(Mouse fibroblasts) cell lines were obtained from NCCS Pune, DMEM
(Dulbecco's Modified Eagle's Medium), FBS (Fetal Bovine Serum), Antibi-
otics (Penicillin and Streptomycin) PBS (Phosphate Buffer Saline) were
purchased from Himedia chemicals. DMSO (Dimethyl sulfoxide) and
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide)
were purchased from SRL Pvt. Ltd. FDA (Fluorescein diacetate), PI
propidium iodide were purchased from Sigma (St. Louis, MO, USA). All
the purchased chemicals were used as received.
2.2. Preparation of silk fibroin solution
The regenerated silk fibroin solution's preparation process is
adapted from the previous work [46] with small yet significant modifi-
cations. Initially, the unwanted sericin removal process, also known as
degumming, was carried out by boiling the silk threads in 0.02 M so-
dium carbonate aqueous solution for a minimum duration of 30 min.
The quality of the RSF depends on the duration of degumming, as higher
degumming might result in the degradation of RSF film [47]. By varying
the degumming time, we have figured out that the optimum time for
degumming, in a flexibility perspective, is 30 min. Higher duration of
degumming resulted in brittle films. In the subsequent experiments,
the degumming time was fixed at 30 min. After degumming, the resul-
tant silk fibers were dissolved in Formic Acid comprising 5% (w/w) Cal-
cium Chloride (CaCl2). The role of CaCl2 solely to aid the initial
dissolution of silk fibers. After that, this solution was cast into a plastic
petri dish to form a thin layer and dry overnight in a fume hood. The un-
wanted calcium chloride is removed after soaking the dried RSF film for
10 h. Subsequently, the dried RSF was reconstituted in formic acid at a
concentration of 13% (w/w) to achieve the desired silk fibroin solution.
2.3. Preparation of regenerated silk composite films
In this work, we combined different biocompatible and biodegrad-
able polymers such as Polyvinyl alcohol (PVA), Polyvinyl pyrrolidone
(PVP), Glycerol, and Chitosan to the final RSF solution of 13% (w/w) as
discussed in the previous section. All the blend solutions were prepared
such that the viscosity of all the blend solutions were equivalent. The
ratio of silk composite films with PVA, PVP, and chitosan was fixed to
be 2% (w/w). Whereas for Silk/Glycerol, the ratio was fixed to be 6%
(w/w). All of the above blend solutions were cast onto glass substrates
separately and dried overnight at room temperature under the fume
hood. After that, all the dried composite films were released from
glass substrates using an ammonium hydroxide solution heated at a
temperature of 90 °C.
2.4. Characterization
2.4.1. Assessment of flexibility enhancement
The flexibility of all the silk composite films in both wet and dry state
was estimated from the tensile properties such as ultimate tensile
strength, percentage elongation at break, and Young's modulus using
INSTRON 5967 UTM equipped with a load capacity of 5 kN. Strain on
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al.
the sample was measured using DIC method with the help of the vic-2D
software. The experimental setup for tensile studies is provided in the
Fig. S5a of Supporting information. Composite file specimens for tensile
tests were in form of rectangular strips of length 50 mm and width
8 mm and three specimens were tested for each condition to get better
statistics. The reasons for flexibility enhancement in the silk composite
films were corroborated using Thermogravimetric analysis (TGA)
study. TGA is performed using a Thermo analyzer Q600 SDT from
Texas instruments, in an alumina crucible, and heating at 10 °C/min
from 30 to 350 °C under nitrogen gas flow of 100 ml/min.
2.4.2. Structural and morphological characterization
The percentage transmittance of all the released films was acquired
using LAB INDIA, Analytical model no UV 3092, thin-film UV–Visible
spectrophotometer in the wavelength range of 200 to 800 nm. The
structural composition of all the silk-blend films was estimated using
Fourier transform infrared spectroscopy (FTIR), X-ray diffractogram
(XRD), and RAMAN spectroscopy. The FTIR data were obtained using
Bruker FTIR with an Attenuated Total Reflectance (ATR) unit attached
to it. The FTIR spectra were recorded in the transmittance mode for 64
scans for a wavelength range of 500 to 4000 cm−1 at room temperature.
A background scan was performed after every successful measurement.
XRD of all the composite films was analyzed using Bruker D8 DISCOVER
X-ray diffractometer using a nickel-filtered Cu Kα radiation source with
a wavelength of 1.54 A°. The XRD intensity data of spin-coated thin silk
and its composite films were collected over a 2θ angle range of 10° to
80° with a step size of 0.05°. The average crystallite size was calculated
for all the prominent peaks using the Debye-Scherrer equation. The
RAMAN spectroscopic data were collected using Bruker SENTERRA dis-
persive micro-Raman spectrometer with a beam incident power of
10 mW. The RAMAN intensity data of spin-coated thin silk and its com-
posite films were collected from the spectral wavenumbers from
500 cm−1 to 3000 cm−1. The average RMS surface roughness (Rq) was
estimated using Bruker's atomic force microscopy (AFM). The average
RMS roughness was estimated from a set of 3 specimens each for both
pure silk (RSF) and Silk/PVA films. The scanning was performed with
an area of 5 μm × 5 μm for every run of the characterization. The images
were recorded in both 2D and 3D methods and analyzed using
Nanoscope analysis software. The optical microscopic images were ob-
tained using Olympus DSX500X digital microscope.
2.5. Cytocompatibility characterization
2.5.1. Cytotoxicity assay
Cytotoxicity of silk composite films was performed using L929 cells.
Silk films were placed in 96 well plates, and L929 cells, a density of
(3 × 103) cells were seeded per well the following incubation for 72 h,
cell viability was determined by MTT assay [43]. The cell number was
estimated by absorption at 490 nm wavelength on an absorbance Mi-
croplate reader (800TS, BioTek, USA).
2.5.2. Live/Dead assay
Cell viability was assessed using FDA/PI staining. L929 cells (3000
cells per well) were seeded on modified silk composite films positioned
in 96 well plates and incubated for eight days. Fresh media was added
subsequently day by day. On the tenth day, media was removed from
the well plate and washed once with PBS [40]. FDA staining solution
was prepared by dissolving 8 μl of 1 mg/ml FDA & 30 μl of PI in DMEM
and added 100 μl of staining solution in each well and incubated for
30 min. After an additional washing step, the cells were analyzed
under a fluorescent microscope (Olympus, Japan).
2.6. Microelectrode array fabrication above Silk/PVA film
Having thoroughly investigated Silk/PVA films' properties, a 32
channel gold microelectrode array pattern with a minimum feature
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International Journal of Biological Macromolecules 176 (2021) 498–509
size of 5 μm was fabricated on a free standing Silk/PVA film to demon-
strate its applicability in the field of bio-integrated electronic device ap-
plications. The fabrication process flow for successful patterning of 32
channel MEA with a minimum feature size of 5 μm is shown in Fig. 1.
First and foremost, p-type SiO2 wafers were cleaned thoroughly using
Piranha solution followed by DI water. After dehydration, the Silk/PVA
blend was spin-coated onto the substrate at the required spin speed
(s), followed by hard baking of Silk/PVA blend film at a temperature of
80 °C. Sequential deposition of Titanium/Gold (Ti/Au) with a thickness
of 3/10 nm was carried using DC sputtering. This was followed by a
spin coating of positive photoresist S1813 and its mandatory pre-bake
step at 115 °C for 1 min. Patterning of S1813 photoresist was carried
out using direct writing by Microtech LW405 laser writer with an opti-
mized exposure dose suitable for Silk/PVA blend films. After exposure,
photoresist development was done in an MF319 developer solution,
and subsequent metal etching was carried out in standard Au etchant
(3:1:2 HCl/HNO3/2H2O) and standard Ti etchant (20:1:1 H2O/HF/
H2O2). Once the MEA pattern was completed on Silk/PVA film, the
final step is to release flexible film along with the patterned MEA. This
was performed by dipping in Ammonium Hydroxide solution heated
at a temperature of 90 °C.
3. Results and discussion
3.1. Structural characterization of silk composites
3.1.1. Raman spectroscopy
First and foremost, the first step post-formation of silk composite
films is to understand the structural and compositional aspects. We car-
ried out Raman analysis to understand the composition of the silk
blends. Fig. 2a compares the Raman spectra of pure silk film and its com-
posite films. All the films have typical peaks at 1083 cm−1, 1228 cm−1,
and 1665 cm−1. These correspond to molecular vibration modes of pro-
teins in regenerated silk fibroin, confirming the harvested silk fibroin.
Spectral peaks at wavenumbers 1228 cm−1 and 1665 cm−1 correspond
to the Silk-II structure of the silk fibroin with regularly aligned crystal-
line ꞵ-pleated sheets [48]. Further, the spectral peak at 1083 cm−1 con-
firms random coils and turns [49]. This observation indicates the silk-
blend films prepared to have both Silk-I and Silk-II polymorphisms.
However, the absence of 1109 cm−1 is indicative of a lack of α-helices.
Thus the films are predominantly of Silk-II in nature. The RAMAN char-
acterization study of individual spectra of pure silk and its composite
films and neat polymer additives PVA and PVP are provided in Figs. S1
and S2, respectively, of Supporting information.
3.1.2. Fourier transform infrared spectroscopy (FTIR)
The FTIR spectrum for various silk composites is shown in Fig. 2b. All
the composites have shown typical peaks at 1230 cm−1, 1513 cm−1,
and 1624 cm−1 correspond to Silk-II structures predominantly with ꞵ-
pleated sheet formation [50]. Additional peaks at wavenumbers
3281 cm−1 attributed to hydrogen bonds [51] and the peak at
690 cm−1 are structures related to the amide-IV region [52,53]. The
quantitative analysis of structural transitions, especially the secondary
structural information of various secondary structures such as ꞵ-sheets,
α-helices, random coils, and turns, can be estimated using Fourier Self
Deconvolution (FSD) method. A detailed description of FSD analysis is
provided in Section S3.1.2.1 of Supporting information. FSD analysis is
carried out to obtain complete secondary structural information from
the Amide-I region (1600–1700) cm−1, which usually are broad and
overlapping, thereby creating difficulty in extracting the individual sec-
ondary structural information [54]. FSD absorbance spectra for pure silk
and its composites are shown in Fig. 3.
The approximate percentage of ꞵ-sheets among various other sec-
ondary structures is tabulated and shown in Table 1. All the films have
shown a higher percentage (above 58%) of ꞵ-sheets attributed to the
RSF extraction method using formic acid as the primary solvent [23].
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al. International Journal of Biological Macromolecules 176 (2021) 498–509

Fig. 1. Fabrication process flow of transparent & conductive MEA above Silk/PVA film.

The enhancement in ꞵ-sheet percentage is primarily due to the com-
plete cleavage of interconnected hydrogen bonds in the crystalline re-
gion by the intense and highly concentrated formic acid and calcium
chloride. Also, as a comparison, FTIR analysis was done on the pure poly-
mer additives such as PVA, PVP, and Chitosan films other than Glycerol,
because pure Glycerol does not attain film formation capability, which is
shown in the Fig. S3 of Supporting information.
Among the composites studied, Silk/PVA has shown the highest per-
centage of ꞵ-sheets (72%), from Table 1. This trend is attributed to the
inherent hydrophilic and hygroscopic nature of PVA. This characteristic
of PVA helps promote the water retention capacity of Silk/PVA as a com-
posite, which further enhances the percentage of ꞵ-sheets, by allowing
fibroin chains to remain mobile throughout the drying process [55].
Here, PVA promotes the structural transition inside the silk fibroin
films by forming more interconnected hydrogen bonds between the hy-
droxyl groups of PVA and amine groups of pure silk fibroin [56]. In this
way, PVA plasticizer helps in the self-assembly of ꞵ-sheets by allowing
more hydrophobic domains to come into proximity [57–60].

Fig. 2. (a) Raman and (b) FTIR spectroscopy comparison of pure silk and its composites.

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D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al. International Journal of Biological Macromolecules 176 (2021) 498–509

Fig. 3. Fourier self-deconvolution absorbance spectra in the Amide-I region (1600–1700 cm−1) for pure silk and its composites indicating various secondary structures like side chains (S),
ꞵ-sheets (ꞵ), random coils (R), and α-helices (α).

3.1.3. X-ray diffraction (XRD) analysis was done on the pure polymer additives such as PVA, PVP, Chi-
To further understand silk fibroin's crystal structure and its compos- tosan films and is shown in the Fig. S4 of Supporting information.
ites, grazing incidence XRD measurements were performed. Fig. 4 de-
picts the XRD plot of pure silk fibroin and its composites, which shows
3.2. Flexibility enhancement study of silk composites
that there is a single unique broad peak located around the diffraction
angles of 2θ = 20.5° other than Silk/PVA, and 2θ = 24.5° for Silk/PVA.
After asserting the compositional and structural characteristics of
This finding suggests that all the prepared films of pure silk fibroin
silk blends through Raman, FTIR, and XRD, the crux of the work,
and its composites are predominantly of Silk-II type, consistent with
i.e., the flexibility enhancement, is studied in detail. Flexibility enhance-
the stated literature on silk fibroin [61]. The diffraction peaks for Silk/
ment of regenerated silk fibroin, especially after dehydration, is critical
PVA composite are slightly shifted towards the right half of the positive
for developing bio-integrated flexible electronic devices utilizing stan-
axis, revealing that the percentage of β-sheets is much higher than that
dard patterning approaches. The extent of flexibility or deformation
of the pure silk fibroin [62,63]. Moreover, to have a more in-depth in-
sightful view, the d spacing for silk composites was calculated from
the XRD plot, and is obtained as 3.7 A° at a 2θ value of 24.5° for Silk/
PVA and is 4.2 A° for remaining composites, which further reveals that
the crystallinity index is more due to the higher percentage of β-
sheets which is consistent with the FTIR as well.
Furthermore, the diffraction peak of both methanol treated films and
as prepared Silk/PVA are very much comparable with the broad diffrac-
tion peak at 2θ = 24.5° [61]. Therefore, the as-obtained results reveal
that the Silk/PVA blended composite has highest ꞵ-sheets, which is
very consistent with the FTIR FSD analysis results. On the other hand,
there is no notable shift in the diffraction peak for pure silk fibroin and
other silk composites such as Silk/PVP, Silk/Glycerol, Silk/Chitosan,
thus revealing that there are no structural transitions as compared
with the prepared pure silk fibroin. Besides, as a comparison, XRD

Table 1
ꞵ-sheet comparison of pure silk fibroin (RSF) and its composites calculated from FSD anal-
ysis of Amide-I region of FTIR absorbance spectra.

Type of silk Pure Silk/PVP Silk/Chitosan Silk/Glycerol Silk/PVA

composite silk

ꞵ-Sheet 58 67.84 55.25 63.12 72

percentage
Fig. 4. XRD plot of pure silk fibroin and its composite films.

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D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al. International Journal of Biological Macromolecules 176 (2021) 498–509

Table 2
Tensile properties of regenerated silk fibroin and its composite films.

Type of silk composite Ultimate tensile strength (MPa) Young's modulus (GPa) Fracture strain (%)

Dry Wet Dry Wet Dry Wet

Pure silk 62.5 ± 0.7 10 ± 1.0 3.4 ± 0.3 0.1 ± 0.1 14.7 ± 0.4 35.7 ± 0.7
Silk/PVP 51.6 ± 0.7 10.8 ± 0.8 2.9 ± 0.2 0.24 ± 0.0 19.5 ± 0.6 28.3 ± 0.4
Silk/Chitosan 65.7 ± 0.5 6.8 ± 1.0 3.4 ± 0.2 0.09 ± 0.1 7.4 ± 1.6 23 ± 1.0
Silk/Glycerol 30.6 ± 0.7 3.4 ± 1.0 1.5 ± 0.3 0.04 ± 0.2 22.7 ± 1.0 28.9 ± 0.9
Silk/PVA 14.3 ± 0.9 3.1 ± 0.4 0.4 ± 0.0 0.03 ± 0.0 44.6 ± 0.9 65.4 ± 0.6

under an applied load was mainly investigated using the tensile proper-
ties. High percentage elongation at break (Fracture strain), lower
Young's modulus (Y.M.), and moderate ultimate tensile strength (U.T.
S) are the essential requirements to be satisfied by a free-standing stable
and flexible film [38]. Fracture strain provides the amount of strain that
a film can withstand without fracture. Young's modulus, the slope of
stress vs. strain curve, gives the material's stiffness and ultimate tensile
strength represents the maximum stress that an object can withstand.
Table 2 summarizes Young's modulus, U.T.S, and Fracture strain of re-
generated pure silk fibroin and its composites. The tensile properties
were investigated using INSTRON UTM and derived the engineering
stress vs. engineering strain behavior using the DIC method [64] to get
accurate data of induced strain under applied tensile stress. The pictorial
representation of strain distribution in the sample was observed using
Vic2D software of the DIC system, which is provided in Fig. S5b of
Supporting information. Tensile experiments were performed on two
sets of samples, wet and dry state. Samples tested in a wet state were
soaked in DI water for 24 h before the test. In comparison, samples
tested in the dry state were dried at room temperature 48 h before
the test. All the experiments were carried out at room temperature
with a 50% R.H. humidity and an initial strain rate of 10−3 per second,
using the screw-driven UTM. The engineering stress vs. engineering
strain curves of regenerated pure silk and its composites in the dry
and wet state were shown in Fig. 5a and b, respectively.
As shown in Table 2, pure silk film in the wet state has shown higher
flexibility with 35.7 ± 0.7% Fracture strain & Young's modulus of 0.1 ±
0.1 GPa, compared to the dry state with only 14.7 ± 0.4% Fracture strain
& higher Young's modulus of 3.4 ± 0.3 GPa, which suggests that pure
silk film is flexible/soft in the wet state and brittle/rigid in the dry
state. The enhancement in flexibility of pure silk film in dry state was
achieved by reinforcing with different biocompatible and biodegradable
polymers such as PVP, Chitosan, Glycerol, and PVA.
Among the composites that were studied, Silk/PVA has met the de-
sired criteria for a stable and flexible film with a higher fracture strain
of 44.6 ± 0.9% and 65.4 ± 0.6%, lower Young's modulus of 0.4 ±
0.0 GPa and 0.03 ± 0.0 GPa & moderate tensile strength of 14.3 ±
0.9 MPa and 3.1 ± 0.4 MPa in the dry and wet state respectively.
Fig. S6 compares stress vs. strain curves of neat polymer additives
PVA, PVP, and Chitosan. Among the neat polymers, pure PVA has
shown a very high fracture strain of 195%, whereas PVP and PVA have
shown a maximum of only 23% and 30% of fracture strain, respectively.
Hence, it can be confirmed that the enhancement in the flexibility of
Silk/PVA film compared to other composites is attributed to the high
fracture strain of pure PVA film, and high water retention capacity of
Silk/PVA film, which will be further verified using thermo gravimetric
analysis in the subsequent sections.
3.3. Thermogravimetric analysis
The thermal stability of all the silk-based composites characterized
by percentage weight loss with an increase in temperature was investi-
gated using Thermogravimetric analysis (TGA). The Thermogravimetric
curve of regenerated silk fibroin is subdivided into three regions. Region
I in the range from 50 °C to 170 °C depicts the water loss, Region II in the
range from 170 °C to 240 °C and Region III above 240 °C depicts the deg-
radation of silk [38]. Fig. 6a shows the TGA analysis of all the silk com-
posites. It is observed that Silk/PVA blend film has shown only 5% of
weight loss at the temperature of 170 °C, end of the region I. However,
all other composite films have shown more than 6.5% of weight loss at
the end of the region I. This observation shows the higher water reten-
tion capability of Silk/PVA film. It showed a similar trend in region II,
which shows that the film is stable. In region III, it leads to the degrada-
tion of all composite films.
The typical temperature range of electronic devices predominantly
falls in Region-I. In this region, Silk/PVA had more water retention ca-
pacity than the other composites, indicates that Silk/PVA retains water
in the dry state and is thus much more flexible than other films and cor-
roborates well with the tensile measurement results. The higher water
retention capacity and stability of Silk/PVA film compared to the re-
maining films is further corroborated using the differential

Fig. 5. Plots of engineering stress vs. engineering strain of the pure silk fibroin and its composite films in (a) dry state, and (b) wet state plotted using DIC method and Instron UTM.

503
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al.
Fig. 6. (a) Thermo gravimetric analysis (TGA), and (b) Differential thermo gravimetric analysis (DTG) of pure silk and its composites.
thermogravimetric (DTG) plots given in Fig. 6b. DTG provides the rate of
change of weight loss at each temperature. It can be clearly observed
that Silk/PVA has shown a shift in the endothermic peak towards higher
temperature in both the regions I (50 °C to 170 °C) and region III (above
240 °C), compared to the pure silk fibroin and remaining silk compos-
ites, which confirms its higher water retention capacity, and higher sta-
bility. For comparison, TGA analysis of neat polymer additives PVP, PVA,
and Chitosan was provided in the Fig. S7 of Supporting information.
3.4. Percentage transmittance
UV–Visible spectroscopy measurements were performed to under-
stand the optical transparency of the pure silk fibroin and its compos-
ites. All the films were prepared by spin-coating their homogenous
solutions onto standard microscopic glass slides, and the process condi-
tions were optimized so that all the films have a thickness of 10 μm. A
plot of transmittance vs. wavelength ranging from 200 to 750 nm is
shown in Fig. 7a. The reference length was chosen as 550 nm to com-
pare the optical transmittance spectra of all the prepared films, as the
Fig. 7. (a) UV–Visible spectroscopy plot of all the prepared films, (b) percentage transmittance comparison at a wavelength of 550 nm, and (c) plot of panel a in the visible region
(400–700 nm).
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International Journal of Biological Macromolecules 176 (2021) 498–509
human eye has the highest sensitivity at that particular wavelength
[65]. Moreover, this wavelength was commonly used in most optoelec-
tronic applications like solar cells and LEDs [66]. At 550 nm, the exact
middle point of the visible spectrum with a wavelength ranging from
400 to 700 nm, all the composites exhibited more than 95% transpar-
ency. Pure silk fibroin film shows excellent transparency of >95%
throughout the visible region and is 99% at 550 nm. Higher optical trans-
parency in both visible and near IR spectral regions is primarily due to
the weak absorption of amino acids in the silk fibroin [67], as depicted
in Fig. 7b. Fig. 7c compares optical transmittance of all the prepared
films in the visible region 400 to 700 nm, which clearly shows that all
the prepared films of pure silk fibroin and its composites have exhibited
higher optical transparency above 85%.
The reduction in transparency of Silk/PVP films in the range ~
(400–525) nm is because PVP causes turbidity of the composite film
during the drying process [68]. Besides, as a comparison, UV–Visible
analysis was performed for the pure polymer additives such as PVA,
PVP, and Chitosan films and is provided in Fig. S8 of Supporting
information.
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al.
Fig. 8. (a) Percentage cell viability, and (b) Live/Dead assay of L929 cells cultured on pure silk and its composite films. (Live/Dead assay: bright field images (first column), FDA staining
(second column), and PI staining (third column) of L929 cells).
3.5. Cytotoxicity assay
In this study, an MTT assay was performed to evaluate the effect of
cell compatibility of L929 (fibroblasts) on pure silk and its composite
films qualitatively. Fig. 8a shows that cell viability is more than 80%
for all the prepared films, indicating that the silk film's surface is suitable
for cell growth. There was no significant difference observed after the
cells were grown on all modified silk films compared with blank control
over three days, n = 8. Based on the results obtained, we found that all
the prepared films of pure silk and its composites have shown favorable
biocompatibility, verified with the reported literature [69,70].
3.6. Live/Dead assay
FDA/PI staining evaluates the viability of L929 cells on all the pre-
pared silk films [70]. As the bright green fluorescence indicates the pres-
ence of viable cells, and the red fluorescence indicates the dead cells.
When we compared all modified silk composite films, they showed
the bright green fluorescence, which demonstrates the cells' viability.
Images also revealed the higher proliferation and spreading of the
cells over ten days of culture (n = 8), as shown in Fig. 8b.
From the analyses as mentioned above, it is evident that Silk/PVA
composite is superior in all aspects, such as higher percentage of β-
sheets (72%), higher flexibility in the dry state (UTS of 14.3 ± 0.9 MPa,
Y.M of 0.4 ± 0.0 GPa, and Percentage elongation of 44.6 ± 0.9%), and
higher transparency (98%) at 550 nm, compared to all other silk com-
posite films. Moreover, Cytocompatibility studies suggest that all the
prepared films of pure silk and its composites have demonstrated
above 80% cell viability and favorable cell growth. Further analysis and
experimentation primarily focus on Silk/PVA film, and comparison, if
necessary, is carried out with pure silk film.
3.7. Atomic force microscopy
One of the critical aspects that a substrate should have is smooth-
ness. This study was performed by carrying out Atomic Force Micros-
copy (AFM) for both the films, pure silk and Silk/PVA spun at a speed
of 1000 rpm. The corresponding thickness of the spun films was mea-
sured to be around 5 μm. Fig. 9 shows the comparison of surface rough-
ness between Silk/PVA and pure silk. The average RMS surface
roughness (Rq) of 1.74 nm for RSF film and 0.76 nm for Silk/PVA
505
International Journal of Biological Macromolecules 176 (2021) 498–509
blend film, superior to that of pure silk film. The enhancement in the
surface smoothness of Silk/PVA compared to the pure silk can be attrib-
uted to the inherent hydrophilic nature of PVA, which promotes the
structural transition from amorphous nature into more crystalline na-
ture comprising well-arranged β-sheets inside the silk fibroin films by
forming more number of interconnected hydrogen bonds between the
hydroxyl groups of PVA and amine groups of silk fibroin [40].
3.8. Chemical durability
Device fabrication on any substrate involves exposure of the sub-
strate to various types of chemicals utilized during patterning and etch-
ing steps. Precise patterning of micron-level patterns above regenerated
silk fibroin is the most challenging task because RSF films are prone to
forming micro-cracks in the film during exposure to these reagents.
The chemical durability studies were carried out on Silk/PVA films to
understand the effects of these reactive chemicals on the composite
films. Here, in this study, we compared the effect of photoresist devel-
oper (MF319), Au etchant (3:1:2 HCl/HNO3/H2O), and Ti etchant
(20:1:1 H2O/HF/H2O2) between pure silk and Silk/PVA blend film, as
Ti/Au are predominantly used for MEA applications. Fig. 10 shows opti-
cal microscopic images of pure silk and Silk/PVA after exposure to
MF319 developer, Au etchant, and Ti etchant for a duration of 60 s.
Silk films either developed cracks (as in the case of MF 319) or be-
came rough (as in Au and Ti etchants). The average RMS roughness of
the pure silk and Silk/PVA films after exposure to commonly used re-
agents such as photoresist developer (MF319), Ti, and Au etchants
was estimated using atomic force microscopy (AFM). The 2D surface
roughness plots of both the films after exposure to the reagents were
provided in the Supporting information (Fig. S9(a & b)). On the con-
trary, the texture and the surface morphology of Silk/PVA films were
not affected much after exposure to reagents. The reason for the
unaltered texture and surface morphology of Silk/PVA films can be at-
tributed to the more crystalline nature of Silk/PVA films comprising
well-arranged β-sheets and by forming more number of interconnected
hydrogen bonds between the hydroxyl groups of PVA and amine groups
of silk fibroin [40].
3.9. Fabrication of 32 channel microelectrode array (MEA)
In order to affirmatively prove the applicability of Silk/PVA as a flex-
ible substrate, as a proof of concept, a 32 channel MEA with fine Au/Ti
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al. International Journal of Biological Macromolecules 176 (2021) 498–509

Fig. 9. Comparison of surface roughness from the AFM images of silk and Silk/PVA films.

patterns of minimum feature size of 5 μm were fabricated on Silk/PVA pattern after the pre-final step of the photoresist strip in an acetone
substrate as per the protocol described in the experimental bath. As shown in Fig. 11a, each square-shaped electrode's width is
Section 2.6. Fig. 11a shows the optical microscopic image of the MEA 25 μm, connected by thin Au/Ti metal lines of thickness 10 nm/3 nm

Fig. 10. Optical microscopic images of (a) silk and (b) Silk/PVA with and without treatment of standards patterning reagents.

506
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al.
Fig. 11. (a) Optical microscopic image of patterned 32 channel transparent MEA before release (b) image depicting the flexibility of released Silk/PVA film with 32 channel transparent
MEA (c) free standing Silk/PVA film, and (d) optical microscopic image of 32 channel transparent MEA above free standing Silk/PVA film.
and width 5 μm. As depicted in Fig. 11b, the flexibility of the dried Silk/
PVA having patterned MEA is demonstrated by rolling the film around a
ballpoint pen, which incidentally also showcases the optical transpar-
ency of the Silk/PVA film. Fig. 11c indicates the region of MEA in the
free-standing Silk/PVA film. MEA patterns are not visible to the naked
eye due to the small feature size. A zoomed version of MEA at a 40×
magnification is shown in Fig. 11d.
4. Conclusions
Regenerated silk fibroin (RSF) films offer splendid mechanical, opti-
cal, and electrical properties favorable towards developing bio-
integrated electronic device applications. The key impediments are the
brittleness of these films in the dry state and vulnerability towards stan-
dard chemicals used in photolithographic patterning. In this work, we
addressed both the limitations by developing regenerated silk compos-
ite films with widely reported biocompatible and biodegradable poly-
mers, such as Glycerol, Chitosan, Polyvinyl alcohol (PVA), and
Polyvinyl pyrrolidone (PVP). Silk/PVA was a promising candidate in ful-
filling the desired objectives among the silk composites that are studied.
It has not only shown the highest percentage of ꞵ-sheets (72%) but also
has shown good flexibility behavior in the dried state, estimated from
the highest percentage of elongation at break of 44.6 ± 0.9%, the mod-
erate ultimate tensile strength of 14.3 ± 0.9 MPa and lowest elastic
modulus of 0.4 ± 0.0 GPa. Silk/PVA remains robust after exposure to
standard reagents, such as Photolithographic developer and Au/Ti
metal etchants. The average RMS roughness (Rq) of Silk/PVA films is
found to be 0.76 nm, significantly improved over pure silk film (Rq =
1.74 nm). The percentage of optical transmittance is found to be 98%
at 550 nm. Moreover, Silk/PVA showed above 80% cell viability verified
using L929 fibroblast cell lines, suggesting that these composite films
are biocompatible. As a proof of concept, we demonstrated the success-
ful patterning of 32 channel optically transparent microelectrode array
pattern, with a minimum feature size of 5 μm above the free-standing
and optically transparent Silk/PVA composite film. Hence, these films
507
International Journal of Biological Macromolecules 176 (2021) 498–509
open up new avenues in the area of flexible bio-integrated electronic
devices.
CRediT authorship contribution statement
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Funding sources
Not applicable.
Acknowledgment
The authors would like to thank innovation hub for Nano-X , IIT Hy-
derabad for furnishing the facilities to complete this work. We are grate-
ful to the Department of Science and Technology (DST), Government of
India for providing the MHRD research fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2021.02.051.
References
[1] S. Ha, C. Kim, Y.M. Chi, A. Akinin, C. Maier, A. Ueno, G. Cauwenberghs, Integrated cir-
cuits and electrode interfaces for noninvasive physiological monitoring, IEEE Trans.
Biomed. Eng. 61 (5) (2014) 1522–1537, https://doi.org/10.1109/TBME.2014.
2308552.
[2] S.K. Ameri, M. Kim, I.A. Kuang, W.K. Perera, M. Alshiekh, H. Jeong, U. Topcu, D.
Akinwande, N. Lu, Imperceptible electrooculography graphene sensor system for
human–robot interface, npj 2D Mater. Appl. 2 (2018)https://doi.org/10.1038/
s41699-018-0064-4.
[3] D. Son, J. Lee, S. Qiao, R. Ghaffari, J. Kim, J.E. Lee, C. Song, S.J. Kim, D.J. Lee, S.W. Jun,
et al., Multifunctional wearable devices for diagnosis and therapy of movement
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al.
disorders, Nat. Nanotechnol. 9 (5) (2014) 397–404, https://doi.org/10.1038/nnano.
2014.38.
[4] H. Lee, T.K. Choi, Y.B. Lee, H.R. Cho, R. Ghaffari, L. Wang, H.J. Choi, T.D. Chung, N. Lu, T.
Hyeon, et al., A graphene-based electrochemical device with thermoresponsive
microneedles for diabetes monitoring and therapy, Nat. Nanotechnol. 11 (6)
(2016) 566–572, https://doi.org/10.1038/nnano.2016.38.
[5] J.-W. Jeong, W.-H. Yeo, A. Akhtar, J.J.S. Norton, Y.-J. Kwack, S. Li, S.-Y. Jung, Y. Su, W.
Lee, J. Xia, et al., Materials and optimized designs for human-machine interfaces via
epidermal electronics, Adv. Mater. 25 (47) (2013) 6839–6846, https://doi.org/10.
1002/adma.201301921.
[6] R. Herbert, J.-H. Kim, Y.S. Kim, H.M. Lee, W.-H. Yeo, Soft material-enabled, flexible
hybrid electronics for medicine, healthcare, and human-machine interfaces, Mater.
(Basel, Switzerland) 11 (2) (2018) 187, https://doi.org/10.3390/ma11020187.
[7] T.R. Ray, J. Choi, A.J. Bandodkar, S. Krishnan, P. Gutruf, L. Tian, R. Ghaffari, J.A. Rogers,
Bio-integrated wearable systems: a comprehensive review, Chem. Rev. 119 (8)
(2019) 5461–5533, https://doi.org/10.1021/acs.chemrev.8b00573.
[8] B. Zhu, H. Wang, W.R. Leow, Y. Cai, X.J. Loh, M.-Y. Han, X. Chen, Silk fibroin for flex-
ible electronic devices, Adv. Mater. 28 (22) (2016) 4165, https://doi.org/10.1002/
adma.201504276.
[9] G. Zheng, Y. Cui, E. Karabulut, L. Wågberg, H. Zhu, L. Hu, Nanostructured paper for
flexible energy and electronic devices, MRS Bull. 38 (2013) 320–325, https://doi.
org/10.1557/mrs.2013.59.
[10] C.-Y. Hsieh, J.-C. Hwang, T.-H. Chang, J.-Y. Li, S.-H. Chen, L.-K. Mao, L.-S. Tsai, Y.-L.
Chueh, P.-C. Lyu, S.S.H. Hsu, Enhanced mobility of organic thin film transistors by
water absorption of collagen hydrolysate gate dielectric, Appl. Phys. Lett. 103
(2013), 23303, https://doi.org/10.1063/1.4813075.
[11] Y. He, J. Sun, C. Qian, L. Kong, J. Jiang, J. Yang, H. Li, Y. Gao, Solution-processed natural
gelatin was used as a gate dielectric for the fabrication of oxide field-effect transis-
tors, Org. Electron. 38 (2016) 357–361, https://doi.org/10.1016/j.orgel.2016.09.017.
[12] V. Raghuwanshi, P. Saxena, S. Rahi, A.K. Mahato, I. Varun, S.P. Tiwari, Solution-
processed flexible organic field-effect transistors with biodegradable gelatin as the
dielectric layer: an approach toward biodegradable systems, ACS Appl. Electron.
Mater. 2 (2020) 3373–3379, https://doi.org/10.1021/acsaelm.0c00648.
[13] J. Ko, L.T.H. Nguyen, A. Surendran, B.Y. Tan, K.W. Ng, W.L. Leong, Human hair keratin
for biocompatible flexible and transient electronic devices, ACS Appl. Mater. Inter-
faces 9 (2017) 43004–43012, https://doi.org/10.1021/acsami.7b16330.
[14] C. Yumusak, T.B. Singh, N.S. Sariciftci, J.G. Grote, Bio-organic field effect transistors
based on crosslinked deoxyribonucleic acid (DNA) gate dielectric, Appl. Phys. Lett.
95 (2009), 263304, https://doi.org/10.1063/1.3278592.
[15] M. Irimia-Vladu, N.S. Sariciftci, S. Bauer, Exotic materials for bio-organic electronics,
J. Mater. Chem. 21 (2011) 1350–1361, https://doi.org/10.1039/C0JM02444A.
[16] C. Pang, J.H. Koo, A. Nguyen, J.M. Caves, M.-G. Kim, A. Chortos, K. Kim, P.J. Wang, J.B.-
H. Tok, Z. Bao, Highly skin-conformal microhairy sensor for pulse signal amplifica-
tion, Adv. Mater. 27 (4) (2015) 634–640, https://doi.org/10.1002/adma.201403807.
[17] M.D. Tang-Schomer, X. Hu, M. Hronik-Tupaj, L.W. Tien, M.J. Whalen, F.G. Omenetto,
D.L. Kaplan, Film-based implants for supporting neuron–electrode integrated inter-
faces for the brain, Adv. Funct. Mater. 24 (13) (2014) 1938–1948, https://doi.org/10.
1002/adfm.201303196.
[18] H. Tao, S.-W. Hwang, B. Marelli, B. An, J.E. Moreau, M. Yang, M.A. Brenckle, S. Kim,
D.L. Kaplan, J.A. Rogers, et al., Silk-based resorbable electronic devices for remotely
controlled therapy and in vivo infection abatement, Proc. Natl. Acad. Sci. 111 (49)
(2014) 17385–17389, https://doi.org/10.1073/pnas.1407743111.
[19] T.P. Nguyen, Q.V. Nguyen, V.-H. Nguyen, T.-H. Le, V.Q.N. Huynh, D.-V.N. Vo, Q.T.
Trinh, S.Y. Kim, Q. Van Le, Silk fibroin-based biomaterials for biomedical applica-
tions: a review, Polymers (Basel) 11 (12) (2019)https://doi.org/10.3390/
polym11121933.
[20] C. Vepari, D.L. Kaplan, Silk as a biomaterial, Prog. Polym. Sci. 32 (8) (2007)
991–1007, https://doi.org/10.1016/j.progpolymsci.2007.05.013.
[21] Q. Lu, B. Zhang, M. Li, B. Zuo, D.L. Kaplan, Y. Huang, H. Zhu, Degradation mechanism
and control of silk fibroin, Biomacromolecules 12 (4) (2011) 1080–1086, https://
doi.org/10.1021/bm101422j.
[22] Q. Lu, X. Hu, X. Wang, J.A. Kluge, S. Lu, P. Cebe, D.L. Kaplan, Water-insoluble silk films
with silk I structure, Acta Biomater. 6 (4) (2010) 1380–1387, https://doi.org/10.
1016/j.actbio.2009.10.041.
[23] I.C. Um, H.Y. Kweon, K.G. Lee, Y.H. Park, The role of formic acid in solution stability
and crystallization of silk protein polymer, Int. J. Biol. Macromol. 33 (4) (2003)
203–213, https://doi.org/10.1016/j.ijbiomac.2003.08.004.
[24] S. Hofmann, C.T.W.P. Foo, F. Rossetti, M. Textor, G. Vunjak-Novakovic, D.L. Kaplan,
H.P. Merkle, L. Meinel, Silk fibroin as an organic polymer for controlled drug deliv-
ery, J. Control. Release 111 (1–2) (2006) 219–227, https://doi.org/10.1016/j.
jconrel.2005.12.009.
[25] G. Nogueira, C. Rodas, C. Leite, C. Giles, O. Higa, B. Polakiewicz, M. Beppu, Preparation
and characterization of ethanol-treated silk fibroin dense membranes for biomate-
rials application using waste silk fibers as raw material, Bioresour. Technol. 101
(2010) 8446–8451, https://doi.org/10.1016/j.biortech.2010.06.064.
[26] B.D. Lawrence, S. Wharram, J.A. Kluge, G.G. Leisk, F.G. Omenetto, M.I. Rosenblatt, D.L.
Kaplan, Effect of hydration on silk film material properties, Macromol. Biosci. 10 (4)
(2010) 393–403, https://doi.org/10.1002/mabi.200900294.
[27] G. Kook, S. Jeong, S.H. Kim, M.K. Kim, S. Lee, I.-J. Cho, N. Choi, H.J. Lee, Wafer-scale
multilayer fabrication for silk fibroin-based microelectronics, ACS Appl. Mater. Inter-
faces 11 (1) (2019) 115–124, https://doi.org/10.1021/acsami.8b13170.
[28] B. Ganesh Kumar, R. Melikov, M. Mohammadi Aria, A. Ural Yalcin, E. Begar, S.
Sadeghi, K. Guven, S. Nizamoglu, Silk-based aqueous microcontact printing, ACS
Biomater. Sci. Eng. 4 (2018) 1463–1470, https://doi.org/10.1021/acsbiomaterials.
8b00040.
508
International Journal of Biological Macromolecules 176 (2021) 498–509
[29] M.A. Brenckle, H. Tao, S. Kim, M. Paquette, D.L. Kaplan, F.G. Omenetto, Protein-
protein nanoimprinting of silk fibroin films, Adv. Mater. 25 (2013) 2409–2414,
https://doi.org/10.1002/adma.201204678.
[30] Konstantinos Tsioris, Hu Tao, Mengkun Liu, Jeffrey A. Hopwood, David L. Kaplan,
Richard D. Averitt, Fiorenzo G. Omenetto, Rapid transfer-based micropatterning
and dry etching of silk microstructures, Adv. Mater. 23 (17) (2011) 2015–2019,
https://doi.org/10.1002/adma.201004771.
[31] Geon Kook, Sohyeon Jeong, So Hyun Kim, Mi Kyung Kim, Sungwoo Lee, Il-Joo Cho,
Nakwon Choi, Hyunjoo J. Lee, Wafer-scale multilayer fabrication for silk fibroin-
based microelectronics, ACS Appl. Mater. Interfaces 11 (1) (2018) 115–124,
https://doi.org/10.1021/acsami.8b13170.
[32] D.V. Santhosh Kumar Gunapu, Jose Joseph, Shiv Govind Singh, Siva Rama Krishna
Vaniari, Selective anisotropic dry etching of piezoelectric silk microstructures
using oxygen plasma ashing, 2018 IEEE Sensors, IEEE 2018, pp. 1–4, https://doi.
org/10.1109/ICSENS.2018.8589957.
[33] M.B. Dickerson, P.B. Dennis, V.P. Tondiglia, L.J. Nadeau, K.M. Singh, L.F. Drummy, B.P.
Partlow, D.P. Brown, F.G. Omenetto, D.L. Kaplan, R.R. Naik, 3D printing of regener-
ated silk fibroin and antibody-containing microstructures via multiphoton lithogra-
phy, ACS Biomater. Sci. Eng. 3 (2017) 2064–2075, https://doi.org/10.1021/
acsbiomaterials.7b00338.
[34] V. Raghuwanshi, P. Saxena, S. Rahi, A.K. Mahato, I. Varun, S.P. Tiwari, Solution-
processed flexible organic field-effect transistors with biodegradable gelatin as the
dielectric layer: an approach toward biodegradable systems, ACS Appl. Electron.
Mater. 2 (2020) 3373–3379, https://doi.org/10.1021/acsaelm.0c00648.
[35] W. Liu, Z. Zhou, S. Zhang, Z. Shi, J. Tabarini, W. Lee, Y. Zhang, S.N. Gilbert Corder, X. Li,
F. Dong, L. Cheng, M. Liu, D.L. Kaplan, F.G. Omenetto, G. Zhang, Y. Mao, T.H. Tao, Pre-
cise protein photolithography (P3): high performance biopatterning using silk fi-
broin light chain as the resist, Adv. Sci. 4 (2017), 1700191, https://doi.org/10.
1002/advs.201700191.
[36] H. Teramoto, K. Nakajima, K. Kojima, Azide-incorporated clickable silk fibroin mate-
rials with the ability to photopattern, ACS Biomater. Sci. Eng. 2 (2016) 251–258,
https://doi.org/10.1021/acsbiomaterials.5b00469.
[37] E.D. Patamia, N.A. Ostrovsky-Snider, A.R. Murphy, Photolithographic masking
method to chemically pattern silk film surfaces, ACS Appl. Mater. Interfaces 11
(2019) 33612–33619, https://doi.org/10.1021/acsami.9b10226.
[38] R.R. da Silva, M. Cavicchioli, L.R. Lima, C.G. Otoni, H.S. Barud, S.H. Santagneli, A.
Tercjak, A.C. Amaral, R.A. Carvalho, S.J.L. Ribeiro, Fabrication of biocompatible, func-
tional, and transparent hybrid films based on silk fibroin and epoxy silane for
biophotonics, ACS Appl. Mater. Interfaces 9 (33) (2017) 27905–27917, https://doi.
org/10.1021/acsami.7b06061.
[39] B.J. Allardyce, R. Rajkhowa, R.J. Dilley, S.L. Redmond, M.D. Atlas, X. Wang, Glycerol-
plasticised silk membranes made using formic acid are ductile, transparent and
degradation-resistant, Mater. Sci. Eng. C 80 (2017) 165–173, https://doi.org/10.
1016/j.msec.2017.05.112.
[40] Y. Wang, X. Wang, J. Shi, R. Zhu, J. Zhang, Z. Zhang, Flexible silk fibroin films modi-
fied by genipin and glycerol, RSC Adv. 5 (123) (2015) 101362–101369, https://doi.
org/10.1039/C5RA19754F.
[41] C.M. Srivastava, R. Purwar, R. Kannaujia, D. Sharma, Flexible silk fibroin films for
wound dressing, Fibers Polym 16 (5) (2015) 1020–1030, https://doi.org/10.1007/
s12221-015-1020-y.
[42] G. Basal, D. Altıok, O. Bayraktar, Antibacterial properties of silk fibroin/chitosan
blend films loaded with plant extract, Fibers Polym 11 (1) (2010) 21–27, https://
doi.org/10.1007/s12221-010-0021-0.
[43] A. Panico, F. Paladini, M. Pollini, Development of regenerative and flexible fibroin-
based wound dressings, J. Biomed. Mater. Res. Part B Appl. Biomater. 107 (1)
(2019) 7–18, https://doi.org/10.1002/jbm.b.34090.
[44] F. Zhang, X. You, H. Dou, Z. Liu, B. Zuo, X. Zhang, Facile fabrication of robust silk
nanofibril films via direct dissolution of silk in CaCl2–formic acid solution, ACS
Appl. Mater. Interfaces 7 (5) (2015) 3352–3361, https://doi.org/10.1021/
am508319h.
[45] C. Zhang, D. Song, Q. Lu, X. Hu, D.L. Kaplan, H. Zhu, Flexibility regeneration of silk fi-
broin in vitro, Biomacromolecules 13 (7) (2012) 2148–2153, https://doi.org/10.
1021/bm300541g.
[46] Z. Liu, F. Zhang, J. Ming, S. Bie, J. Li, B. Zuo, Preparation of electrospun silk fibroin
nanofibers from solutions containing native silk fibrils, J. Appl. Polym. Sci. 132 (1)
(2015)https://doi.org/10.1002/app.41236.
[47] K. Nultsch, L.K. Bast, M. Näf, S. El Yakhlifi, N. Bruns, O. Germershaus, Effects of silk
degumming process on physicochemical, tensile, and optical properties of regener-
ated silk fibroin, Macromol. Mater. Eng. 303 (12) (2018), 1800408, https://doi.org/
10.1002/mame.201800408.
[48] O.K. Gasymov, A. Aydemirova, O. Alekperov, R.B. Aslanov, K. Khalilova, N. Gasimov,
N. Mamedov, I. Mamedova, S. Babayev, N. Gasanov, IR ellipsometry of silk fibroin
films on Al nanoislands, Phys. status solidi c 12 (6) (2015) 628–630, https://doi.
org/10.1002/pssc.201500005.
[49] Y.-Q. Zhang, W.-D. Shen, R.-L. Xiang, L.-J. Zhuge, W.-J. Gao, W.-B. Wang, Formation of
silk fibroin nanoparticles in water-miscible organic solvent and their characteriza-
tion, J. Nanopart. Res. 9 (5) (2007) 885–900, https://doi.org/10.1007/s11051-006-
9162-x.
[50] B.-H. Lv, W. Tan, C.-C. Zhu, X. Shang, L. Zhang, Properties of a stable and sustained-
release formulation of recombinant human parathyroid hormone (RhPTH) with chi-
tosan and silk fibroin microparticles, Med. Sci. Monit. 24 (2018) 7532–7540, https://
doi.org/10.12659/MSM.911203.
[51] A. Barlian, H. Judawisastra, A. Ridwan, A.R. Wahyuni, M.E. Lingga, Chondrogenic dif-
ferentiation of Wharton’s Jelly mesenchymal stem cells on silk spidroin-fibroin mix
scaffold supplemented with L-ascorbic acid and platelet rich plasma, Sci. Rep. 10
(2020), 19449, https://doi.org/10.1038/s41598-020-76466-8.
D.V.S.K. Gunapu, Y.B. Prasad, V.S. Mudigunda et al.
[52] H. Zhang, L. Li, F. Dai, H. Zhang, B. Ni, W. Zhou, X. Yang, Y. Wu, Preparation and char-
acterization of silk fibroin as a biomaterial with potential for drug delivery, J. Transl.
Med. 10 (2012) 117, https://doi.org/10.1186/1479-5876-10-117.
[53] X. Hu, D. Kaplan, P. Cebe, Determining beta-sheet crystallinity in fibrous proteins by
thermal analysis and infrared spectroscopy, Macromolecules 39 (18) (2006)
6161–6170, https://doi.org/10.1021/ma0610109.
[54] F. Zhang, R. Yang, P. Zhang, J. Qin, Z. Fan, B. Zuo, Water-rinsed nonmulberry silk film
for potential tissue engineering applications, ACS Omega 4 (2) (2019) 3114–3121,
https://doi.org/10.1021/acsomega.8b03542.
[55] H. He, R. Cai, Y. Wang, G. Tao, P. Guo, H. Zuo, L. Chen, X. Liu, P. Zhao, Q. Xia, Prepa-
ration and characterization of silk sericin/PVA blend film with silver nanoparticles
for potential antimicrobial application, Int. J. Biol. Macromol. 104 (2017) 457–464,
https://doi.org/10.1016/j.ijbiomac.2017.06.009.
[56] C. Niu, X. Li, Y. Wang, X. Liu, J. Shi, X. Wang, Design and performance of a poly(vinyl
alcohol)/silk fibroin enzymatically crosslinked semi-interpenetrating hydrogel for a
potential hydrophobic drug delivery, RSC Adv. 9 (70) (2019) 41074–41082, https://
doi.org/10.1039/C9RA09344C.
[57] J. Zhong, M. Ma, J. Zhou, D. Wei, Z. Yan, D. He, Tip-induced micropatterning of silk
fibroin protein using in situ solution atomic force microscopy, ACS Appl. Mater. In-
terfaces 5 (2013) 737–746, https://doi.org/10.1021/am302271g.
[58] J. Zhong, M. Ma, W. Li, J. Zhou, Z. Yan, D. He, Self-assembly of regenerated silk fibroin
from random coil nanostructures to antiparallel β-sheet nanostructures, Biopoly-
mers 101 (2014) 1181–1192, https://doi.org/10.1002/bip.22532.
[59] J. Zhong, X. Liu, D. Wei, J. Yan, P. Wang, G. Sun, D. He, Effect of incubation tempera-
ture on the self-assembly of regenerated silk fibroin: a study using AFM, Int. J. Biol.
Macromol. 76 (2015) 195–202, https://doi.org/10.1016/j.ijbiomac.2015.02.045.
[60] M. Ma, J. Zhong, W. Li, J. Zhou, Z. Yan, J. Ding, D. He, Comparison of four synthetic
model peptides to understand the role of modular motifs in the self-assembly of
silk fibroin, Soft Matter 9 (2013) 11325–11333, https://doi.org/10.1039/
C3SM51498F.
[61] H. Zhang, L. Li, F. Dai, H. Zhang, B. Ni, W. Zhou, X. Yang, Y. Wu, Preparation and char-
acterization of silk fibroin as a biomaterial with potential for drug delivery, J. Transl.
Med. 10 (1) (2012) 117, https://doi.org/10.1186/1479-5876-10-117.
509
International Journal of Biological Macromolecules 176 (2021) 498–509
[62] I.C. Um, H. Kweon, Y.H. Park, S. Hudson, Structural characteristics and properties of
the regenerated silk fibroin prepared from formic acid, Int. J. Biol. Macromol. 29
(2001) 91–97, https://doi.org/10.1016/S0141-8130(01)00159-3.
[63] J.L. Whittaker, N.R. Choudhury, N.K. Dutta, A. Zannettino, Facile and rapid ruthenium
mediated photo-crosslinking of Bombyx mori silk fibroin, J. Mater. Chem. B 2 (2014)
6259–6270, https://doi.org/10.1039/C4TB00698D.
[64] M. Quanjin, M.R.M. Rejab, Q. Halim, M.N.M. Merzuki, M.A.H. Darus, Experimental in-
vestigation of the tensile test using digital image correlation (DIC) method, Mater.
Today Proc. 27 (2020) 757–763, https://doi.org/10.1016/j.matpr.2019.12.072.
[65] M. Liu, Y. Zhang, C. Wu, S. Xiong, C. Zhou, Chitosan/halloysite nanotubes
bionanocomposites: structure, mechanical properties and biocompatibility, Int. J.
Biol. Macromol. 51 (2012) 566–575, https://doi.org/10.1016/j.ijbiomac.2012.06.
022.
[66] K. Min, M. Umar, H. Seo, J.H. Yim, D.G. Kam, H. Jeon, S. Lee, S. Kim, Biocompatible,
optically transparent, patterned, and flexible electrodes and radio-frequency anten-
nas prepared from silk protein and silver nanowire networks, RSC Adv. 7 (2017)
574–580, https://doi.org/10.1039/C6RA25580A.
[67] A. Balčytis, M. Ryu, X. Wang, F. Novelli, G. Seniutinas, S. Du, X. Wang, J. Li, J. Davis, D.
Appadoo, et al., Silk: optical properties over 12.6 octaves THz-IR-visible-UV range,
Mater. (Basel, Switzerland) 10 (4) (2017) 356, https://doi.org/10.3390/
ma10040356.
[68] P. Molyneux, Water soluble poly-N-vinylamides: synthesis and physicochemical
properties YE Kirsh John Wiley & Sons, Chichester, 1998, Polym. Int. 48 (5) (1999)
426, https://doi.org/10.1002/(SICI)1097-0126(199905)48:5<426::AID-PI163>3.0.
CO;2-%23.
[69] S.H. Kim, Y.K. Yeon, J.M. Lee, J.R. Chao, Y.J. Lee, Y.B. Seo, M.T. Sultan, O.J. Lee, J.S. Lee, S.
Yoon, I.-S. Hong, G. Khang, S.J. Lee, J.J. Yoo, C.H. Park, Precisely printable and biocom-
patible silk fibroin bioink for digital light processing 3D printing, Nat. Commun. 9
(2018), 1620, https://doi.org/10.1038/s41467-018-03759-y.
[70] S. Manchineella, G. Thrivikraman, K.K. Khanum, P.C. Ramamurthy, B. Basu, T.
Govindaraju, Pigmented silk nanofibrous composite for skeletal muscle tissue engi-
neering, Adv. Healthc. Mater. 5 (2016) 1222–1232, https://doi.org/10.1002/adhm.
201501066.