Development of robust, ultra-smooth, flexible and transparent regenerated silk composite films for bio-integrated electronic device applications

Abstract

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

Introduction

Flexible bio-integrated electronic devices play a significant role in applications involving soft and curved biological systems. These applications include basic measurements of electrophysiological signals [1,2], drug delivery for advanced therapy [3,4], human-machine interfaces [5,6], to name a few. Traditional Silicon or Gallium Arsenide based devices are fundamentally rigid and planar. In contrast, the biological 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 movements and associated biological processes. The successful development of flexible and stretchable bio-integrated wearable systems requires utmost care and attention during the materials design of both active devices and supporting substrate [7]. In this regard, researchers explored a wide range of synthetic and natural polymers towards the development 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 devices [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 garnered special attention as a potential candidate for bio-integrated electronic devices such as sensor skins [16], brain-machine interfaces [17], and biomedical diagnosis & therapy [18] owing to their appealing properties such as natural abundance, superior mechanical properties, biocompatibility, 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 regenerated 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 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 reported 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], [31], [32]] to create 2D micron/nano resolution patterns of silk films. Dickerson et al. [33] have utilized multiphoton lithography printing to create 3D silk structures. Several groups have modified silk fibroin/sericin structure to act as a photoresist in creating a variety of high-resolution patterns [[34], [35], [36]]. Murphy et al. [37] have introduced a photolithographic masking method to pattern silk film surfaces chemically. 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 mechanisms 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, essential 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 adverse effects on optical transparency, surface smoothness, and biocompatibility. We assessed the enhancement of flexibility & chemical durability while retaining the optical transparency and surface smoothness of RSF by combining different biocompatible & biodegradable polymers 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 substantiated by standard structural, optical, and material characterizations such as RAMAN spectroscopy (RAMAN), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction study (XRD), Optical microscopy, Atomic force microscopy (AFM), and UV visible spectroscopy (UV–Vis). We estimated 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 fibroblasts (L929 cell lines). Finally, we verified the improvement in the critical criteria for its potential use in conformal bio-integrated electronics by fabricating 32 channel MEA having electrodes with a minimum feature size of width 5 μm and thickness 10 nm onto an optically transparent free-standing Silk/PVA film.