Over the past few decades, Bombyx mori silk fibroin has become a ubiquitous material for applications ranging from biomedical devices to optics, electronics, and sensing, while also showing potential in the food supply chain and being re-engineered as a functional material for architecture and design-related applications. Its widespread use derives from its unique properties, including biocompatibility, edibility, optical transparency, stabilization of labile compounds, and the ability to controllably change conformation and degrade in a programmed way. This review discusses recent and pivotal silk-based devices in which the presence of silk brings added value in terms of functionality, as demonstrated in a broad variety of fields. First, it gives an overview of silk's natural structure and main properties in terms of cross-linking, biocompatibility, and biodegradability to provide the reader with the necessary toolbox to fully make use of silk's multifaceted properties. Then, multifunctional silk-based devices are discussed highlighting the advantage of using silk over more traditional materials. Representative devices from both established and emerging applications for silk are examined. Finally, a roadmap for the next generation of silk-based devices is laid out.
For centuries, silk has been used as a textile luxury good as well as a biomedical material due to its lustrous sheen and biocompatible properties, respectively. Silk reinvention at the laboratory scale initially focused on (1) biomedical applications, in the form of drug delivery devices, scaffolds for cell cultures, implants, and stabilization of labile compounds, to only later expand to other disciplines with applications in the fields of (2) biocompatible optics, as photonic and diffractive nanostructures, waveguides, and resonators, (3) resorbable electronics, as a transient and biodegradable substrate for antennas and metamaterials, and (4) sensing, as pollutant and health monitoring wearable devices. More recently, silk's use has been investigated in relation to (5) the food supply chain both as a crop booster and a food protective coating while being (6) re-engineered as a technological material for architecture and sustainable design products. Currently, silk is considered as a full-rounded platform as it is being used as a support (substrate for resorbable electronics), an interface (drug delivery devices monitored via an optical response), and as a carrier of function and chemical/biological activity.
This widespread use of silk arises from the protein's multifaceted properties and from the versatility of processing techniques and formats that can be applied to it. In particular, the way functions can be embedded in silk is twofold: either (1) via chemical modification, doping, and tuning of the protein's properties or (2) by assembling proteins in a specific form that confers responsiveness to the final construct. The combination of these two approaches allows for the fabrication of multifunctional, responsive platforms that are not necessarily achievable using more inert, traditional materials. To fully harness silk's potential, it is crucial to have a thorough understanding of the physical origin of its properties and of how to control and optimize them. Only in this way, it is possible to diversify silk's application range and, especially, develop platforms with additional functionality.
This review is structured into two sections. The first section presents a high-level analysis of the hierarchical structure of silk and the properties of regenerated silk fibroin. It then focuses on established silk fibroin formats discussing the effect of the silk form on the final function of the manufactured constructs. The aim of this section is to provide the reader with a comprehensive toolbox that explains properties, established fabrication techniques, formats, and the potential of silk as a technological material. The second section covers recent and pivotal applications of silk fibroin in a broad variety of fields focusing on optics, electronics, sensing, biomedical platforms, food supply chain, and technological design. This section aims at highlighting applications where the use of silk provided additional functionality, thus contributing to the fabrication of next-generation silk-based devices.
II. SILK PROTEIN
While silk and silk-like materials are produced by several insects, this review focuses on silk extracted from the cocoons of the domesticated silkworm, Bombyx mori. Use of silk from other sources, including other arthropods, or from recombinantly expressed silk proteins face their own unique challenges and are outside the scope of this review. This section concentrates on the isolation of silk fibroin from B. mori cocoons, silk's polymorphs, biodegradation, and biocompatibility.
A. Silk composition and isolation
Most silk utilized in scientific, medical, and commercial endeavors is derived from cocoons of B. mori domestic silk moths. These cocoons are constructed from a continuous fiber strand that each silkworm excretes from its pair of silk glands and spins into a fiber woven around the pupating larva to form a multi-layer structure.1 The fiber itself is a dual-core fiber, with two continuous cores of fibroin protein, one from each silk gland, held together by glue-like sericin protein3,542 [Fig. 1(a)]. Each fibroin fiber core has a rounded triangular shape with an equivalent diameter of ∼10–13.7 μm and a sericin coating with thickness in the range of ∼1–2 μm.40 These two types of proteins form a natural fiber composite with sericin acting as a bulk matrix and fibroin as inner reinforcing fibers.5 The fibroin proteins are expressed as a heavy chain (∼390 kDa) and a light chain (∼25 kDa) bridged by a disulfide bond, both non-covalently linked to the glycoprotein P25 (∼30kDa).6,7 The heavy chain proteins are comprised predominantly of long domains of the repeated GAGAGS and GAGAGY amino acid motifs interspersed with short, disordered linkers.8 The repetitive sections form crystalline domains that are hydrophobic and tend to coalesce and form intra- and inter-chain links with numerous hydrogen bonds in the backbone. The formation of these hydrogen bonds both restricts the freedom of the chain and prevents bonding with water molecules, resulting in the native protein being insoluble in water.9
While fibroin can be extracted directly from the spinning glands of the silkworm larvae, this is a difficult and, so far, unscalable procedure that is predominantly employed for fundamental research purposes; therefore, silk is more commonly extracted starting from raw cocoons. The processing stages of fibroin extraction can be largely summarized in three steps: degumming, dissolution, and regeneration, followed by an optional step of annealing/post-processing [Fig. 1(b)]. At each stage, various factors can be leveraged to influence the end material properties of the fibroin from the molecular level (e.g., molecular weight, dispersity) to the bulk material level (e.g., strength, elasticity, porosity, degradability).
The extraction process typically begins with degumming, which consists of washing away sericin and other undesirable cocoon components (endotoxic and immunogenic larval residues) to leave only the fibroin. Degumming is particularly important for using silk in a biomedical context since this process dissolves sericin, which has been traditionally (but disputably) reported as immunogenic.11,12 There are several layers of sericin protein surrounding the fibroin cores: the innermost layer is soluble in boiling water, the middle layer is soluble in 120 °C water, and the outer layer can be dissolved in boiling alkaline aqueous solution.13 Various methods can be employed to simultaneously remove all these types of sericin from the fibroin, including boiling the cocoons in an alkaline solution of sodium carbonate or calcium hydroxide,14 using surfactants such as sodium dodecyl sulfate,15 or even acidic solutions such as boric or citric acid.16 With the sodium carbonate method, a minimum of 5 min of boiling is required to fully extract the sericin proteins;17 however, many protocols employ longer boiling periods as a facile way to control the length of the regenerated fibroin molecules and, therefore, the mechanical, chemical, and biological properties of fibroin. This can be useful in various applications such as tuning drug release profiles or degradation rate of films.18,19 Employing the sodium carbonate solution degumming method, the minimum 5 min boiling duration yields fibroin molecular weight (MW) averages upward of 300 kDa, whereas an hour duration yields fragments of 50 kDa or lower, with the dispersity increasing with longer degumming times.17,20 The molecular weight of fibroin within this range may be controlled reproducibly by accurate timing of the degumming process. It is also possible to degum silk at lower temperatures (50–65 °C) using proteolytic enzymes such as papain to preferentially attack the hydrophilic sericin.21–23 While this method has been used in some textile applications, it is rarely used in research and medicine as it is very difficult to achieve complete degumming and often results in fibroin with reduced molecular weight, despite the reduced temperatures.
After degumming, the purified dry fibroin can be transformed into a water-based solution by disrupting fibroin's hydrophobic crystalline structure. While the hydrogen bonds that allow fibroin's crystalline structure to form are strong when in aggregate, they are individually weak and, thus, can be disrupted with the addition of chaotropic agents such as LiBr, CaCl2, or CeCl3.24,543,544 Once the hydrogen-bonds are disrupted, inter-polymer linkages are broken, and these highly hydrophobic structures become hydrophilic, resulting in the polymer becoming water soluble. Once in solution, the chaotropic salts may be removed by dialysis or desalted by the use of size exclusion columns to yield a protein solution in pure water.10,25 The resulting protein solution is semi-stable as the chains spontaneously begin reforming hydrogen bonds, which will eventually result in the formation of a hydrogel or solid precipitant. The practical stability time varies from a few days to several months when stored at 4 °C depending on the size of the fibroin fragments and on their concentration.26 It is also possible to dissolve silk in ionic liquids and in organic solvents such as formic acid and hexafluoroisopropanol (HFIP); the greater volatility of these solvents makes them attractive for fabrication techniques that require very fast drying and can be useful for non-aqueous applications. However, as both HFIP and formic acid are corrosive and HFIP is toxic, thorough washing is required if the final silk products are intended to interact directly with living systems.24 The main biochemical, electrical, optical, and thermal properties of regenerated silk fibroin are reported in Table I.
|Property .||Value .|
|Heavy chain6||391 kDa|
|Light chain6||25 kDa|
|Silk I (35% crystallinity)28||3.5|
|Silk II (50% crystallinity)28||4.4|
|Density35||1.348 g cm−3|
|Amorphous silk (100–0.02 kHz)29||5.2–7.3|
|Silk I (100–0.02 kHz)29||4.9–6.7|
|Silk II (100–0.02 kHz)29||4.6–6.1|
|Dielectric breakdown strength|
|Amorphous silk30||0.07 MV cm−1|
|Silk II (50% crystallinity)30||2 MV cm−1|
|Electrical resistivity31||1015 Ω cm−1|
|Refractive Index (λ = 633 nm)32,33||1.54|
|Birefringence (λ = 633 nm)33||0.007|
|Extinction coefficient (λ = 276 nm)34||1.13 cm–1 g−1 L|
|Thermal conductivity (native fiber, relaxed)36||0.042 W m−1 K–1|
|Glass transition temperature|
|Silk (amorphous)37||178 °C|
|Silk I (30% crystallinity)38||215 °C|
|Silk II (50% crystallinity)38||227 °C|
|Decomposition onset temperature48||230 °C|
|Property .||Value .|
|Heavy chain6||391 kDa|
|Light chain6||25 kDa|
|Silk I (35% crystallinity)28||3.5|
|Silk II (50% crystallinity)28||4.4|
|Density35||1.348 g cm−3|
|Amorphous silk (100–0.02 kHz)29||5.2–7.3|
|Silk I (100–0.02 kHz)29||4.9–6.7|
|Silk II (100–0.02 kHz)29||4.6–6.1|
|Dielectric breakdown strength|
|Amorphous silk30||0.07 MV cm−1|
|Silk II (50% crystallinity)30||2 MV cm−1|
|Electrical resistivity31||1015 Ω cm−1|
|Refractive Index (λ = 633 nm)32,33||1.54|
|Birefringence (λ = 633 nm)33||0.007|
|Extinction coefficient (λ = 276 nm)34||1.13 cm–1 g−1 L|
|Thermal conductivity (native fiber, relaxed)36||0.042 W m−1 K–1|
|Glass transition temperature|
|Silk (amorphous)37||178 °C|
|Silk I (30% crystallinity)38||215 °C|
|Silk II (50% crystallinity)38||227 °C|
|Decomposition onset temperature48||230 °C|
B. Silk polymorphs and cross-linking
Fibroin has several stable crystalline polymorphs characterized by different secondary structures in the bulk, which result in a variety of different properties. Silk I is the metastable state of silk found in the silk gland of B. mori, while the silk II polymorph is present in native silk fibers, as they are produced by the silkworm, and is defined by a high degree of anti-parallel pleated β-sheets.39,40 Most artificial curing methods involving silk fibroin solution cause the fibroin to adopt a silk II structure, and both random-coil and silk I materials spontaneously reorganize to silk II under ambient conditions. Silk I is characterized by high β-turn content and bulk materials made of this polymorph typically show lower strength, modulus, and glass transition temperature than silk II materials (Table I). Silk I solids can be obtained by slow drying of the silk solution, precise pH, and concentration control in foams, or by the inclusion of certain plasticizers such as polyethylene glycol (PEG) oligomers or glycerol.41 Silk III is a rarely observed polymorph of silk that is typically limited to films formed at air/water or oil/water interfaces.42 In this last crystal structure, the fibroin chains adopt a left-handed threefold helix aligned parallel to the surface, likely as the result of the surfactant nature of aqueous silk.43,44 Silk fibroin can also be dried and isolated in an amorphous (random coil) conformation that remains water soluble after drying, which can prove useful in processing, storage, and certain applications.47
Silk fibroin extracted directly from the silkworms' gland has uniform molecular weight and composition.6,45 Regenerated silk fibroin, on the other hand, is subject to variability based on the regeneration and processing conditions; specifically, degumming significantly impacts the molecular weight of the fibroin. This, in turn, affects properties such as the strength and modulus of any final constructs,46 while the methods used to make silk water insoluble determine the mechanical, chemical, and biological features of the final material.47–49
The water solubility of regenerated silk solid can be tailored to match a variety of processing needs. Native silk is water-insoluble due to its highly crystalline secondary structure; however, regenerated silk is readily soluble in water in its disordered conformation. Broadly, the processes that make regenerated silk water-insoluble are recrystallization and chemical cross-linking. The former, also referred to as physical cross-linking, is the reformation of the hydrophobic crystalline domains [β-sheets, Fig. 1(c)] and can be reversed by the same chaotropic agents used for dissolution; the latter is achieved by the formation of intra- and inter-molecular covalent chemical bonds or bioconjugate linkages that are generally irreversible.
Recrystallization occurs spontaneously under most conditions but can be greatly accelerated by the addition of organic solvents, including methanol48 and ethanol,545 heating,50 slow drying,47 sonicating,51 shearing,52 electric current,53 high salt concentration,54 chemical modification of tyrosine residues,55 and casting from formic acid solution.56 Any form of recrystallization done in the liquid state results in a silk gel, while those done in a solid state generally maintain the shape and form of the solid before recrystallization.46 If maintenance of the fine structure is needed, a common method of solid-state recrystallization is solvent-vapor annealing.49,197 In this process, silk is placed in a temperature-controlled chamber and exposed to water or alcohol vapors. Highly crystalline silk II structures can be obtained by using higher temperatures, longer treatments, and using methanol as the annealing vapor instead of water. To produce water insoluble silk I solids, water vapor and low temperature must be used (∼4 °C), otherwise the silk undergoes a transition to silk II. Intermediate temperatures (4–40 °C) result in a combination of silk-I and silk-II structures, while higher temperatures (>40 °C) produce crystalline structures purely in the silk-II conformation. Further increase in temperature merely increases the amount of crystallinity.49
Alternatively, chemical cross-linking can be performed to create an amorphous water-insoluble solid.546 A useful silk cross-linking site is the tyrosine residue, which is exceptionally abundant in fibroin when compared to most proteins, comprising 5% of all residues. Tyrosine can be dimerized by the action of enzymes such as horseradish peroxidase (HRP), mushroom tyrosinase, and laccase as well as certain species of photoactive compounds such as riboflavin and ruthenium (II) tris-bipyridyl dication.57 The backbone of silk can also be readily cross-linked by fixative aldehydes such as glutaraldehyde.58 Additionally, there are sufficient amine and carboxylate groups present to allow cross-linking with carbodiimides such as the water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).59 Finally, considerable research has focused on functionalizing silk with reactive groups that can work as cross-linking sites, such as methacrylate moieties for radical polymerization60 and azide-alkyne pairs for click chemistries.61
Fibroin yarns have found common usage in medicine as sutures for centuries. Results of allergenic and foreign-body responses to these sutures have shown these yarns to be highly biocompatible and immunologically comparable to other natural and synthetic suture materials.11 When considering the material more broadly, B. mori silk fibroin has also been consistently shown to be non-toxic, non-immunogenic, and fully resorbable with no harmful degradation products.62–65 Like other proteinaceous materials, silk and its breakdown products are potential type 1 allergens; there are, however, very few reported cases of allergic responses to fibroin, and thus, fibroin is considered to have low allergenic potential.66 As such, silk fibroin yarns have been used for making hypoallergenic garments aimed at relieving symptoms of diseases such as atopic dermatitis.67
In biomedical research, fibroin has been studied extensively and found to be well tolerated in various formats, implantation sites, and time scales. For instance, silk-based electrospun artificial blood vessel implanted subcutaneously in rats showed a mild inflammatory reaction.68 Over the course of 15 days, a fibrotic capsule formed around the implant and macrophages could be found inside the capsule; however, no lymphocytes could be found implying that the inflammatory response had not escalated to a systemic immune response. Additionally, host cells had managed to bypass the capsule and had homogenously colonized the implant. Fibroin hydrogels have also been reported to be well tolerated in the brain.69 After injection of a fibroin hydrogel in the mouse forebrain, motor and learning functions stabilized to baseline levels in 1 week. At no point were these functions significantly different from phosphate-buffered saline (PBS) controls; additionally, CD45 positive cells indicative of inflammation returned to background levels in 4 weeks. The long term tolerability of fibroin implants has also been experimentally validated.70 In this case, a series of scaffolds with variable porosity and processed either in water or HFIP were implanted in rats and observed different time points up to 1 year. Inflammatory marker expression levels (IL-4/6/13, TNF-α, IFN-γ) were elevated in all scaffolds after 8 weeks but diminished as the degradation proceeded. After 1 year, all water-processed scaffolds degraded completely, most HFIP processed scaffolds were significantly eroded, and the expression for most markers was below detectable levels in fully degraded scaffolds, indicating no prolonged immune reaction from the degradation products.
Sericin is the second most prevalent component in silk cocoons, and it is historically considered as having high allergic potential. Native silk (sericin-containing) sutures caused moderate to severe inflammatory responses and allergic reactions, which were mitigated by using degummed silk.11 Early immunological experiments seemed to indicate this was from proteins in the 20–220 kDa range, thus identifying sericin.12 Newer studies with purified and isolated sericin suggest that this protein does not cause inflammation71 and possesses a similar level of biocompatibility to fibroin.72 These older results could be explained by the presence of an immunogenic contaminant in the sericin, possibly lipopolysaccharides (also called endotoxins), that were subsequently removed during degumming along with the sericin.72 These newer revelations have led to a revitalized effort to explore the potential uses of sericin in cosmetic73 and biomedical applications, such as therapeutic nanoparticles,74 wound dressings,75 and neuron-loaded hydrogels for nerve repair.76
Silk fibroin readily biodegrades in vivo, and its degradation time can be tuned from seconds to years. The in vivo degradation time for silk materials varies by format and processing and, within each format, by crystallinity.77 Amorphous or non-crystalline silk remains water soluble, so implantation of any such silk causes the implant to undergo near-instant structural degradation of the fibroin components. This fast degradation can be exploited to create transient mechanical supports, such as bioresorbable adhesives79 and dissolvable substrates for highly conformal electrodes.78 In this latter case, the silk support mechanically stabilized flexible metal/polyimide electrode meshes as they were implanted into the brain; upon contact with the wet brain tissue, the silk fibroin reinforcement dissolved and allowed the electrodes to freely conformally adhere to the contours of the brain. Silk's rapid dissolution also increased the efficiency of transdermal drug delivery in microneedle patches.80 Here, the non-crystalline fibroin-based microneedles were sufficiently strong and stiff to penetrate murine skin and dissolved within 60 s in a gelatin skin model. When loaded with insulin, the encapsulated insulin retained 78% relative bioavailability and 82% relative pharmaceutical availability compared to insulin administered by intraperitoneal injection. The silk remained water soluble and the microneedles delivered upwards of 99% of their cargo after 20 days of dry, room-temperature storage.
The ability to control the silk degradation time allows tuning of the overall silk-based device functional lifetime, which can span from a few minutes to a few years.81 Fibroin that has been recrystallized does not instantly degrade, and the degradation rate is controlled by numerous factors, including the degree of crystallinity, the polymorphism of crystal, and the meso-/micro-structure of the solid. As the degree of crystallinity can be easily tuned, this property has been used to program the degradation of various transient optical devices, with higher crystallinity leading to longer degradation times.77 Some noticeable examples include silk implants that can naturally be proteolytically cleaved after delivering therapeutics82 and silk-based quick-response (QR) codes that can be safely discarded after use without negatively affecting the environment as humidity degrades both the encrypted message and the substrate itself.83 Despite this, other applications find altering crystallinity to be an undesirable method of tuning degradability since low crystallinity silk typically spontaneously crystallizes with time,84 and the strength and modulus of the material also depend on this parameter. The degradation time increases as the enzyme/cell access to the silk structure is restricted, so materials with higher density and thickness and lower porosity have a longer functional lifetime.85 Silk degradation rates can also be tuned by blending it with other polymers; slower rates are observed for the incorporation of more stable polymers such as polycaprolactone,86 while faster rates for the addition of more labile polymers such as collagen.87 Finally, the degradation time can be increased by covalent cross-linking techniques that create highly stable bonds such as genipin88 or carbodiimides.89
Native and regenerated fibroin is primarily degraded in vivo by the action of general proteases such as proteinase K and trypsin-like proteases90 and has relatively few recognition sites for more specific remodeling proteins such as matrix metalloproteinases (MMPs). Additionally, these MMP-cleavage sites are hidden in the dense hydrophobic crystalline domains and are, thus, inaccessible to MMPs.90 To improve specific remodeling, these cleavage sites can be added into silk fibroin structures by the addition of MMP cleavable peptides that can physically cross-link with the fibroin91 or fibroin may be blended with a polymer such as gelatin that naturally contains a high abundance of these sites.92 Both methods resulted in much faster in vivo degradation of the composite structures. Alternatively, the relatively few MMP sites can be digested more rapidly by active induction of macrophages to secrete more remodeling proteins. This strategy was demonstrated by mixing silk with the lipophilic molecule 4-hexylresorcinol (4HR), which preferentially bonded to the hydrophobic crystalline domains of the silk.93 This molecule was shown to induce increased expression of various MMPs from macrophages in vivo, resulting in faster degradation of native silk sutures. After implantation in a mouse model, 4HR-treated silk sutures lost 40.5% of their total mass in 11 weeks, compared to untreated silk sutures which lost only 8.5%.
E. Silk formats
Silk fibroin can be converted into many different formats, each through a specific set of processing methods94 driven by targeted functions for various applications. Among the many investigated silk forms, the most relevant ones from an engineering point of view are discussed here with a focus on the properties associated with the specific transformation of the material.
Fibroin films [Fig. 2(a)] can be optically clear, flexible, and tough, making them excellent substrates for 2- and 2.5D technologies. Silk films can be fabricated by spin coating,95–100 spray coating,101,102 drop casting,78,100,104–106 and dip coating.103,107 Most silk fibroin films are formed in a disorganized and water-soluble state, which is desirable for transient applications. Fibroin films can also be made insoluble with a variety of techniques, with vapor annealing being the most common as it maintains the clarity and the surface topology of the film.49 The films surface roughness can be tailored to the specific application with higher surface roughness usually desirable to increase cells adhesion on silk films, while a lower roughness generally improves technical functions such as optical properties or suitability for deposition/device transfer. Techniques, such as methanol exposure and incubation, can tune the surface roughness from less than 1 nm to above 75 nm root mean square.108,109 If films are cast or coated onto a surface that is both textured and wettable, the texture can be conformally transferred to the surface of the film, making it an easy method to create nano- and micropatterned substrates.110–112 Films can also result from drying silk hydrogels formed via electrogelation, a process that can be exploited to create arbitrary 3D structures.113 Solid silk films can subsequently be processed to obtain complex architectures through a variety of techniques, including dip-coating, transfer printing,78 screen printing,114 embossing,111,115 and photolithography.116–119 In the case of photolithography, silk polymorphism allows for the fabrication of both positive and negative patterns in 2D film formats starting from a single material, as silk natively acts as a resist for electron beam (e-beam) and deep-UV lithography techniques. Unmodified crystalline silk fibroin has a strong absorbance near 200 nm,120 which can be excited by an argon fluoride eximer laser (λemission = 193 nm) to cause photodegradation of the exposed regions, which then become water soluble yielding a positive tone resist.119 Similarly, crystallized silk films can be made soluble in selective regions by e-beam exposure (100–125 keV, 2250 μCcm−2) and removed with pure water. A water-soluble film may also be treated by e-beam with higher dosages (25 000 μCcm−2) to insolubilize the exposed region for a negative tone resist.116
2. Micro- and nanoparticles
Fibroin micro- and nanoparticles [Fig. 2(b)] can be fabricated through techniques such as spray-drying,121,122 milling,123,124 dissolution/sonication,125 microfluidics,126–128 and phase separation.129–131 Silk-based particles make excellent candidates as vehicles for drug delivery, targeted therapies, and imaging contrast agents (Sec. VI). Additionally, composite nanoparticles can be produced forming, for instance, core–shell structures that can exploit the biofriendly nature of silk to minimize the toxicity of cargo and serve as a platform for further modification.132–136
Silk scaffolds comprise hydrogels and solid materials that can act as support matrices for the development of tissues and their structure can accommodate a second material (e.g., dyes, growing cells, or therapeutics). Silk sponges are solid types of scaffolds with a high degree of porosity [Fig. 2(c)] that are fabricated by combining porogens with silk solutions, followed by the removal of the porogens, which leaves air voids.137 Water soluble solids, such as salts138 and sugars,139 and polymers, such as polyvinyl alcohol, can easily be sifted to select a particular pore size and are all suitable as sacrificial porogens for silk scaffolds. The presence of these hygroscopic materials is often sufficient to initiate recrystallization if high MW silk is used; otherwise, alcohols can be used to insolubilize the structure. After the physical cross-linking of the silk is completed, these porogens can be removed by immersion in water. Ice can also be used as a porogen for silk as the ice crystals size can be controlled by the freezing rate and their aspect ratio can be increased by directional freezing. The ice can then be removed by lyophilization and the resulting silk scaffold turned insoluble by vapor annealing or alcohol/solvent curing.140 Similarly, polymers, such as polystyrene and poly(methyl methacrylate) (PMMA), can be used as porogens for silk scaffold,141,142 as they can be easily removed using solvents, such as toluene, that do not dissolve or degrade fibroin. Depending on the porous silk scaffold fabrication process, porogens can be either randomly dispersed through the silk block or arranged in a regular lattice leading to, in the latter case, the fabrication of silk photonic crystals that can be used, for instance, as matrices for drug delivery143 or for tissues regeneration.141,142
Raw fibroin fibers may be used directly after degumming as traditionally done in the textile industry and for the production of sutures.144 However, the production of silk fibers from the regenerated solution offers finer control over the final fiber morphology as well as over the fibers' properties. Regenerated fiber production is commonly carried out via wet spinning, dry spinning, or electrospinning. If the regenerated fibroin solution is expelled into a solution that causes silk's recrystallization, continuous hydrogel is formed in a process known as wet spinning.145–151 The crystalline hydrogel retains the nozzle cross-sectional shape and dimension and can be dried to yield a continuous fiber. Alternatively, a highly concentrated solution can be extruded directly in air to produce a fiber, which must then be dried.2,4,152,153 In this dry-spinning process, the final fiber morphology is largely determined by the drying conditions and by the composition of the spinning solution. Finally, aqueous solutions of silk fibroin, silk fibroin and PEG, or silk fibroin in formic acid can be electrospun by applying a large electrical field (E > 500 V/cm) between a silk-laden emitter and a conductive collection plate.60,68,154–159 The electric field stretches the fiber and causes it to rapidly dry in flight to produce fibers with a small diameter (Ø < 1 μm), depending on viscosity and electric field strength, often in a nonwoven mat arrangement [Fig. 2(d)].160,161
Hydrogels are water-based cross-linked 3D formats formed by cross-linking silk fibroin while still in solution, trapping the water in the silk matrix [Fig. 2(e)]. They typically contain between 1% and 30% solid content by weight. The low density and generally soft nature of these materials combined with their biodegradability make them ideal for biosensing and engineering of soft tissues.162–166 Similar to fibroin film fabrication, it is possible to generate hydrogels with high optical clarity that can be used for biological optical applications such as corneal augmentation.167–170 If loaded with electrolytes, these materials readily conduct electricity and, thus, make attractive substrates for sensing and ion-based electronics.171–173 By exchanging the water in a silk hydrogel with a solvent such as ethanol, critical point drying can extract the liquid and form an ultralow density aerogel structure.174,175
Silk's selective gelation enables the 3D printing of silk fibroin hydrogel constructs. Controlled gelation can be achieved in several ways, including gelling baths, light-mediated gelation, and mixing with secondary cross-linkable polymers. Gelling-bath 3D printing rapidly forms hydrogels by exploiting the interaction of fibroin solutions with solvents, such as methanol176 and concentrated salt solutions.177 Specifically, the aqueous silk ink is extruded into a solution, where it gels rapidly after exiting the nozzle and can quickly retain its shape and support subsequent layers. The gelling bath can alternatively act enzymatically, as the silk ink can be mixed with an enzyme, such as HRP and printed into a bath of dilute hydrogen peroxide, which HRP reacts with to catalyze the tyrosine-tyrosine cross-linking of the fibroin.178 Slower methods of gelation, such as spontaneous gelation, can be used if the fibroin is printed into a laponite clay/PEG suspension, which provides mechanical support and confinement for the fibroin ink during gelation.179 The laponite/PEG mechanical support also allows completely freeform printing of silk as no support structures are needed for the printing process itself. Light-mediated 3D printing techniques are compatible with fibroin, as native fibroin can be selectively cross-linked by photooxidative molecules such as riboflavin180 or ruthenium.181 Alternatively, silk fibroin can be chemically functionalized with radical-polymerizable groups like methacryloyl moieties,60 or thiol-ene click pairs182 and, when mixed with a photoradical generator, can be polymerized by exposure to UV–light. Finally, the fibroin can be mixed with other polymers, such as alginate,183 gelatin,184 and methacrylates,185 to access alternative gelation strategies of ionotropic, thermal, and photoradical gelation, respectively. Alginate composites have an additional benefit in that the fibroin can be independently cross-linked by enzymatic action after printing and the alginate extracted via chelation to leave pure fibroin structures. Noteworthy usages of these 3D printing techniques include bioprinting, hierarchical material fabrication, and 2.5D printed silk-composite leathers (Secs. VI D and VIII C).
6. Bulk solids
Bulk solid silk structures are usually obtained by drying silk hydrogels causing them to undergo a gel-solid transition and become homogeneous, transparent, and amber-colored solids [Fig. 2(f)].46 The hydrogel may be molded and result in a solid of the same shape, however, as these solids are formed from hydrogels that generally contain upwards of 70% water by mass, substantial shrinkage must be accounted for [Fig. 2(g)].46 Once solidified, the solid can achieve compressive strength up to 225 MPa, a compressive modulus up to 6 GPa, and flexural shear stress at failure up to 120 MPa.46 Applying these mechanical attributes to biomedical devices, silk solid cylinders were machined into screws that achieved pullout forces from bone phantom up to 160 N for #1–72 sized silk screws. As with other silk formats, silk solids demonstrate physical properties that vary with the degree of degradation of the protein, with mechanical properties such as strength decreasing with decreasing molecular weight. Bulk silk solids have thermoplastic properties similar to silk films, so heat and pressure can be used to imprint microstructures on the scale of micrometers at a temperature of 60 °C and can also experience plastic deformation when static loads are applied at temperatures above 80 °C. These silk solids can store and stabilize biomolecules (such as HRP) as is the case with other solid silk formats. As alternative processes to achieve silk solids, degummed silk fibroin can be lyophilized, redissolved in HFIP, and cast to form bulk solids,187 and both degummed silk fibroin as well as silk containing sericin can be dissolved, lyophilized, and then hot-pressed into similarly engineered solids.188,189
Regenerated silk fibroin has been successfully used as the building block for the fabrication of optical devices due to the combination of silk's optical and chemical properties (Table I). In particular, silk's high transparency in the visible range32,33,190 combined with its abundance of functionalization sites and the easy room-temperature aqueous processability enabled its widespread use in the optics and integrated optics field. Silk's low roughness, nanoscale processability, and mechanical durability further allowed the fabrication of silk-based optical devices with nanoscale resolution. Notably, silk aqueous solution makes it ideal to be used as an active matrix to disperse secondary polar materials, therefore, expanding the functionality of silk-based devices with the introduction of additional responses, such as plasmonic resonances191 and fluorescence,116 as well as both organic and inorganic functional molecules.192
This section provides an overview of the most recent silk-based optical devices. In particular, it discusses devices in which the combination of the silk form and its function allows expanding the usability of established optical platforms to other fields, such as the biomedical and encryption worlds (Fig. 3). Silk's versatility arises from leveraging conformational changes and functionalization with additional materials. Silk's nanoscale conformability, aqueous processability, and thermal and enzymatic degradation enable its use also as a sacrificial template193 for the fabrication of sophisticated and complex three-dimensional optical devices, though these applications will not be treated in depth here.
A. Photonic structures
The use of silk fibroin for photonic devices has been pioneered by the fabrication of a wide range of standard optical components by simple yet effective templating and patterning of silk films at the micrometric and nanometric scale. The fabrication of silk-based devices, including microlens arrays,190 hydrogel lenses,194 zone plates,195 waveguides,196 as well as diffraction gratings,197 demonstrated the use of silk as a technological optical material and led to the implementation of more complex and multifunctional optical platforms that promoted the substitution of glass and synthetic-polymers optical components counterparts. Nowadays, silk fibroin is used to fabricate photonic structures that generate an optical response as a consequence of light interference with the nanostructure in which silk is molded; silk's polymorphism enables the photonic devices to be responsive (e.g., to humidity and gases) and variable in time and their variability can be directly quantified through measurable shifts in their optical response.198
1. 1D photonic crystals
Silk fibroin can be efficiently assembled into one-dimensional (1D) photonic stacks in which, for example, the alternation of pure silk and silk doped with titanate nanosheets provides the necessary index contrast to have selective reflection (Δn = 0.26, nsilk = 1.56, ntitanate = 1.82, at λ = 500 nm).199,200 Silk's inherent response to external stimuli, such as water, causes variations in the stacks that affect the photonic device optical response; thus, changes in structural color can be used as the transduction mechanism200–202 (see Sec. V E). For the silk/silk-titanate, 1D stack changes in relative humidity (RH) from 10% to 80% affect the photonic stack by swelling the silk layers due to water uptake, therefore, inducing a reversible redshift and a bandwidth broadening of the transmitted light that can be traced to the relative humidity surrounding the device.200 Silk fibroin has also been used to modulate the self-assembly of colloidal systems, such as cellulose nanocrystal suspensions, by affecting the electrostatic interactions underlying the helicoidal self-assembly process and ultimately tuning the spectral response of the obtained 1D photonic crystals.203
2. 2D photonic crystals
2D silk photonic crystals with both positive and negative patterns have been fabricated by using all-water based electron beam lithography by leveraging silk's polymorphism116,204 [Fig. 4(a)]. In addition, the variation of the lattice constant (Λ = 500–700 nm) combined with different effective refractive indexes of the positive and of the negative patterns, led to structural coloration spanning from dark blue to yellow. Notably, features down to 30 nm in diameter were fabricated, with the resolution of the lattices being determined by the silk's molecular weight and crystallinity degree; specifically, higher molecular weight led to better mechanical performances while crystalline silk resulted in higher resolution, due to the lower amount of bound water. 2D silk aperiodic crystals (quasi-crystals) have also been fabricated using electron beam lithography205 and transfer imprinting;206 in this case, the presence of critical modes generated by the aperiodic pattern allowed for increased sensitivity compared to 2D ordered crystals up to the detection of single protein monolayers.
3. 3D photonic crystals
3D photonic crystals based on silk fibroin have been fabricated using a combination of templating and self-assembly manufacturing techniques. Silk inverse opals (SIOs) were first fabricated by casting silk solutions on 3D fcc scaffolds of PMMA spheres, followed by the removal of the synthetic scaffold by dissolution.191 By using spheres with diameter ∼250–350 nm, the so-formed SIOs displayed a partial photonic bandgap (pPBG) which generated reflection in the visible range191 [Fig. 4(b)] and which could be tuned during the fabrication process via engineered defects insertion207 and through variations in the periodic unit leading to SIO superlattices.208 Alternatively, the addition of stilbene to silk fibroin during the inverse opal fabrication process,191 followed by cross-linking, led to a bendable and conformable silk hydrogel inverse opal.209 This is a straightforward strategy to create mechanically responsive and biocompatible 3D photonic crystals that can be interfaced with in vivo tissues, e.g., used for ocular implants. The polymorphic state of silk determines the variability of the photonic lattice; in contrast to SIOs fabricated with silk in the crystalline phase,191 SIOs made with silk in the amorphous phase showed additional functionality by being spatially programmable to display multiple photonic responses within the same material by locally inducing structural changes in the silk protein. This photonic reconfiguration can be introduced by exposing silk to either a water vapor (WV)/methanol rich atmosphere (controlled crystallization) or to UV radiation (controlled degradation) through shadow masking198 (Fig. 3). Notably, this patterning methodology is high resolution, contact-free, and highly beneficial to promote the use of SIOs for optics-based biomedical platforms due to the compatibility with biological water-based environments and the low UV radiation, respectively. Local variations of silk's conformation led to variations in the SIO lattice and thus in the pPBG. In particular, exposure to WV for a few seconds (1–5 s) induced irreversible silk conformational rearrangements from random coil to β-sheets that lead to the formation of physically cross-linked crystalline domains; this shrinking was observed to be uniform but anisotropic, being limited to the vertical direction  of the opal. For instance, a yellow reflecting SIOs (λreflectance = 590 nm) exposed to WV, progressively shifted its reflection to green (λ = 530 nm), blue (λ = 450 nm), and violet (λ = 385 nm), for exposure times of 1, 3, and 5 s [Fig. 4(c)]. Exposure to low dosage UV radiation (λ = 254 nm, intensity = 76 μW cm−2) leads to a similar blueshift of the pPBG, though over a much longer timescale (0.5–3 h); this photonic variation is induced by a non-uniform anisotropic shrinkage of the opal due to the photodegradation of the silk fibroin, where the outer layers of the SIO are more exposed to the UV source and, therefore, more affected than the internal ones.198
SIO's photonic polymorphism allows for the design of biological materials for encryption-based applications that often require local patterning along with reconfiguration capabilities. These can be obtained by combining preassembled reconfigurable (amorphous) SIOs with inkjet printing.83 The local patterning is achieved by inkjet printing solvents that lead to the dissolution of the synthetic opal, therefore, creating a material where the structural response is present only in specific regions (Fig. 3). The spectral reconfiguration is obtained, instead, by directly inkjet printing MeOH/water on SIOs to induce silk fibroin local uniform shrinking.198 By doing so, it is possible not only to increase the complexity of the photonic response (by adding location-dependent individual spectral responses), but also to encode information into the material by digitally defining multispectral lattices, thus leading to a truly multifunctional silk-based device for encryption, storage, and security applications.83 One notable example of this technology is the fabrication of environmentally dependent and multilevel encryption quick-response (QR) code-based optical devices [Fig. 4(d)]. In this case, the first encryption layer consists of the QR code itself, created by patterning the direct opal with inkjet printing, thus locally removing the photonic crystal to generate the macroscopic pattern. The second encryption layer consists of a 3-digit structural color key obtained by tuning the spectral response of the SIO by exposure to WV for various times as to generate three regions with progressively blue-shifted structural color with respect to the untreated regions of the SIO which can be read as differences in RGB intensities. In addition to these functionalities, the QR code can be programmed to be an environmentally dependent encryption device, simply by tuning the cross-linking degree of the silk fibroin, so that it degrades after a specific time or upon exposure to WV.
The utility of SIOs can be further expanded by integrating additional optical effects either by partially changing the form of the SIO at the microscale or by directly incorporating another optical function in them by doping with optically functional elements. This ability to create devices with co-located functions and optical interplay is demonstrated by hierarchical opals (HOPs) in which 2D microscale and 3D nanoscale patterns are co-located within a single biocompatible structure.210 The conformal self-assembly of SIOs (3D photonic structure) on pre-patterned diffractive 2D optical elements (DOEs) was obtained through a combination of self-assembly, layer-by-layer assembly, and top-down transformation techniques. In terms of optical interplay, the far-field intensity distribution of HOPs depends on the DOE properties (surface periodicity), while the reflected and transmitted contributions depend on the SIO's properties (lattice periodicity, effective refractive index, number of layers). In particular, when the SIO's stop band matches the laser wavelength used to illuminate the HOPs, the SIOs increases the DOE's reflected far field diffraction intensity proportionally to the number of layers of the SIOs, while the DOE's topography decreases the angular-dependence of the SIO's reflected color.210 These structures with coexisting optical responses can, thus, be used to create sensing devices with a dual response such as a pPBG shift associated with variations in the SIOs and a far-field diffraction pattern related to the DOE periodicity and integrity.210
Metallic nanoparticles [Figs. 4(e) and 4(f)] and fluorescent dyes are among the most notable examples of optically functional dopants for SIOs,191,211 whose signal can be strongly enhanced by the careful design of the SIO. In particular, by designing SIOs so that their pPBG matches with the absorption range of gold nanoparticles, it is possible to control the material absorption (λabsorption ∼ 545 nm) and promote local plasmon resonance-induced heating; for instance, green reflecting SIOs (λreflectance = 546 nm) show a relative increase in temperature of ∼20 °C compared to blue-reflecting SIOs (λreflectance = 512 nm).191 Similarly, SIOs have been reported to enhance the fluorescence of a dye that they are doped with. To achieve this effect, it is necessary to tune the pPBG of the SIO so that λpPGB > λemission. In the case of SIO (λreflectance ∼ 885 nm) doped with Rhodamine 6G (λemission = 553 nm), the enhancement factor increases with the number of layers forming the SIO and can reach up to 50 for a 10-layers SIO, which is particularly suited to increase the energy conversion efficiency of solar cells.211
Silk's array of functionalization sites offers opportunities to employ SIO as direct sensors with analyte specificity. Binding events of the molecules of interest within the nanostructures, previously properly functionalized to display the required binding sites, results in a change in the effective refractive index of the SIO which produces a spectral variation of the reflected signal, thus working as a colorimetric sensor for which the transduction mechanism is based on variations of structural coloration. This strategy has been explored in the context of antibody–antigen interactions,212 as discussed more in detail in Sec. V.
B. Diffractive optical elements
Silk conformability at the micrometric and nanometric scale and excellent optical transparency in the visible range allows also for the fabrication of diffractive optical elements (DOEs), whose simplest form is linear diffraction gratings; DOEs project a far-field pattern when illuminated by monochromatic light, and their diffraction efficiency depends on the pattern design (i.e., critical dimension and height of gratings) as well as on the refractive index of the grating and of the surroundings. Starting from a liquid silk fibroin solution, soft-lithography approaches enable the fabrication of free-standing silk DOEs with low roughness (<10 nm rms) and control of the pattern morphology down to 125 nm.190 Alternatively, silk gratings can be fabricated directly on solid silk films exploiting thermal reflow, which uses a combination of heat and pressure to imprint periodic nanostructures on nanometric-smooth silk monoliths.46
Both monochromatic and multichromatic (MC) DOEs have been successfully fabricated with silk. For the former, a single pattern is stored in the DOE, while for the latter, multiple patterns (commonly referred to as channels) can be imprinted in the silk film, each readable by a laser with a different wavelength, allowing multilevel encryption.82 MC-DOEs operate at multiple wavelengths either independently or synergistically, enabling independent spatial and temporal manipulation of information in terms of far-field pattern. From a spatial point of view, arbitrary patterns can be imprinted in MC-DOEs enabling different shape and intensity of the far-field diffraction patterns; while from a temporal point of view, each channel can be programmed to have different crystallinity, thus making the projected pattern temporally dependent. In particular, the lower the crystallinity of the channel, the faster the degradation, thus various information can be preserved for specifically tailored times82 (Fig. 3).
Therefore, silk fibroin DOEs allow for information manipulation through three different modalities: information concealment, information re-appearance, and information destruction. For the former two operation modalities, silk is used as a programmable and tunable sacrificial coating of a second DOE element with a similar refractive index, while for the latter, silk forms the DOE itself.82 Information destruction and revealment can be done stepwise meaning that by spatially tuning the crystallinity of the silk, only specific channels can be deactivated/exposed at a certain time [Figs. 4(g) and 4(j)]. Information concealment works, instead, by covering a DOE engraved in glass with a uniform layer of silk; the refractive index matching between glass and silk (nglass ∼ 1.52, nsilk = 1.54) nullifies the DOE effect, thus protecting the message concealed in it [Fig. 4(h)]. The message can be subsequently revealed either directly, by washing away the silk layer, or remotely, by tuning the silk crystallinity as to program its degradation time214 [Fig. 4(i)].
Silk DOEs can also be used to spatially and temporally modulate electronic circuits provided these are fabricated with an electroactive component, such as cadmium sulfide photoresistors, and are coupled to a silk DOE. Upon illumination of the circuit by shining on it the DOE-produced far-field pattern, the photoresistor resistance can, thus, be locally modulated.215 This approach can be used to either regulate the overall voltage output of the circuit or, in a more sophisticated approach, to reconfigure the circuit design by spatially activating only specific regions through the precise redistribution of light on the circuit [Fig. 4(j)]. In both cases, the photoresists illuminated by the DOE undergo a decrease in the resistance from MΩ to kΩ and thus switch from the off state to the on state. This strategy enables embedding within a single circuit different operation modes (e.g., adder and integrator) that can be independently activated using the silk DOEs, and it constitutes an example of a wireless, optically activated reconfiguration device.215
Dynamic diffraction gratings have also been reported for bilayer PDMS-silk systems able to form a microscale wrinkle pattern during the cooling step of the fabrication protocol by exploiting variations in the mechanical properties (elastic modulus) of the two constituent materials. Specifically, the programmed wrinkle pattern can be dynamically controlled by exposure of the structure to water/methanol vapors, UV radiation, or by electricity-induced local heating; the molecular rearrangement of silk releases the compressive stress accumulated in the wrinkles and makes the bilayer transition to a wrinkle-free and more transparent structure.216
As previously discussed for 3D photonic crystals, silk-based DOE can also be engineered to display multifunctional optical responses by, for instance, coupling with a fluorescent dye,217 conformally growing a SIO on it,210 or by using silk-azobenzene for a photoswitchable optical response, which also leads to an increased diffraction efficiency for higher laser pump intensities.218 Some critical applications of silk DOEs can be found in relation to sensing and implantable optical devices as discussed in detail in Secs. V E and VI B. In both cases, silk's ability to embed active molecules within the bulk of the DOE device while retaining their activity has turned out to be crucial.214 Both application fields are based on variations of the medium surrounding the DOE (e.g., from air to glucose204) and/or on variations of the DOE composition and/or structure (as a consequence of water-induced swelling214 or bonding to another chemical/biological species82) that induce an angular redistribution of the scattered light for all grating orders, thus altering the far-field diffraction pattern and creating a label-free, structural color-based transduction mechanism.190,204,214
C. Whispering gallery mode microresonators
Optical microresonators, or optical microcavities, are ubiquitous in modern optics as they can significantly reinforce light–matter interactions; they have been developed in many forms such as Fabry–Pérot etalons, photonic crystals, or whispering gallery mode (WGM) resonators. In particular, WGM resonators are an emerging class of optical microresonators capable of tightly confining light beams into axially symmetric microstructures, which show exceptional properties, such as high quality (Q) factors (∼109) and small mode volume.219 WGM resonators have been studied as outstanding platforms for lasers,220,221 sensors,222–224 and nonlinear optics.225–228 For these applications, the cavity host is generally made of inorganic compounds, including III–V semiconductor materials and silicon compounds.229 Though inorganic materials are compatible with semiconductor processes that provide reliable and precise results, they are usually not flexible, biocompatible, and biodegradable, thus limiting the use of WGM resonators in biological regimes. To address these challenges, WGM resonators made from biocompatible and biodegradable materials have been suggested in the recent decade.222,230 Among many available biomaterials, regenerated silk fibroin has been demonstrated to be an attractive candidate for all protein-based WGM resonators with excellent resonance performance due to silk's high refractive index (n ∼ 1.5432,33,190) mechanical strength, and broad compatibility with existing fabrication methods. These factors improve total internal reflection of light inside the WGM cavity and enable various WGM resonator shapes with perfect circularity and atomic surface smoothness.
On-chip silk microtoroids have been traditionally fabricated using solution casting techniques222 [Fig. 5(a)]. Silk microtoroids with a diameter of ∼100 μm can have a Q factor as high as 105 [Fig. 5(b)], proving that they can sufficiently replace inorganic microtoroids.229 Due to silk's large thermal expansion coefficient, silk microtoroids could be efficiently utilized in thermal sensing applications with sensitivity is as high as -1.17 nm/K, which is about eight times larger than that of silica-based WGM resonators.231–233
Facile functionalization of silk fibroin with optical gain dopants offers an opportunity to expand the use of high Q silk resonators to lasing applications [Fig. 5(c)], as recently reported for free-standing silk microdisk lasers functionalized with Rhodamine 6G (Rh6G) and Rhodamine B (RhB) dyes, respectively.221 To fabricate silk WGM microlasers with a smooth convex profile, silk droplets were placed under an incandescent lamp to accelerate the evaporation rates, thereby suppressing the coffee ring effect. Notably, RhB doped silk disks with a diameter of 500 μm showed narrow lasing emission above the threshold pumping energy density of 10.8 nJ/μm2 [Fig. 5(d)]. As the diameter of the silk microdisks decreased (700, 500, and 250 μm), the dominant emission peak shifted toward lower wavelengths and lasing thresholds were increased.
1. Metal-insulator-metal resonator based on Fabry–Pérot etalons
Metal-insulator-metal (MIM)s resonators based on Fabry–Pérot etalons are resonators made of an insulating layer sandwiched between two parallel planar metal mirrors. In the MIM resonator, incident photons are reflected between the metal mirrors and then strongly confined into the structure. MIM resonators optical resonances can be easily manipulated by changing the gap distance between the mirrors and the refractive index of the insulating sandwiched layer. Due to responsive optical performance and cost-effectiveness, MIM resonators are being spotlighted in biological and chemical sensing.234,236–238
The use of silk as the insulating layer allows overcoming some of the limitations of conventional MIM resonators, which are usually based on rigid materials for the insulating layer, and whose physical properties (e.g., refractive index and film thickness) are not easily changed by analytes bonding, therefore, limiting the sensing capability. Once again, silk's tailorable mechanical properties as well as optical transparency contribute to the development of highly responsive and biocompatible MIM resonators, which would, otherwise, be difficult to obtain with conventional materials.
One notable example of silk's versatility for MIM resonators consists of transmission color filters made of ultrathin (90–150 nm) crystalline silk films sandwiched between two parallel optically thin silver layers.234 These silk MIM resonators were fabricated using traditional fabrication techniques, yet showed additional functionality due to the presence of a silk hydrogel as an insulating layer. As the insulating film thickness has a dominant influence on the resonance wavelengths, the fabricated silk MIM resonators serve as humidity-responsive bandpass filters due to silk's intrinsic ability to absorb water and reversibly change the layer thickness. Specifically, the exposure of the MIM color filters to a wet environment caused water absorption in the silk hydrogel, which increased the insulating layer volume and redshifted the resonance wavelength; practically, the filter transmitted light progressively switched from blue to green, and red [Figs. 5(e) and 5(f)].
Similarly, silk MIM resonators could serve as color absorbers,238 by fabricating an asymmetric resonator with one of the two parallel metal mirrors being optically thicker than the other to block the light transmission. Due to this unique structure, the incident light can be localized and strongly absorbed in the silk insulating layer. Changes in the thickness and the refractive index of the silk layer induce variations in the resonance wavelengths. Similar to the Fabry–Pérot interferometer, the optical responses of the silk MIM resonator were tuned with the angle of incident light relative to the resonator surface. The silk MIM color absorbers were used as a refractometric sensor to detect glucose concentrations.
D. Plasmonic silk structures
The combination of metallic materials with regenerated silk fibroin has been long-exploited due to the ability to integrate form and function, whereby silk fibroin has been often used as a sustainable, biodegradable, and biocompatible scaffold while the metallic nanoparticles provided the plasmonic response. As a scaffold, silk fibroin is highly promising due to its polymorphic behavior and flexibility when molded in 2D/3D structures; this allows metallic nanoparticles to be dispersed in non-rigid formats, such as films,191,239 hydrogels,240 and aerogels.241,242 The plasmonic response can be introduced in silk-based optical devices by doping silk fibroin with metallic ions,201 directly mixing silk fibroin with metallic nanoparticles,191,242 by depositing tens of nanometer-thick metallic coatings (e.g., Ag/Au film) on pre-assembled optical devices,191,239 or by nanotransfer printing.206,243 Quite a few hybrid silk-plasmonic devices have been fabricated using gold as the metallic component due to its biocompatibility and strong affinity for silk's amino acids which leads to fully biocompatible devices with good adherence between gold and silk.206,243
Silk-plasmonic materials have been mostly fabricated with sensors and photothermal heating effects as target applications. A notable example consists of the combination of a 3D photonic crystal, silk inverse opals, with a 2D plasmonic structure, a nanometric (70 nm thick) conformal silver layer, to obtain a refractive index sensor with a sensitivity of 200 000 nmΔ%T/RIU.239 By taking into account variation in the spectral response given both by the photonic and the plasmonic crystal a higher sensitivity is obtained compared to a purely plasmonic or purely photonic system.239 For photothermal applications, one of the simplest yet most versatile strategies consists of using silk-Au nanoparticle inks to print functional patterns whose thermal response can be finely tailored by varying the number of layers of the ink.244 In addition, Au nanoparticle doped aerogels and SIOs locally heat up due to the photonic crystal-induced enhanced absorption.191,239,242 Due to silk's biocompatibility and degradability, these hybrid gold SIOs can be used in medical devices that help in reducing the infection of inflamed tissues through localized heating.191
E. Silk optical waveguides
Biocompatible and biodegradable optical waveguides have been of strong interest in bioimaging and therapy fields due to their ability to deliver light into a small volume within the tissue. Conventional optical fibers made of glass have offered a method for the delivery of light into the body with low absorption and scattering loss. Though glass waveguides have many benefits, such as biologically inertness and cost-effectiveness, their physical properties, such as stiffness and non-biodegradability, are not suitable for tissue and in vivo applications. In this regard, flexible, biocompatible, and biodegradable optical guides with low propagation loss are required.
Silk fibroin has been investigated as an attractive material for biocompatible optical waveguides due to its superior properties, including biodegradability, optical transparency, and mechanical strength. In addition, silk's refractive index in the visible range (n ∼ 1.54) ensures light guiding in water-based environments (n ∼ 1.33) having a higher refractive index than the surrounding. To deliver light with low propagation loss, the silk waveguides should also retain uniform transverse dimensions and display a defect-free surface. The direct-ink writing technique was found to be ideal to fabricate waveguides with such properties, as it allowed the printing of silk optical waveguides into programmed patterns on glass substrates196 and the stabilization of their shape by exposure of the printed guide to a methanol-based environment; this induced rapid crystallization and thus a quick structural transition from random coils to stiff β-sheets. The developed silk waveguides show, therefore, consistent transverse dimensions, lack of discontinuities, and smooth surfaces. Both optical properties and geometrical features contribute to optical loss as low as 0.25 and 0.81 dB/cm for straight and curved silk waveguides, respectively [Fig. 5(g)].
Though these waveguides show low propagation loss, the printed silk optical fibers on the rigid substrate still have limitations for in vivo application. To overcome this challenge, free-standing step-index optical waveguides were fabricated by encapsulating a silk film core within a silk hydrogel cladding235 [Fig. 5(h)]. In this case, the core's refractive index (n ∼ 1.54) is higher than the cladding layer (n ∼ 1.34), mostly composed of water, allowing strong guidance and propagation of light through the core. The so-fabricated step-index waveguide has flexibility and biodegradability suitable for in vivo applications. The light propagation loss along 8 cm long waveguides was as low as 2 dB/cm on average, which is comparable to that of other polymeric waveguides (0.02–5 dB/cm). The ability to guide light in biological tissues was demonstrated by inserting the free-standing silk step-index optical fiber into bovine tissue and by verifying the incident light being guided in the biological tissue along with the core of silk waveguides [Fig. 5(i)].
F. Silk-based light actuators
Magnetic silk light-actuators are an example of an untethered optical technology based on the use of a polymorphic host material doped with optically absorptive elements with low Curie temperature,245 which results in localized and reversible movements. The principles at the base of this actuation mechanism are the (1) optically induced demagnetization caused by a local increase in temperature and (2) an out of plane gradient in the density of the magnetic particles which creates anisotropy in the mechanical properties of the structure, thus favoring bending toward the magnetic nanoparticles-rich side of the scaffold.
For a CrO2 doped silk fibroin cantilever, a bending movement toward a permanent magnet can be obtained by exposing the composite to a magnet which generates an attractive force toward CrO2 particles; localized heating causes the demagnetization of the CrO2 particles once T > TCurie, CrO2 ∼ 122 °C, therefore decreasing the cantilever attraction toward the magnet and thus resulting in a smaller actuator deflection. The bending is reversed to the initial state by removing the heating source which induces a decrease in the cantilever temperature and thus an increase in its magnetization which translates into stronger deflection. The actuation process depends on the physical parameters of the composite (shape, CrO2 loading) with thermal relaxation times that can be as low as few seconds (0.66 ± 0.04 s) for a 24-μm-thin 50% w/w magnetic silk fibroin film, under an illuminating power of 190 mW. As the heat-induced demagnetization is localized, this allows for a highly precise actuation that can target only specific parts of the device.
Multifunctional silk-based optical actuators have been recently fabricated in the form of bilayer structures of PDMS and of gold nanoparticles (Au-NP) doped SIO that can be activated by controlling the light propagation within the bilayer system.213 The actuation mechanism is based on the large difference in the thermal expansion coefficients between the Au-silk composite and PDMS; this difference is enhanced by localized photothermal heating obtained by engineering the SIO lattice (λreflectance = 533 nm) so to match the absorbance peak of gold nanoparticles (λabsorption = 527 nm) promoting a longer light–matter interaction in the photonic crystal. The bending extent of the SIO/Au-NP/PDMS composite is side dependent; a strong deflection is obtained when light impinges on the PDMS first: this is due to the fact that the nanostructure given by the SIOs modulates the incoming light by reflecting into the composite (and thus toward the Au-NP rich layer) the wavelengths corresponding to the pPBG thus increasing the amount of light that can reach the Au-NP to create local heating. By precise design of the location and shape of the patterned SIO regions, it is possible to locally program the light propagation into the bilayer system so that the actuator performs a broad variety of movements such as symmetrical bending, outer bending, twisting, and unsymmetrical folding. Similarly, the angular dependence of the SIO reflected light is exploited to create reversible phototropic movements of the SIO/Au-NP/PDMS composite [Fig. 4(e)]. For illumination from the SIO side, the displacement increases strongly for small illumination angles, while later decreasing for higher illumination angles. This system can be coupled to solar cells for a higher efficiency solar energy harvesting platform, due to the composite bending and following the light source, behaving like a light self-tracking system, which keeps the angle between the light source and the composite fixed as the light source moves [Fig. 4(f)].
G. Silk materials with tunable transparency
Light scattering properties of materials are usually dependent on the material's structure, and it is not straightforward to reconfigure them once a material form has been defined. Silk's polymorphism comes in handy when it is necessary to tune the scattering properties of a device on demand. By fabricating bilayer systems of regenerated silk fibroin on PDMS it is possible to program the system to display microscale wrinkles at the surface by exploiting differences in the elastic modulus of the two materials; these wrinkles render the material opaque (high scattering) and can be reversibly erased by exposing the composite to cycles of heating and cooling (that release the mechanical stress accumulated in the material) or they can be permanently erased upon exposure to water/methanol vapors rendering the material fully transparent (low scattering). Patterned wrinkle surfaces can be obtained by shadow masking the silk-PDMS composite, enabling encoding of messages in the bilayer structure which can be revealed only under specific environmental conditions. Similarly, electrically responsive silk-based devices can be achieved by integrating the bilayer silk wrinkle structure with a voltage responsive element (e.g., ITO, indium tin oxide), which can tune the scattering properties.216
Silk fibroin has a combination of properties that make it suited to a variety of applications in electronic interfaces, including optical transparency, high permittivity, tunable degradation, biocompatibility/degradability and mechanical robustness (Table I). These properties enable applications both as a passive substrate or encapsulation and as an active dielectric component. Silk fibroin has been used as a substrate for transient electronics, transparent conductors, triboelectric and battery devices, and gate dielectric for active components such as organic field-effect transistors (OFETs) and memory devices.
A. Bioresorbable substrates and packaging for transient electronics
The biocompatibility and tunable degradation properties of silk fibroin led to the development of transient electronics, namely, devices that can be fully degraded or bio-resorbed after they have completed their function. These devices have potential applications in temporary implantable sensors, disposable green electronics, and wearable devices. In these devices, silk acts as either a proteolytically degradable substrate for the active electronic layer or as a dissolvable substrate for mechanical support.
The development of electronic devices on silk films has typically involved transfer printing devices fabricated using conventional micro/nanofabrication techniques to the film substrate. For this fabrication technique, a silk film can either be cast on the patterned wafer and peeled off with the device, or a soft polydimethylsiloxane (PDMS) stamp can either be used to transfer the devices from the wafer to silk films.246 Transient complementary metal-oxide-semiconductor (CMOS) devices on silk films were demonstrated using thin-film semiconductor materials (Si), conductors (Mg), and dielectric (MgO) materials. This allowed for integrated transistors, inductors, and diodes to be encapsulated in silk with controllable degradation times from few minutes to days in phosphate-buffered saline (PBS) depending on packaging conditions and film thicknesses247 [Figs. 6(a) and 6(b)]. Controlling the crystallinity and creating multilayer air–silk interfaces was shown to modulate the degradation time of packaged electronic devices.248 Further control over the degradation rate of individual elements in the device enables functional, programmed transformation of these transient electronic devices. For instance, this was demonstrated by incorporating metals with different dissolution rates (e.g., Mg vs Fe) as well as patterned protective oxides (MgO) into devices to transform the function of logic gates or alter the frequency of an antenna during degradation.248
Current fabrication methods for incorporating electronic structures on silk have typically been limited to transfer printing and shadow mask deposition, limiting the scale and ease of fabrication of devices. Recently, direct, multi-layer patterning of electrical devices on silk films was demonstrated using an aluminum hard mask as a protective layer during fabrication.99 In this process, a 15 nm Al layer was deposited and patterned via photolithography and then etched in buffered hydrofluoric acid. The silk film could then be etched, patterning the film, or metalized via liftoff techniques. Direct, wafer-scale fabrication of drug-loaded, patterned neural microelectrode probes, resistance/capacitance (RC) devices, and aperiodic bioactive silk films were also demonstrated via this technique.99,206
B. Dielectric for active electronics
The transparency, flexibility, solution processibility, and dielectric properties of silk fibroin (k ∼ 5–7) led to the development of active electronic devices incorporating silk as the gate dielectric. Notably, the dielectric performance of silk films is highly dependent on their crystalline state. Specifically, an increase in inter-chain hydrogen bonding due to high β-sheet content improves the performance of silk fibroin as a gate dielectric.30,252 Pentacene thin-film-transistors (TFTs) fabricated using highly crystalline, methanol-treated silk as the gate dielectric showed low hysteresis, high bias stability, and low turn-on voltages.30
Silk has also been demonstrated in resistive memory devices (RRAM). In RRAM, a metal-insulator-metal (MIM) capacitor structure is used. By applying an electric field across the MIM capacitor, the cell can be switched between a high resistance state (OFF) and a low resistance state (ON) due to transport changes in the insulator matrix. In these devices, the silk film acts as an active component allowing diffusion of metal ions to form an electrochemically reversible conduction path between the metal layers on either side of the thin film. Memory devices with metal layers (Mg, W) on either side of a 120 nm spin-coated silk film were demonstrated with 2 V (SET) and –2 V (RESET) states, 105 ON/OFF ratio, and reversible switching shown for 100 cycles.253 The devices additionally show complete degradation within 24 h in PBS, demonstrating potential for nonvolatile memory in transient electronic applications. Recent work demonstrated the mesoscopic functionalization of the silk dielectric layer with Ag/BSA (bovine serum albumin) nanoclusters, increasing the speed of the devices and lowering the set/reset voltages to 0.3 V/–0.18 V.254 Multi-level memristor devices have also been proposed using silk fibroin/cadmium selenide quantum dot layers or silk fibroin-reduced graphene oxide (rGO) layers.255,256 Memory devices can be further integrated into arrays of memory devices based on a crossbar geometry.257,258 In particular, wafer-scale fabrication of flexible memristor arrays was recently demonstrated, enabling high density (20 × 20 μm2) cells with patterned, 20 nm thick, silk dielectric layers249 [Figs. 6(c) and 6(d)].
C. Conductive films and e-skin
Methods to create conductive, flexible films are important for the next generation of implantable devices, wearable electronics (e-skin), and optoelectronic devices. The high transparency (>90%), high mechanical strength, and flexibility of silk films enable their use as high-performance substrates. This was demonstrated by transfer printing a silver nanowire (AgNW) mesh to silk films through a casting and peeling method, creating conductive films (12 Ω sq−1) with high transparency (80%).259,260 This technique was used to fabricate transparent organic light-emitting diode (OLED) devices and organic solar cells. The conductive films had lower surface roughness than indium tin oxide (ITO) films. This method proved to be an inexpensive and simple fabrication process for transparent electronic devices on silk films. Further patterning of the AgNW layer enabled transparent resistors and radio frequency antennas for food sensors.261 The conductive layer was flexible and resilient as devices showed high fatigue stability with minimal change in resistance (<1%) after 10 000 bending cycles. The addition of plasticizers (glycerol) allowed for the fabrication of stretchable (60%), transparent, and conductive films which were utilized for strain sensing.262 Alternately, the addition of 4% w/w polyurethane were also shown to be useful as a highly stretchable (up to 300%) substrate for electronic skin applications.250 In this study, AgNF (silver nanofiber) and PtNF (platinum nanofiber) networks were transferred to the substrate for simultaneous temperature sensing and heating162 [Figs. 6(e) and 6(f)]. Conductive textiles incorporating AgNW and transition metal carbide/carbonitride nanosheets (MXenes) onto silk textiles using a vacuum spray technique have also been applied as electromagnetic interference (EMI) shielding and humidity sensors with exceptionally low sheet resistances of 0.8 Ω sq−1.263
Calcium-modified silk alters the mechanical properties of silk through metal-chelate bonding and water coordination (6–8 molecules per Ca2+) to create elastic films that can conformally bond to skin for epidermal electronics. Degummed silk was dissolved in formic acid/CaCl2, then the formic acid was evaporated, creating films with a range of mechanical properties depending on the weight ratio of CaCl2 and the humidity of the environment. This method was first demonstrated as an adhesive for capacitive and electrocardiogram (ECG) skin sensors.264 Further tailoring of the methods enabled transfer printing of Au nanotroughs onto silk films doped with calcium, creating elastic conductive films.265 In this study, tuning of the humidity allowed for both transfer printing at higher stiffness (∼100 MPa) and attachment to the skin (∼1 MPa). This film was demonstrated as an elastic low impedance electrode with a strong interface with skin when maintained in high humidity environments. Additionally, the use of a mixture of graphene ink, silk, and calcium enabled electronic-tattoos with self-healing properties demonstrated for strain, ECG, and temperature measurement applications251 [Figs. 6(g)–6(i)].
Alternatively, transparent conductive films have also been fabricated through the pyrolysis of electrospun silk mats, which transformed the β-sheet structure into a partially graphitized, nitrogen-doped, polyaromatic carbon structure.266 This process led to a transparent conductive film on PDMS with up to 80% transmittance which could be used for durable pressure sensors.
D. Energy devices
Energy devices incorporating silk as the encapsulation, active layer, or carbon precursor have been developed as novel materials for wearable or implantable energy storage.
Triboelectric nanogenerators (TENG) are a promising technology to convert mechanical energy into electricity for wearable or implantable electronics. Numerous TENG devices incorporating silk fibroin have been developed, as silk exhibits a strong electron-donating tendency, making it useful as a positive triboelectric material.267–270 In combination with common processing techniques to form high surface roughness materials (structured films, electrospun mats), silk-based TENG devices show high power outputs.271 Power densities of 22 W m−2 with 395 V output voltages were obtained using a sandwich of microstructure silk films and PTFE.272 For use in implantable electronics, silk-encapsulated TENG devices have also been demonstrated based on silk and Mg as a transient power source267,273 [Figs. 7(a) and 7(b)].
Silk has also found uses in packaging/encapsulation of batteries and fuel cells and as a component in a polymer electrolyte. Implantable, transient, biocompatible batteries were developed based on thin-film magnesium and a silk-choline nitrate ionic electrolyte.274 The batteries functioned for up to 3 hours in PBS with tunable degradation properties based on metal thickness and number of silk encapsulation layers and could be fully proteolytically degraded in 45 days. Separately, silk encapsulation was demonstrated as a method of increasing the lifetime of biofuel cells as a stabilization and encapsulation layer, as well as a film-forming additive for enzymatic fuel cells based on flexible carbon nanotubes (CNT).275
As a bio-renewable carbon source with high nitrogen content, silk fibroin textiles and films have also been explored as a templated carbon precursor for electrodes in energy storage devices. Graphitization of raw silk treated with metal salts (Zn, Fe) produce defect rich, nitrogen-doped (4%–6%), highly porous activated carbon electrodes (SBET: 2500 m2 g−1) containing 2D carbon nanosheets. Lithium-ion batteries and supercapacitors with high energy density have been demonstrated using this technique.276 Carbonization of silk films containing nanocarbon additives has also been used to develop flexible Zn-air batteries with a silk-based air cathode with high stability, good rechargeability and improved efficiency toward the oxygen-reduction reaction277 [Figs. 7(c) and 7(d)]. Additionally, carbonization of commercial silk textiles has also shown promise as a hierarchical structure for wearable sensors and battery electrodes. Vertically oriented molybdenum disulfide nanosheets grown on carbonized silk textile (MoS2/CSilk) demonstrated high efficiency as the anode in Li-ion batteries owing to its combined 3D/nano structure278 [Figs. 7(e) and 7(f), Sec. V A].
Integration of silk films with electromagnetic metamaterials (e.g., split ring resonators) enables the fabrication of conformal sensing devices with applications in food quality monitoring, humidity monitoring, and in vivo sensing. In these devices, thin film metallic resonators are patterned on silk films to create structures with resonances that can be tuned to be sensitive to changes in the geometry of the silk substrate (e.g., bending, swelling), permittivity of the substrate due to infiltration of the film with solutes, and permittivity of the surrounding environment. These sensors have been demonstrated to monitor food spoilage based on the resonant response of the devices caused by dielectric and conformational changes of the food item.280 Similarly, THz resonators were patterned on silk films demonstrating humidity sensing based on the modulation of silk's permittivity with humidity.281 Additionally, recent work reported the successful fabrication of implantable THz resonator-patterned, drug-loaded films for real-time monitoring of drug release from the silk matrix during degradation279 [Figs. 7(g) and 7(h)]. A metamaterial approach was also used to demonstrate passive sensing in the oral cavity using a broad-side coupled split ring resonator surrounding a silk film282 (see Sec. V). In brief, changes in permittivity and thickness of the silk film hydrated with saliva allowed for differentiating ionic (e.g., salt) and nonionic (e.g., sugars) analytes in saliva-based on the loss and frequency variations in resonance.
Recent research efforts to develop sensing devices focused on the adaptation of miniaturized designs based on relatively mature technologies dedicated to the detection of physical parameters such as displacement, rotation, pressure, and temperature, to name a few. Chemical analytes contained in fluids (i.e., biological samples, environmental water, and gases) are overlooked as possible targets despite the valuable information they convey about the state of health of individuals and their surrounding environment. Nonetheless, the approach to implement physical and chemical sensors and their intertwined connections has increasingly exploited the very pliable properties of biomaterials. In particular, silk has a unique versatility as it can be used in the form of pristine substrates and as interactive solid formats upon tuning of the silk fibroin solution; examples of the former case include pliable textile sections and flexible threads, while films, hydrogels, and sponges constitute responsive solids. The recent advances in fabrication techniques coupled to silk's versatile chemical structure empower the realization of novel sensing configurations that rely on the transduction of optical and/or electrochemical signals.283–287 These transduction mechanisms can be used for applications that range from environmental monitoring to conformable wearable configurations enabling the development of novel human-machine interfaces.288 This section focuses on the use of silk as an inert textile substrate that can be easily functionalized to obtain wearable sensing devices251,289 and it explores the potential of regenerated silk fibroin sensing devices. Furthermore, it discusses the use of regenerated silk fibroin as adherent soft matrix290,291 that encapsulates and preserves sensing functionalities. Finally, it contextualizes the power of regenerated silk fibroin for the fabrication of novel free-standing optical sensing devices.
A. Silk fabrics as substrates of sensing units
Silk fabrics combined with other functional elements are ideal candidates for smart textiles that can sense their surrounding environment.292–294 First, silk-based textiles can be transformed into conductive interfaces. In general, electrical conductivity above 1 S cm−1 is typical of conductive materials suitable to build devices that embed conducting and/or semiconducting layers.295 With an electrical conductivity of only 10−15 S cm−1, B. mori silk fibroin is highly insulating, as dictated by a nonconjugated molecular backbone not supported by alternating double bonds. Nevertheless, after functionalization with a range of materials, which span from conductive polymers to electrically conductive nanostructured materials, silk textiles can be converted into electrically conductive interfaces used as a base layer for the successive development of electronics/sensing textiles.296,297
Electrically conductive polymers (e.g., PPy, PANI, PTh derivatives such as PEDOT) can be transferred to silk fabrics via in situ polymerization.298 Electrically conductive nanostructured materials, e.g., carbon, aluminum, copper, gold, silver, or iridium, in the form of nanocrystals, nanoparticles, nanowires, nanotubes, and nanosheets (such as CNTs,299 graphene nanosheets, metal nanowires297,300) have been used extensively to functionalize silk fabrics via coating techniques ranging from dip-coating, screen-printing, and polymer-assisted metal deposition to evaporative deposition and sputter coating.301
Graphene oxide (GO) based materials were first transferred to silk fabrics by dip-coating and they strongly adhered to silk fibroin via strong interactions based on intermolecular hydrogen bonding, hydrophobic–hydrophobic, and polar–polar interactions.302 They were first spray-coated on top of conductive interdigitated electrodes made on silk fibroin masks to accurately detect human respiration rate. Moreover, GO active layers effectively differentiate normal, deep, and fast breathing repeatedly with up to 2500 repetitions of bending and twisting not affecting the sensing performances. GO layers can conformally coat various patterns to achieve different esthetics providing an easy approach for the fabrication of textile-based wearable electronic interfaces.303
GO is, however, usually transformed into reduced GO (rGO) by a thermal reduction process292 that removes oxygen functionalities but preserves the main β‐sheet structures of silk fabrics; this reduction process guarantees the final material flexibility along with resistivity and conductivity of the order of 3.28 kΩ cm−1 and 3.06 × 10−4 S cm−1, respectively, that can meet electron conductive requirements for wearable electronics.304 rGO was used as the base layer of wearable sensors for pressure and strain that enabled human motion monitoring:305 pulse sensitivities of 0.4 kPa−1 for measurement ranges as high as 140 kPa were recorded293 and a maximum Gauge Factor (GF) of 124 in the strain range of 10% could be measured.305 rGO was also used to functionalize silk textiles that monitored human arterial pulse, respiration, and throat vibrations in real-time, as for instance, during coughing and singing, while also demonstrating voice recognition.306 An additional layer of ZnO nanorods electrodeposited on top of the same rGO layer enabled the monitoring of mechanical deformations.306 Thermal reduction carried out at temperatures as high as 400, 500, and 600 °C also induced gas sensing properties on rGO-silk substrates, as verified by targeting NO2. In this case, the best sensing performance was recorded for rGO-silk substrates reduced at 400 °C and was dictated by the large surface area and optimum pore size of the functionalized interface; the highest gas sensing response was 24%, recorded for 10 ppm at room temperature and showed stability during sample bending.307 Despite the promise of this technology, it remains a challenge to improve selectivity and extend the system for multi-gas detection.
Silk fibroin textiles can be further converted into conductive graphitic nanocarbon by thermal treatment of the substrate which leads to carbonized silk fibers (CSF). β‐sheet crystallites in the silk polypeptides can be aromatized or cyclized into sp2‐hybridized carbon structures using environmentally friendly and less expensive synthetic approaches that do not require the use of toxic chemicals and that can be easily scaled-up.308 CSFs exhibit good cytocompatibility and better electrical properties compared to silk fibroin interfaces functionalized with graphene, with CSF thin films resistivity being an order of magnitude lower than silk fibroin-graphene composites'.308 CSFs were first embedded and tested in preliminary studies targeting energy conversion and storage devices.309
The same strategy was adopted to implement wearable strain sensors.309,310 Silk textiles have a hierarchical structure, imposed by weaving techniques (i.e., plain, satin, twill, georgette), that can influence strain patterns. Therefore, strain sensors based on plain-woven silk fabrics exhibited a large strain sensing range, more than 500%, and high sensitivity with GF of 9.6 for strain within 250% and GF of 37.5 for strains in the range of 250%–500%.309 Silk georgette-based strain sensors showed, instead, a sensing range of ∼100% and ultrahigh sensitivity with GF of 173.0 for strains in the range of 60%–100%.310 The distinct hierarchical structures of carbonized silk fabrics respond differently to external strain stimuli dictated by variations at the microstructural level. This phenomenon was exploited for the fabrication of flexible pressure sensors based on CSF that were manufactured on the fingers area of a glove and on tights/stockings to successfully track limbs' motion in real-time.311 Better sensing performances were, however, obtained by embedding biologically inspired hierarchical structures on top of CSF. For instance, the faceplate of sunflowers was reproduced by growing vertically aligned molybdenum disulfide (MoS2) nanosheets in situ on carbonized silk fabric (MoS2/CSF).278 MoS2/CSF enabled the detection of pulse waves and voice vibrations278 with improved sensitivities dictated by the increased surface roughness of the metallic layer fabricated on top of the wearable pressure sensing device [Figs. 8(a)–8(c)].
B. Silk fibers as substrates of sensing units
Fabrics are usually functionalized on a large scale but there is sometimes the need to diversify, distribute, and isolate sensing units with miniaturized patterns. Silkworm fibers are durable, good heat conductors, insulating, and biocompatible. Therefore, they are regarded as excellent materials for flexible electronics that can undergo modification and incorporation into fabrics through textile technologies. Smart fibers can be converted into conductive units using the same materials and approaches described in Sec. V A. Graphite-coated silk fibers find applications in the implementation of flexible and wearable strain sensors for motion detection.312 Metal nanomaterials were also combined with fibers or yarns but more sophisticated coating approaches, such as polymer-assisted metal deposition, were used to guarantee adhesion and improve stability and the consequent conductivity of metal-coated fibers.313 A conjugated polymer/polyelectrolyte complex, poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT:PSS), was also employed to obtain conductive silk yarns which found application as flexible thermoelectric devices as p-type legs.314
Due to the difficulties in successfully functionalizing and post weaving the sensing units, the use of smart fibers and yarns is not yet widespread. However, multifunctional and multidimensional sensors for temperature and pressure detection were reported based on silk fiber coiled yarns: the yarns functionalized with sensing mix consisting of carbon nanotubes and the ionic liquid [EMIM]Tf2N reached a temperature sensitivity of 1.23% °C−1 and a high-pressure sensitivity of 0.136 kPa−1, with a low relaxation time of 0.25 s, and high recycling rate of 5000. The fibers were weaved together to create a unique combination of textile sensors. Temperature and pressure can be sensed independently using a pressure detection mechanism based on the capacitance change of each cross‐point of two yarns, with a position precision of 1 mm2. The same configuration was efficaciously tested in the format of a smart glove, an integrated multimode/multidimensional electronic textile with high position resolution that can be easily translated into removable electronic skins for humanoid robotics, biomedical prostheses, and electronic skins315 [Fig. 8(d)]. A similar approach was followed to design ultrasensitive dual-mode sensing devices for temperature and pressure266,316 that did not meet the standards set by the silk coiled yarns315 in terms of design and overall sensing performances [Figs. 8(d)–8(f)].
Aside from natural silk fibers, regenerated silk nanofibers can be designed and combined with carbon nanotube yarns to obtain flexible and lightweight conductive silk wires. In this case, the silk yarns were sewn into a t-shirt and tested as wireless electric energy transmission lines.317 Regenerated functionalized silk nanofibers that can maintain both mechanical and sensing performances are still in their infancy318 but the advances in fabrication and functionalization techniques indicate a trend toward widespread research ramp up in the coming years.
C. Silk fabrics and fibers as chemical sensing units
To expand the applications of flexible sensors beyond the monitoring of physical parameters, it is important to devise strategies for detecting analytes in readily accessible environmental and biological fluids. Fluids are routinely analyzed through standard analytical techniques (e.g., atomic absorption spectroscopy, ion chromatography, gas chromatography) that are relatively expensive and inevitably provide only discontinuous one-time measurements, since they are currently not suitable for miniaturization. Recent advances in materials science and fabrication methodologies are opening routes toward enhanced miniaturization. Simultaneously, more efficient fluidic handling techniques, integrated within textiles, may provide improvements in robustness and mechanical durability of chemical sensors in these formats.319
Electrochemical sensors detect continuously and in real-time variations in the analyte of interest via electrical signals such as changes in current, potential, or impedance. Smart bandages or pad-like configurations applied on the skin were the pioneering devices that naturally evolved into fashionable appealing smart garments for which silk substrates played a key role.
Preliminary work focused on fabricating flexible glucose sensing devices based on carbonized silk fabric coated by multi-walled carbon nanotubes (MWCNTs/CSF). MWCNTs/CSF were decorated by electrodeposited Pt microspheres that hold in position the glucose oxidase (GOx). Pt microspheres and GOx synergically interact to allow glucose detection: glucose reacts with GOx that produces D-glucono-1,5-lactone and H2O2 and the latter is then detected by the Pt microspheres. The resultant sensor showed a good sensitivity of 288.86 μA mM−1 cm−2, due to a large contact area and stable connections between MWCNTs/CSF, but with an unclear sensing range (0–5 mM, missing limit of detection).320 The sensors perform well under bending and show good over time stability (15 days). Instability is usually dictated by the degradation of enzymatic activities that pushed research studies toward the implementation of enzyme-less electrochemical detection approaches based on the design of nanomaterials with unique structures. Sensing performances of enzyme-less detection approaches were explored using electrodes in wearable formats, such as CSF.321–323 For instance, cuprous oxide nanoparticles were embedded in carbon spheres and directly deposited on carbonized silk fabrics (Cu2O NPs@CSs/CSF) for glucose detection.321 The Cu2O NPs@CSs/CSF substrates were soft, with flexibility similar to that of carbon cloth, and they showed good electrical conductivity due to the large contact area and stable connection between the CSF and carbon spheres. The obtained non-enzymatic sensor also exhibited high selectivity, a low detection limit of 0.29 μM, improved sensitivity of 462.6 μA mM−1 cm−2 but with unclear time stability.321 Hybrid substrates consisting of rGO functionalized CSF substrates were also used for glucose detection.322 In this case, Cu nanoflowers were electrodeposited on top of rGO/CSF surfaces: the sensing unit showed improved sensitivities of 6613.3 μA mM−1 cm−2 and 1541.7 μA mM−1 cm−2 for concentration ranges of 0.05–4.0 and 4.0–7.0 mM, respectively, due to the presence of Cu nanostructures that increased catalytic activity, selectivity, and stability in air for 15 days.322 Enzyme-less approaches generally improved performances in terms of sensitivity and sensing ranges but they did not overcome the stabilization issues, highlighting the need to explore different strategies.
Sensor operating under mechanical stress generally preserved their sensing functions overall but additional improvements may be reached by implementing the detecting units directly on individual yarns or fibers. This technology is still in its early stages, but preliminary studies focused on manufacturing miniaturized electrochemical sensors onto biocompatible silk yarns. Specifically, silk yarns were coated with conducting inks and sensing reagents for glucose and hemoglobin electrochemical detection before being handloom woven as electrodes into patches to generate multiplexed sensing arrays. Hydrophilic and hydrophobic yarns were used to design the fluidics and to restrict sample flow to a small sensing area. These glucose sensors performed well but accuracy and repeatability needed to be improved, for instance by implementing increased automation in the fabrication process. These sensing units were speculated to have shelf life of over 1 year after packaging. The production of the present multiplexed configuration may be scaled-up easily through weaving, enabling the realization of electrochemical distributed interfaces at decreased production costs.324
Despite the quantitative information continuously harvested from complex biological fluids using distributed electrochemical sensing units, these techniques present a set of drawbacks, such as the need for constant power supply, electrical signal drifting over time, and noise affecting the electrical connections, that are easily overcome resorting to optical sensing devices. Optical sensing interfaces can be implemented through direct transfer (e.g., via dip-coating, screen-printing, inkjet printing) or direct chemical functionalization of silk fibers and textiles. The surface of silk fabrics can be easily functionalized exploiting very fine chemical attaching techniques that target exposed functional groups, though with only a few reported examples so far;325 one example consists of nonwoven silk fabrics chemically modified with fluorescent probes that allowed for selective and sensitive detection of Zn2+ variations.325 The present approach is promising and the resulting device can be potentially implanted in vivo although more studies are needed to evaluate stability overtime within complex biological matrices. The scale-up of functionalization techniques to cover large surface areas is another opportunity that needs to be better addressed in future research studies.
D. Regenerated silk fibroin based sensing units
Regenerated silk fibroin constructs can be exploited for the fabrication of sensing interfaces that directly adhere and interact with the functionalized surfaces and their surrounding environment. These interfaces are available in different formats, including transparent films, hydrogels, inks for inkjet and screen-printing, are biocompatible, processed in water-based environments, and are characterized by mechanical properties and degradation dynamics that can be tuned by controlling the degree of crystallinity (β-sheets content) of the protein structure. Interfaces based on regenerated silk fibroin can easily conformally adhere to curvilinear objects and biological surfaces like organs and tissues, enabling the realization of conformable, wearable, and implantable devices.302,326
1. Active substrates based on regenerated silk fibroin
Devices based on regenerated silk fibroin found application as sensing units for environmental monitoring in the form of carbon-coated silk fibroin scaffolds for nitroaromatic explosives detection in vapor samples, via impedimetric measurements327 and fluorescent copper nanoclusters (CuNCs) stabilized in silk matrices that responded to pH variations in water samples.328
Film formats, instead, proved their efficacy to entrap and stabilize enzymes such as urease,329 heme protein structures,330 and antibody331 complexes. These active films were deposited on top of conductive electrodes (i.e., glassy carbon for urease329 and heme,330 gold interdigitated for antibody331) and they enabled the electrochemical detection of urease,329 nitric oxide,330 and hepatitis C,331 respectively. Stand-alone films were employed as transparent immobilization vehicles of luminescent transducers based on lanthanides (e.g., europium complexes) employed in the evaluation of ammonia vapors332 or coupled to antibody reactions for hepatitis C virus detection.333 Silk films were also deposited on top of planar terahertz metamaterials configurations334 and they were evaluated in label-free biosensing applications335 or as humidity sensors.281 Spin coated, sub-micron thick silk films were tested, instead, as colorimetric humidity sensors: they showed reversibility, high sensitivity to vaping, and they can be easily translated into active anti-counterfeit labeling.96 Similar configurations may be easily adapted for contactless measurement of breathing patterns.336
The sensing devices described in Refs. 96,281,327–333, and 335 delivered good overall performances but they were free-standing devices tested on the bench with standard analytical instruments in laboratory-controlled conditions. Silk films can, instead, adapt to the interface they are transferred onto and can be processed to maintain active behaviors. More specifically, epidermal interfaces based on ultrathin and flexible silk fibroin-based electronics represent the next generation of wearable sensors enabling the simultaneous monitoring of physical (e.g., mechanical forces, temperature, humidity) and biochemical signals (e.g., ionic content, glucose, dopamine, virus, bacteria). To enhance user comfort, it is, therefore, essential that a stable, nonallergic contact is established on the skin surface to ensure reliable measurements with a high signal-to-noise ratio. Silk-based biocompatible adhesives were developed following different strategies that involve micro structuring silk fibroin thin films to make them easily removable,337 mixing silk with other naturally available biocompatible adhesive materials (e.g., silk fibroin gels combined with polydopamine),338 or chemically modifying the protein structure.251,264,339,340
Calcium (Ca)-modified silk fibroin adhesives based on random coils cross-linked by Ca ions form metal-chelate complexes that exhibit excellent physical adhesive characteristics (e.g., peel strength > 800 N m−1), empowered by the ability of CaCl2 to capture water molecules from the surrounding environment. The same adhesives have excellent peel strength and ionic conductivity, enabling accurate monitoring of underlying electrophysiological signals.281,289 Furthermore, the chelating agent CaCl2 is biosafe and flame resistant.341 The latter property was exploited to make an adhesive layer that was employed to develop flame-retardant materials, temperature sensors, and fire alarms.342 All the devices demonstrated transparency, conductivity, excellent stretchability, and self-healing properties. Ca-modified silk fibroin adhesives were further functionalized with conductive gold (Au) nanowires, metalized Au wrinkled structures,340 and graphene layers251 to assess their performance as supporting adhesives of adherent electronic interfaces339 in the format of self-healable electronic tattoos, commonly known as E-tattoos. The graphene-based E-tattoo responded to multiple stimuli, including strain, humidity, and temperature, and self-healed after mechanical damage through water addition which restored hydrogen and coordination bonds at the fracture interface.251 Ca-modified silk fibroin adhesive layers do not cause skin irritation after 10 days of lamination unlike their commercial Ag-AgCl based hydrogels counterparts. Furthermore, they have high stability and conformability under various mechanical deformation modes and they display lower noise amplitude, enabling a wide range of applications such as electrocardiogram monitoring sensors,338 capacitive sensors based on Ag nanowires interdigitated electrodes to evaluate environmental humidity variations and different breathing conditions,343 and strain262 and pressure sensors262,344 based on stretchable Ag nanofibers262 or Ti3C2Tx MXene nanosheets344 for tactile,262 motion,262,344 and sound detection.344
The sophistication level of E-tattoos was further increased by combining passive capacity sensors used to detect humidity variations accounting for breathing states and novel printed silk fibroin-based triboelectric nanogenerators on the same device.268 The biomaterial-based nanogenerators harvested the biomechanical energy produced by the human body's motion generating a power area density of 412 μW/cm2, which was sufficient to support most of the electronics built on the wearable interface.
Recently, more research studies focused not only on the evaluation of physical parameters but also on the design of skin adherent E-tattoos tuned for chemical biomarkers detection, defined here as chemically sensing tattoos or Chem-Tattoos. Most of these devices were made of PEDOT:PSS electrode arrays photolithographically micropatterned using sericin as the carrier matrix onto regenerated flexible, bioresorbable silk fibroin films.326,345 The PEDOT:PSS arrays were then functionalized with different sensing solutions based on silk fibroin if enzymatic stabilization was needed.326,345,346 This allowed the development of devices that detected the temperature of human skin,347 analytes such as glucose326,345 and dopamine,348 and vascular endothelial growth factor (VEGF)346 available in biological fluids and/or tissues. This repertoire of biomarkers can be easily expanded modifying the top sensing layer directly exposed to variations of analytes. Chem-Tattoos showed good sensing performance indicated by a stable linear operation during proteolytic biodegradation and they generally worked for up to 2 weeks. This concept of fully organic devices can, therefore, provide a pathway for the development of the next-generation, easy-to-use, and degradable flexible biosensors. Chem-Tattoos can be considered a useful transient tool for the monitoring of analytes in vivo both inside and at the body surface for a controlled time before complete degradation. However, it must be emphasized that the performance of these PEDOT:PSS devices326,345,348 were tested in-vitro and they did not undergo real-time testing as chemically active human-machine interfaces.
Seminal work targeting in vivo contexts proposed three different strategies that allowed in vivo monitoring of biological fluids (Fig. 9). The first strategy consisted of a Chem-Tattoo which adapted passive dielectric sensing configurations into active, conformable interfaces adherent to tooth enamel282 [Figs. 9(d) and 9(e)]. The interlayer material consisted of either a porous silk film or a modified PNIPAM hydrogel that dictated the selectivity and sensitivity of the device and that accounted for the detection of pH, salinity, and alcohol salivary content in real-time. This device can be tuned to detect a variety of biomarkers and the millimeter construct can adhere to a variety of interfaces allowing real-time monitoring of different biological fluids.282
The second strategy was based on a multilayered silk patch fabricated via matrix-assisted sacrificial 3D printing methods.349 It consisted of various microfluidics channels that provided a liquid channel for body fluid transport and sensing (immunological assays for human cancer markers CEA and AFP) and of graphene microcircuits that worked as a resistance variation-based motion sensor. This wearable platform possesses excellent tensile properties, self-healing ability and biocompatibility and it can also be adapted for environmental monitoring.349 The third strategy demonstrated the use of inkjet printing244 and screen-printing350 techniques to encapsulate and transfer biologically active molecules (e.g., pH indicators, enzymes, antibiotics, growth factors, and antibodies) and dopants (e.g., nanoparticles) embedded into silk fibroin inks onto a variety of substrates, such as plastics, glass, paper, Petri Dishes, and textiles in the format of T-shirts [Figs. 9(a)–9(c)]. The properties of the ink additives were preserved after printing244,350 and the functionalized surfaces were readily accessible for real-time detection of biological fluids (e.g., sweat)350 and contaminated surfaces244 via colorimetric readings encoding distributed information.
2. Active hydrogels based on regenerated silk fibroin
As highlighted for active films based on silk fibroin, the adhesion of sensing units to the surface undergoing real-time monitoring remains a challenge. Different silk-based adhesives were developed for film sensing formats to overcome those issues but, recently, flexible configurations designed as functionalized hydrogels found application in certain experimental conditions. Differently from films, hydrogels have the advantage to include sensing and adhesive properties in the same configuration, with no need for the separate design and fabrication of multifunctional layers post-assembled in a unique device.
Although the silk fibroin-based sensing configurations described for film formats exhibit excellent mechanical properties, they may still be below the requirements of adhesion for practical applications. Different reinforcing strategies were investigated to increase structural and functional performances of silk-based hydrogels, focusing especially on conductivity. Nanocomposites based on carbon materials (e.g., graphene, carbon fibers, carbon aerogels, amorphous carbon, carbon black, CNTs) proved their efficacy in preventing catastrophic failure.288,351 Therefore, recent research studies focused on the combination of GO with silk fibroin for the fabrication of hydrogels that achieved conspicuous improvements both in the conductive and mechanical properties, which is desirable for biomedical applications.162,352,353 Tough hydrogels were obtained exploiting a photo-catalytical reaction that triggers the covalent cross-linking of amorphous structures and tyrosine residues of silk fibroin. Additional physical cross-linking points in the silk fibroin–GO hydrogel are provided by GO, which greatly improves overall mechanical properties.353
A similar approach was exploited to fabricate soft conductive hydrogels made of polyacrylamide (PAM), GO, silk fibroin, and PEDOT:PSS;162 these hydrogels have excellent elasticity, stretchability, and compressibility, a wide sensing range (strain of 2 − 600%; pressure of 0.5 − 119.4 kPa), and undergo fast recovery from large deformations. This configuration is distributed on the skin and allows distinguishing different body signals such as joint movement, facile gesture, pulse, and breathing.162 CNTs were also used as conductive media163 embedded in a flexible and stretchable silk-based hydrogel cross-linked via enzyme catalyzed reactions that showed better flexibility than the GO/silk based hydrogels. The devices were tested in vivo to evaluate motions such as fingers and knee displacements and bio-signals like vocal cords vibrations during speech.163
However, carbon-based materials do not represent the only option for the realization of effective conductive hydrogels that can work as movement and pressure detectors. Silk was recently combined with ionic conductors354 that have the additional capabilities of converting hydrogel structures in hybrid sensing and actuating devices; this is extremely appealing in the fabrication process of artificial muscles, artificial skins, and triboelectric generators. For instance, biocompatible ionic hydrogels based on poly(vinylalcohol) (PVA), silk fibroin, and borax demonstrated stretching performances first of their kind reaching a strain larger than 5000%, self-healing abilities, tunable conductivity, high water retention, and strong adhesion.355 The ionic hydrogel had impressive performance when stored in an open environment (2 days at 25 °C, 65% RH) since it maintained high water content up to 75%, and retained the fracture strain above 4300% ensuring long-term stability in vitro.
Few research studies, however, focused on the implementation of hydrogel-based chemical sensors. Preliminary work was carried out on the development of cytocompatible and degradable phenol red-silk tyrosine cross-linked pH sensing hydrogels via enzymatic reactions.164 Phenol red was chemically attached to the hydrogel structure enabling the realization of leak-proof, biocompatible hydrogels that allow pH detection via absorbance and fluorescence measurements in fibroblasts cell cultures. This configuration may be converted into a self-reporting unit that accounts for pH variations during in vitro experiments in cellular microenvironments and bioreactors.164
Silk fibroin based hydrogels were also combined with embedded Au nanoclusters (AuNCs) to detect H2O2 variations via fluorescence quenching.356 This type of device was tested in biological fluids (e.g., fetal bovine serum) displaying a limit of detection of 0.072 mM, selectivity, stability over time, and biocompatibility. AuNCs-silk fibroin hydrogels have potential for clinical diagnosis356 and the same technology can be adapted for the development of wearable colorimetric hydrogels for noninvasive detection of a variety of analytes available in biological fluids. Novel devices that allow manipulating plasmonic resonances show some potential to monitor water,357 different analytes in vivo and in real-time.234,240 A gold resonating nanoabsorber was based on a silk fibroin hydrogel that worked both as insulating spacer and substrate. Specifically, glucose molecules were absorbed by the silk spacer and changed the physical parameters of the device causing the tunability of the plasmonic resonances. Analytes' variations can be monitored in real-time and the same configuration can be adapted for in vivo detection240 but it seems that the translation into wearable platforms is complex.
There are, however, different factors that hamper the wide-spread diffusion of hydrogels in the format of E- and Chem-Tattoos sensing configurations for practical applications. First, the drying and hardening of the substrates, dictated by the loss of the solvent, require constant storage in water-based environments. Many research groups are, therefore, focusing on the implementation of hydrogel sensing configurations that can retain their intrinsic properties for a longer time and the addition of ionic liquids to regenerated silk fibroin mixtures gave promising results so far.173 Second, two factors that dictate the long-term working stability of hydrogels implemented for soft electronics are self-healing and the consequent preservation of electronically conductive properties of the restored structure. Different methods were explored to obtain hydrogels with self-healing properties in recent years and the most effective ones are based on the incorporation of dynamic covalent (chemical cross-linking, creating disulfide and acrylhydrazone bonds) and non-covalent reactions (physical cross-linking, e.g., hydrophobic or supramolecular interactions, ionic bonding, and hydrogen bonding) into the hydrogel network.171
Last but not least, most designs often neglect the need for biodegradable and biocompatible hydrogel-based wearable devices. Recent research efforts focused on tuning biosensors biodegradability as a function of the final application.358 Novel configurations improve the overall biocompatibility by embedding antibacterial agents359 that make them a poor substrate for bacterial survival causing inflammation cascade reactions.
E. Optical silk fibroin based sensing units
Photonic crystals can be physically and chemically tuned for a variety of sensing applications that exploit the interaction of these nanostructures with light via interference, diffraction, and scattering. Photonic structures commonly available for markers detection either expand and contract upon application of external stimuli or undergo a variation of their effective refractive index as a result of fluid infiltration or chemical interactions. Both effects induce visible color changes and affect the device's spectral responses, thus working as the sensing device's transduction mechanism.360
Due to its transparency and mechanical properties, regenerated silk fibroin has been used to fabricate free-standing structurally colored films, usually in the form of inverse opals (SIO).191 The silk inverse opals can be converted into stretchable films via chemical cross-linking209 that found application as multi-analyte sensors. A library of functionalized SIOs was created to enable the realization of colorimetric immunosensors by embedding antibodies, red fluorescent protein, and β-subunit of cholera toxin. The specific antigens-antibody interaction within the nanostructure was accounted for by changes in reflectance spectra monitored with portable equipment and is one of the first examples of structural color used for the transduction of chemical information.212 SIOs were also evaluated as humidity sensing units whereby visible color changes were dictated by the expansion of silk matrices due to vapor/fluid absorption.361 Controlled SIO anisotropic structures offered a rapid and irreversible response to water vapor, providing single-shot measurements of the surrounding humidity and working as irreversible devices.198 Reversible readings were, instead, recorded using multilayered films made of silk and silk-titanate 1D photonic crystals that showed colorimetric responses when exposed to relative humidity variations and to human breath (use case scenario).200 The promise of this device depends on the biocompatibility of titanate nanosheets, which may hinder its implantability. In contrast, SIOs offer finer spectral tuning and enable the implementation of libraries of naturally inspired photonic structures. These may be further empowered by multi-analyte sensing functionalities through chemical functionalization or encapsulation of sensing molecules within the material. Several research strategies can improve the availability of structural color sensing devices and enable testing in real environmental conditions. Therefore, attention should be focused on obtaining accurate sensing readings in real-time, scaling-up production of structural color-based devices in reproducible fashion, and exploring new detection mechanisms induced by structural variations such as stimuli-responsive surface roughness362 via wrinkle216,363,364/crease365 formation at the surface of materials.
The intrinsic properties of silk fibroin-based films enable the implementation of other nanopatterned optical sensing elements in the form of smart diffraction gratings.214 These optical elements were fabricated simply by doping the silk matrix (mixing and casting, without the need for chemical reactions) with active sensing molecules190 like hemoglobin192,366 (e.g., oxygen sensor), HRP367 (e.g., colorimetric assays for glucose and lactate detection), and pH indicators.190,192 Consistent diffraction properties were observed for the doped silk gratings compared to the pure silk and, most importantly, the silk stabilization properties ensured that all these active components retained their activity when embedded in the silk matrix and kept at room temperature up to a few months. The optical relevance of these structures resides in their ability to work as self-analyzing devices as effectively demonstrated by free-standing hemoglobin-doped silk fibroin gratings embedded in a microfluidic cell and exposed to varying concentration of oxygen. The oxygen content in the fluid in the cell could be determined by monitoring spectral response of the hemoglobin entrapped in the grating.192 The high control that can be achieved in generating nanostructured geometries makes the silk diffractive optical element more sensitive to the expected small changes in the index of refraction dictated by targets variations in the sample being monitored. Silk provides structural integrity but it also embodies the multi-functional roles of stabilizing matrix (Sec. VI A),368–371 of optical transducer (due to high-fidelity nanostructured films that enable direct information readout), and degradation controller, therefore, paving the way toward the fabrication of active transient optical interfaces for in vitro and in vivo applications.
Optical sensing devices were also developed in waveguide formats that can manipulate and transport light in a controlled fashion. Commercially available standard multimode and long-period grating optical fibers (OF) were dip-coated with silk fibroin based layers enabling humidity372 and organic vapors (e.g., methanol)373 detection, respectively. The methanol sensing OF373 was tested against different alcohols: isopropanol did not cause any variation in the recorded signal but ethanol seemed to cause signal shifts accounting for a certain device sensitivity. The pristine silk coating layer was in its amorphous (silk I) water soluble state; exposing the material to alcohols can induce the transformation from silk I to crystallized silk II configuration. The sensing OF showed, however, reversibility suggesting that only a small number of crystallized structures were formed during methanol detection raising questions on the durability and reproducibility of the sensing unit. A more selective approach was followed by doping the silk layer with the fluorophore 5,6-carboxynapthofluorescein (CNF), which allowed optical measurement of pH by a ratiometric fluorescence method. The sensing optical fiber was tested in vivo in a mouse model of hypoxia and enabled pH detection in real-time showing a continuous drop in the subcutaneous pH in the mouse lumbar area as hypoxia developed.374
When targeting biomedical applications directly interfaced with living cells, tissues, and organs, it is crucial to choose OFs that can easily biodegrade inside the host environment. Silk fibroin is an ideal candidate and it was thoroughly characterized and used for the fabrication of biodegradable and physically transient OFs, as discussed in Sec. III E.196,235,375–377 These biocompatible configurations have not been yet fully tuned and functionalized for physical and chemical sensing. Despite a few examples being available in the literature, transient silk OFs with sensing capabilities were fabricated by electrospinning silk-based solutions with two optically active organic dyes (i.e., sodium fluorescein and riboflavin).378 Sodium fluorescein was used for hazardous and volatile hydrochloric acid vapors detection, while riboflavin is a nutrient that needed to be delivered into biological tissues. The resulting fluorescent silk nanofibers maintained the molecules activities and successfully exhibited sensing and delivery performances.378 The use of biocompatible and biodegradable polymers to guide light would open new opportunities for biologically based modulation and sensing, with unprecedented potential in targeting biomarkers as well as analytes correlated with environmental monitoring.196
VI. BIOMEDICAL PLATFORMS
Silk as a biopolymer has attracted particular attention largely due to its excellent material properties, biocompatibility and biodegradation, and the ambient aqueous processing conditions that preserve the bioactivity of the payload. These features, together with silk's capability of stabilizing bioactive molecules and its versatility to be processed into different formats have inspired a myriad of different applications in drug delivery, imaging, and tissue engineering. Sections VI A–VI D explore the biomedical applications of silk fibroin, including tunable drug delivery systems, customizable active platforms capable of tracking drug release, biohybrid systems, and bioengineered-oriented tissue layers with the physical and biological cues necessary to achieve the desired function.
A. Stabilization of labile compounds
Biological entities and labile molecules are known to be sensitive to physical and chemical stresses that may occur during manufacturing, shipping or storage, and which lead to instabilities (e.g., chemical modification, precipitation or aggregation in solution, and loss of function).379 Refrigeration and lyophilization are historically the most common methods to preserve the stability of these labile entities. However, such methods require specific laboratory settings, are costly, and do not always allow for the preservation of native functionalities. Silk is inherently stable to changes in temperature and moisture for a long period of time.370,380 Stabilization of labile molecules within silk materials can be achieved via encapsulation, by simply mixing the molecule of interest with silk solution, or also via covalent immobilization, by modifying the silk chain's amino acid residue through coupling reactions (e.g., diazonium, carbodiimide, or glutaraldehyde) or enzyme-mediated grafting reactions. Silk stabilization is complex and relies on the interplay of different mechanisms, comprising hydrophobic and electrostatic interactions, which stabilize the native structure of analytes, and the formation of microenvironments around analytes, which protect labile molecules from changes in pH through a buffering effect.379,381,382 Furthermore, encapsulation in a silk matrix reduces the molecular mobility of biological entities, which is associated with loss of native structure and functions, and protects the labile cargo from environmental stresses [Fig. 10(a)].369,382,383 The stabilizing properties of silk fibroin have been observed on a variety of labile molecules, such as enzymes, small molecules, proteins, antioxidants, growth factors, nucleic acids, plasma components, and cells, as schematically represented in Fig. 10(b).
Storage/encapsulation of labile entities in a silk matrix is, in general, straightforward and cost-effective, since the manufacturing process can be entirely water-based, and thereby compatible with different biomolecules. For example, in order to preserve catalytic activity, carbonic anhydrase was embedded in hydroxyapatite-silk microparticles which retained 80% of initial activity at 110 °C after 1 h treatment.384 Several other enzymes can also benefit from silk's protective features, including glucose oxidase, lipases, and ribonucleases.382
Furthermore, silk's properties allow combining the stabilization and the drug formulation steps during drug development. Because of silk's versatility and tunability, various formats, from injectables to implants, can be prepared without relying on fabrication conditions which can potentially degrade incorporated proteins and small molecules such as harsh temperature, pressure, or chemicals. In addition, many common stabilizers already used in drug formulation (e.g., proteins, sugars, salts) are compatible with silk and can provide additive or synergistic enhancement to the compound's stability when combined in dried formats.388 For instance, various potent human immunodeficiency virus (HIV) inhibitors were encapsulated in silk lyophilized disks, which managed to stabilize the protein cargo after 14 months of storage at 25, 37, and 50 °C [Ref. 389; Fig. 10(b)]. Although lyophilized formats may be susceptible to disaggregation, during handling and usage, this strategy has great potential beyond medical or laboratory settings and especially when cold-chain conditions are not practical. Similarly, the stability of contraceptive drugs such as levonorgestrel into a silk-based transdermal patch was ensured for 30 days during accelerated aging studies at 40 °C and 75% RH.390
Another therapeutic class that can benefit from silk's protective effect are vaccines, which are known to be unstable to changes in temperature and require controlled storing and shipping temperature. Recently, air-dried thin silk films were explored for the inactivated polio vaccine as an alternative to current products which need to be stored between 2 and 8 °C [Fig. 10(b)]. This formulation maintained 70% poliovirus D-antigen potency after storage for nearly three years at room temperature, vastly outperforming the stability of the existing liquid vaccine.391 However, the adoption of silk fibroin as a common excipient like gelatin, which retains the issue of being an animal-derived excipient, will require additional studies and validation processes to ensure no allergic responses are generated after use.
The added value of silk as a stabilizing biomaterial comes into play when its material versatility is considered, thus expanding its role from a stabilizer to a universal platform with tailored features. Indeed, this stabilizing effect has been observed both in solution368 and in dried state382 even with different formats: whether as film, microneedle, lyophilized matrix, or 3D composite, silk can ensure the integrity of entrapped biomolecules at room temperature and under stress conditions. Furthermore, silk fibroin can be processed by various advanced fabrication methods such as photolithography, molding, and inkjet printing, thus allowing for a variety of stabilizing formats to be developed [Fig. 10(d)]. For example, silk has been engineered into 3D constructs to obtain machinable solid formats with predesigned functions, in the form of implantable screws46 [Fig. 10(c)]. This work showed that silk ensured enzyme stability even after mechanical stresses during machining and polishing of the silk monoliths into screws. Specifically, silk preserved the enzymatic activity of HRP in PBS at 37 °C for at least 14 days, compared with the nonencapsulated counterpart which rapidly lost 50% of its activity in ∼4 h.46 Similarly, rabbit Immunoglobulin G (IgG)-doped silk microstructures were manufactured via multiphoton lithography which maintained the antibody's recognition functionality in the presence of fluorescently-labeled antirabbit IgG387 [Fig. 10(f)]. In another work, silk fibroin was used to improve the physical stability of emulsions and preserve the activity of HRP for long term storage (6 weeks)392 as well as to ensure enzymes and antibiotics stability during inkjet printing.244 The unique attribute of silk's biological stabilization has also been coupled to silk's optical features to function as a transducer and monitor encapsulated biological functions without the need for any further chemical modification. For example, human hemoglobin was entrapped in a silk film by simply mixing it with the silk solution;190 further casting into a diffraction grating mold led to a functional biomatrix that retained the properties of hemoglobin and was able to respond to the oxygenation state of the external environment192 [Fig. 10(e)].
The ambient aqueous processing conditions used during the preparation of regenerated silk fibroin allow for applying its stabilizing effect to more complex biological entities, such as cells, while ensuring their viability. In the effort to fabricate high-tech biohybrid devices that can be activated on-demand, chloroplasts were entrapped in a bendable, water-insoluble silk film to obtain a photosynthetic material activated after exposure to visible light [Fig. 10(b)].385 This work demonstrated that silk was superior in preserving the photocatalytic activity of chloroplasts under various storage conditions for several days in comparison with polyvinyl alcohol. In a more recent study, silk fibroin was combined with a bacterial cellulose matrix to develop a functional cell-based biosensor.393 In this case, the encapsulation in a transparent silk matrix conferred recombinant E. coli cells protection against UV exposure while preserving the cell metabolic potential.
Finally, a novel interest is emerging in leveraging the exceptional potential of regenerated silk for the stabilization of proteins and nucleic acids in point of care devices (PoC), whose wide-range adoption and distribution has been hindered by biomarker instability. In this regard, initial studies have evaluated the impact of silk on the stability of DNA and RNA. Silk-coated filter papers preserved DNA at high temperatures (37 and 45 °C) for more than 40 days and under UV irradiation for more than 10 h.394 In addition, air-dried silk matrices ensured RNA stability for 2 weeks at 45 °C with a 4–8-fold improvement over the stability imparted by air drying without silk protection.395 In this case, a purification step to optimize the RNA recovery yield was required, but it is a common procedure for RNA analysis from complex matrices and it can be performed with inexpensive commercial purification kits. Interestingly, silk matrix encapsulation can also be utilized to stabilize biomarkers in complex biological fluids, such as blood, urine, and saliva [Fig. 10(b)].386,547
Silk's ability to stabilize biological entities, together with its remarkable properties, motivates its potential applications in drug delivery, sensing devices, and high-tech bio-hybrids devices. The possibility to stabilize genome, transcriptome, and proteome further broadens the applicability of silk fibroin in PoC devices for disease testing, in a forensic setting, as well as in biobanking. However, scalable production techniques for both silk fibroin solution and silk-based formats will be needed to expand its usage on a broader scale.
B. Therapeutics delivery
From a pharmaceutical standpoint, silk-based systems provide a reservoir for active therapeutic ingredients while tuning their physicochemical properties and their pharmacokinetics, enabling specific targeting at the site of action, modulating intracellular transport and thus improving treatment efficacy and patient quality of life. Like other protein-based polymers, and in contrast with some synthetic polymers, silk has the advantages of environmental sustainability and cost-effectiveness. In addition, it is nontoxic, non-antigenic, and it degrades into amino acids that are well absorbed by the human body. Silk dosage forms can be processed in aqueous conditions and at room temperature without the need for harsh manufacturing conditions that could damage the incorporated active ingredient. Furthermore, its mechanical properties, degradation rate as well as drug release rate can be refined by controlling the processing conditions, which impact the β-sheet crystalline structure and, by combining silk with other biomaterials, it is possible to tune the mechanical properties of the formulation and create a variety of drug delivery platforms.396–398,548
Active ingredients can be entrapped, adsorbed, or encapsulated into silk matrices, which allow for delivering both hydrophilic and lipophilic molecules. This can be done by simply mixing silk solution with active molecules, or incorporating therapeutics onto pre-formulated drug delivery systems. [Figs. 11(a-i) and 11(a-ii)].404–406 Moreover, silk consists of a diverse range of amino acids with functional groups including amines, alcohols, phenols, carboxyl groups, and thiols that simplify the attachment of different biomolecules or ligands, thus allowing for targeted drug delivery while providing additional tunability for drug release rates [Fig. 11(a-iii)].405,407 Silk-based carriers have been formulated as nanoparticles,399 microparticles,408 hydrogels,400,409 aerogels,175 films,405 lyophilized sponges,410 and fibers,411–413 as individually discussed below [Figs. 11(b)–11(h)].
Microparticles (Ø = 0.1–100 μm) and nanoparticles (Ø = 1–500 nm) have been widely used in drug delivery applications owing to their large surface area, enhanced permeability, and targeting ability [Fig. 11(b)]. A broad spectrum of manufacturing strategies has been used to generate silk nanoparticles,414 including solvent precipitation,415,416 supercritical fluid technology,417 microfluidic techniques,392 or via a modified dissolution process of silk fibers that enables in situ particle formation.399 Silk nanoparticles generally present good drug loading capacity and favorable pH-dependent release profiles.388,416 Furthermore, solid tumors can be targeted with nanoparticles due to their leaky vasculature and high vascular density, which results in the enhanced permeability and retention effect (EPR). In addition, the modification of the nanoparticle surfaces with suitable targeting groups allows for active drug delivery to specific sites of action. Targeted drug delivery is the ideal choice for drugs that must have a local effect, induce systemic side effects, or to increase cellular uptake efficiency, thereby enhancing the therapeutic effect.418–420 In this regard, PEGylation of silk fibroin nanoparticles has been exploited to improve colloidal stability and to tailor drug release and carrier degradation.407,421 Interestingly, under simulated venous blood flow, the silk and PEGylated silk fibroin nanoparticles also showed low inflammation when compared to silica nanoparticles.422
Hydrogels are widely used in drug delivery, owing to their facile fabrication methods and potential injectability. Silk can form hydrogels with good biocompatibility, mechanical strength, and tunable degradation rate.423–425 Many different triggers have been used to control the silk sol-gel transition, including pH, sonication, phase separation, and direct electric current.426,427 In addition, by enzymatically cross-linking silk fibroin with other polymers, it is possible to obtain elastomeric hydrogels with combined properties.428–430 What is more, silk hydrogels show shear thinning behavior, which makes them ideal for injection and minimally invasive procedures, and have also shown potential for sustained release of chemotherapeutics over a long time, thereby reducing the need for frequent dosing and increasing patient's compliance [Fig. 11(c)].409
Films [Fig. 11(d)], wafers, lyophilized foams [Fig. 11(g)], and electrospun fibers [Fig. 11(f)], are silk-based solid carriers that have been used for local delivery due to the ease of modifications in terms of release kinetics, mechanical strength, and size. Sustained-release studies with antibiotics and chemotherapeutic drugs demonstrated silk's ability to incorporate molecules with different physicochemical properties and to provide controlled release using a diverse set of silk formats.391,409 Furthermore, a sustained release is ideal for molecules that have a short serum half-life. By tuning silk-based solid formats, it is possible to achieve sustained release via zero-order release kinetics.431 Recently, it was demonstrated that for local treatment with chemotherapeutic-loaded silk fibroin the drug release follows a diffusion process and that treatment efficacy can be improved by decreasing diffusion distances.432 The versatility of silk-loaded reservoirs to deliver tailored amounts of chemotherapeutics locally was also evaluated. A controlled release rate was achieved by dip-coating drug-loaded wafers into a silk fibroin solution, thus creating a silk shell around the wafer. Concentration and number of layers altered the chemotherapeutics release rate and initial burst, thus enabling to reach drug dosages that were previously found toxic.433 Silk lyophilized foams loaded with different drugs (vincristine and cisplatin) have been used for co-delivery of chemotherapeutics and have been found to be more effective at suppressing tumor cell growth compared with a single-agent treatment in neuroblastoma.434 Antibacterial silk foams were successfully prepared by loading the hydrophobic ciprofloxacin or the correspondingly hydrophilic hydrochloride salt in a silk matrix. The release of the antibiotic was modulated between 1 and 200 days based on the loading format and the antibiotic form included [Fig. 11(g)].403 Modulation of the solubility of the silk films through regulation of β-sheet content has also been used to achieve short-term release (4–8 h) of Interferon-gamma (IFN-γ).435 Furthermore, by chemically conjugating IFN-γ to silk films through disulfide bonds, a longer-term release of 10 days was achieved. In another work, silk-based lyophilized disks were also evaluated for the sustained release of the protein 5P12-RANTES, a potent HIV entry inhibitor.436 In this case, water annealing and layering of silk solution on silk lyophilized disks were used to attenuate the initial burst.
Implantable drug delivery systems are highly valuable since they provide an alternative to multiple dosing and improve compliance issues. Ideally, they should also degrade after implantation to avoid the second surgery otherwise required for the removal. In this perspective, the use of silk becomes relevant since it is bioresorbable and it integrates with the surrounding tissue after implantation without major adverse effect. Orthopedic devices with improved mechanical properties were prepared via an aqueous-based process used to incorporate bioactive molecules such as BMP2 and P24 peptide directly during the fabrication process [Fig. 11(e).401 Furthermore, by incorporating ciprofloxacin in the same device, it was possible to provide local antimicrobial prophylaxis without significant systemic exposure.401 The intermolecular interactions between the active drug and the silk matrix are critical for controlling diffusion-based release mechanisms. In addition, in contrast to small molecules, biotherapeutics have a larger, more complex structure which needs to be preserved to elicit the in vivo pharmacological activity. Silk's intrinsic stability to environmental stresses provides silk-based formats with the advantage of stabilizing labile biotherapeutics, such as growth factors or vaccines, enabling manufacturing, shipping, and storing at room temperature for molecules that would, otherwise, need constant refrigeration.
Microneedle arrays are a recently developed sustained release platform that is gaining attention in vaccine delivery [Fig. 11(h)], embodying the advantages of coexistence of form (microneedles) and function (therapeutics). Microneedle patch delivery systems provide for shelf-stable dosage forms without the need for refrigeration and form depot release reservoirs in the dermal space to modulate the drug release kinetic. Furthermore, microneedles are minimally invasive, thereby resulting in a patient-friendly and painless alternative to hypodermic needles for drug administration. Silk microneedles have been used to deliver vaccines against influenza, Clostridium difficile and Shigella, which resulted in the generation of humoral immune responses, although lower than those generated by the injected controls with the same dose formulation. It is possible that the therapeutic effectiveness was affected by an incomplete elution of the dose from the patch, thus reducing the immune response.437 More recently, a needle-free immunization system was developed by using a silk microneedle loaded with stabilized HIV gp140 trimer immunogen and adjuvant, which enabled MW-dependent release over time, the enhancement of multiple aspects of humoral immunity, and the induction of long-lived plasma cells.438 Even small molecules have been incorporated in silk microneedles to promote the long-term release and avoid daily drug administration.439 For instance, microneedles are useful as contraceptive devices, since they achieve sustained drug release for months.390
Silk is also a versatile biopolymer that enables the integration of specific functionalities, thereby enabling the design of transient devices with multiple capabilities, such as stimuli-responsive devices or drug delivery systems for tracking drug release or device degradation rate, while ensuring biocompatibility and in vivo degradation. For example, magnetically responsive, drug‐loaded silk fibroin nanoparticles have been developed by seeding silk with Fe3O4440 [Fig. 12(a)]. Under the effect of a magnetic field, it was possible to guide these silk nanoparticles to the tumor area, thereby promoting drug accumulation in the site of action and improving antitumor response [Fig. 12(b)]. In another work, silk fibroin was combined with nanodiamonds in the form of microspheres loaded with the chemotherapeutic doxorubicin, which were able to release the drug in a controlled manner while enabling fluorescent tracking inside biological structures.441 Silk inverse opals (SIO) have also been developed for drug delivery applications, due to the possibility of binding the drug to the silk and loading the active molecule in the air voids created by the inverse opal structure.143 In this case, the degradation of the silk-based photonic lattice leads to a time-dependent signal variation that could be leveraged to monitor drug delivery, as well as the silk structure interaction with a degrading enzyme (e.g., proteinase K) or with a growing tissue.142 Furthermore, silk‐based multichromatic diffractive optical elements (MC‐DOEs), which can work at multiple wavelengths either in sequence or simultaneously on-demand, can be doped with active molecules and work as a customizable bioactive platform with different functionalities. For example, by loading multiple drugs in different channels of the silk MC-DOEs and by monitoring the change in time of the reflected far-field diffraction pattern, it has been possible to achieve real-time tracking of the therapeutic released.82 In a different work, silk-DOE have been leveraged as skin patches for antibiotics delivery. In this case, by tracking the variation of the reflective diffraction patters of a silk-DOE loaded with penicillin, the drug release and the degradation process could be monitored. Furthermore, the device effectively promoted wound healing after application on a Staphylococcus aureus infected wound in a rat214 [Fig. 12(c)]. Finally, free-standing microprism arrays (MPAs) have also been used for drug delivery applications. They were prepared using a molding approach, by casting silk fibroin on a master mold to replicate the pattern.442 Due to the designed pattern, the replicated silk microprism could reflect light along the incident path. Once functionalized with doxorubicin (DOX), the MPA could be used to track the drug release from the formulation, since the changes in reflectivity followed both the burst release and the sustained-release phases. Furthermore, as the DOX-doped silk reflector degraded over time in the proteinase solution, the reflectance ratio gradually decreased, indicating the drug release and the MPA partial dissolution [Figs. 12(d) and 12(e)]. The backscattered signal could also be monitored after in vivo implantation in the dorsal region of a mouse, thereby opening the path for a variety of transient drug delivery systems with new optical functionalities which could enable more precise monitoring of drug release and imaging in vivo for sustained delivery systems.
For clinical use of polymers and for regulatory approval, requirements such as the fulfillment of Good Manufacturing Practices (GMP) and sterilization need to be considered. Contrary to most biopolymers, silk is compatible with common sterilization methods, including high-pressure steam (121 °C for 15 min), dry heat (180 °C for 30 min), ethylene oxide (55 °C for 4 h), or exposure to disinfecting agents (70% aqueous ethanol or an antibiotic–antimycotic solution) with minimal effect on the silk film mechanical properties.443 However, the effect of the sterilization protocol depends on several factors (e.g., type of dosage form), and therefore it is important to consider possible modifications on specific silk-based formats as well as the sterilization effect on the embedded active drug during drug formulation.110
Silk fibroin-based materials are promising as optical components for novel medical imagining modalities and can also be used to augment current imaging technologies. Existing imaging modalities may fail to detect signs of illness due to poor contrast between diseased and healthy tissue.444 Therefore, there has been growing effort to specifically target disease states with dyes or contrast agents that allow for more reliable early diagnosis. The biocompatibility and processability of silk fibroin make it an appealing vehicle to transport these dyes and it has further been shown that silk fibroin can also augment their optical445 and biological446 properties. As discussed below, fluorescent dyes,422 carbon quantum dots synthetized from silk,447 and magnetic resonance contrast agents448 are among the most investigated routes to use silk for both noninvasive in vitro and in vivo imaging modalities.449,450
1. Fluorescent silk materials
Silk fibroin modified to fluoresce finds applications in various medical contexts. It often serves as a vehicle that may either be taken up by cells for live cell imaging or for visualizing the degradation of silk fibroin-based implants in vivo. Several techniques for creating fluorescent fibroin have been developed in recent years: chemical modification of the fibroin, functionalization with small molecule fluorescent dyes, conjugation/entrapment of fluorescent proteins and nanoparticles, and hydrothermal carbonization of silk fibroin to carbon quantum dots have all produced materials for a variety of medical imaging techniques. Alternatively, a surprisingly simple way of producing fluorescent silk materials consists of feeding the developing silkworm larvae on a diet of food doped with fluorescent dyes like rhodamine (Rh),451 fluorescein,452 hexaphenylsilole453 or nanoparticles like carbon nanodots.454 These materials are absorbed by the larva and later deposited in the cocoon silk with hydrophilic dyes segregating preferentially in the sericin layer and hydrophobic dyes segregating preferentially into the fibroin [Figs. 13(a) and 13(b)]. These dyes are stably but non-covalently bound to the fibroin such that the degummed fibers retain a high degree of fluorescence.
Recently, several methods have been established for using silk-fibroin as a feedstock for carbon quantum dots (SF-CQDs). These colloidal materials, similar to their heavier metal counterparts, are strongly fluorescent, have moderately high quantum yields, resist photobleaching and photo blinking, and are readily functionalized, yet do not contain any potentially toxic heavy metals. Preparation of SF-CQDs requires controlled and pressurized heating of the fibroin above the decomposition temperature to facilitate selective carbonization of the fibroin chains. One means of achieving this controlled heating is by using microwave irradiation.447,450 For instance, microwave treatment at 200 °C for 20–240 min generates water-dispersible nanoparticles (Ø ∼ 6 nm) with emission peaks tunable from 425 to 550 nm as the excitation shifted from 300 to 500 nm [Fig. 13(e)] and a quantum yield of 15% compared to rhodamine B. This thermal synthesis process enriched particles with hydroxyl and carboxyl functional groups, making them strong candidates for further functionalization. These materials were readily taken up by cells in vitro for uses in live-cell imaging, caused no acute toxicity or immunogenicity in vivo, and were rapidly cleared from the body.
The hydrophobic domains of the SF network are also ideal for trapping synthetic fluorescent particles such as quantum dots (QDs), nano-diamonds (NDs),445 and silver clusters.456 SF-entrapped nano-diamond colloidal suspensions can be synthesized in a co-flow fluidic process. The fluorescent particles are particularly interesting due to their relatively long excitation wavelengths (∼500 nm) and their point-like emission that allows imaging down to the diffraction limit [Fig. 13(g)]. The silk NDs exhibited high mobility and diffusion in aqueous environments due to the hydrophilicity of the coatings. Beyond acting as a simple carrier for the NDs, the NDs entrapped in silk also exhibited 2–4 times emission enhancement compared to bare NDs. More traditional nanoparticles such as silver clusters and cadmium telluride (CdTe) nanoparticles can be augmented with silk to allow for interfaces with living tissues. In particular, entrapment in silk fibroin can significantly reduce the toxicity of heavy metal compounds like CdTe by tightly enclosing them, allowing these ordinarily toxic materials to be used in biointerfaces.446 Notably, the emissive properties were retained and could also be easily tuned across the visible spectrum by varying the nanoparticle diameter (Ø = 3.0–4.5 nm) [Fig. 13(f)]. These materials demonstrated suitability for two-photon fluorescence microscopy: as the longer excitation wavelength was less scattered by tissue, deeper imaging into tissues than ordinary fluorescence was possible.449
2. Magnetic resonance contrast agents
Beyond the fluorescence methods listed above, biominerals and biocompatible metals can be conjugated to silk nanoparticles to create contrast agents for magnetic resonance (MR) imaging modalities. Fibroin nanoparticles can be synthesized in sizes ranging from 70 to 1000 nm making them useful vehicles to exploit the EPR effects of some tumors.457 This concept was exploited to create fibroin nanoparticles that had been “mineralized” by magnetic manganese oxide as well as loaded with a phototherapy agent to create theragnostic nanoparticles.455 The EPR effect caused the accumulation of these particles in a breast cancer tumor model in mice, which could be readily visualized by their brightening under MR imaging [Fig. 13(d)]. The contrast was further enhanced by additional brightening in the acidic conditions created by the tumor microenvironment [Fig. 13(c)]. Once the tumor was located by MR imaging, it could be immediately treated by activating the photodynamic therapy component with transdermal near IR irradiation. Similarly, as magnetite nanoparticles are known MR contrast agents, nontoxic magnetite/fibroin core–shell nanoparticles were fabricated and found well-tolerated by cells in vitro.448
D. Tissue engineering
Silk fibroin materials are among the most commonly used for tissue engineering and regenerative medicine, with examples of silk formats being used for almost every solid tissue in the human body. Recent examples include bladder,458 bone,141,459,460 cornea,167,180,461 cartilage,166,462,463 dental pulp,464,465 ear drum,466 intestine,467,468 kidney,469 heart,470,471 liver,472,473 nervous tissues (brain474–476 and spinal cord/peripheral nerves477–479) skeletal muscle,480,481 and tendon.482,483 Silk is particularly well suited for tissue engineering and regenerative medicine because of its nontoxicity which implies that it can entirely degrade and resorb while being non-immunogenic and minimally inflammatory.11 This is ideal as an implanted tissue would not have any scaffolding that would need to be surgically removed after implantation. Furthermore, as apparent also from the other sections of this review, silk materials can be easily processed into a variety of formats, such as hydrogels, fibers, sponges, and films, that are well matched to the mechanical properties of the tissue being mimicked/substituted while providing topographical cues that guide cellular differentiation and maturation.484
1. Mechanical matching
A key consideration for tissue engineering is the mechanics of the extracellular material being mimicked, as material properties like elastic modulus and viscoelastic properties significantly bias stem cells toward certain growth lineages.484 Silk films and fibers are known for their especially high elastic modulus of up to 7 GPa in the native fiber, which is stiffer than most bodily tissues and largely limited to 2D tissue engineering. Softer 3D structures like silk hydrogels and sponges can have elastic moduli in the range of 1–250 kPa,487,488 and up to the 1–10 MPa range if reinforced.463 The common fabrication techniques for creating films, fibers, hydrogels, and sponges (Sec. II E) produce materials that comfortably encompass the native moduli of soft bodily tissues such as brain (<1–7 kPa),246 adipose, glandular tissues, kidney, and liver (1–25 kPa),489 along with muscle, peripheral nerves, and tendon (25–100 kPa).246
For instance, to use silk sponges and hydrogels for bone tissues growth, it is necessary to increase their elastic modulus above 25 kPa; in this regard, fabrication methods that allow for highly ordered and homogenous materials show promise to improve the final structure and biological function. For instance, low density aerogels of highly ordered pure silk fibroin inverse opals fabricated from direct opals templates showed a modulus about three times higher than the one of more disordered porous scaffolds obtained with the traditional salt-leeching method (∼12 vs 4 kPa), despite showing similar computed pore-wall moduli (540 and 830 kPa, respectively).141 Mesenchymal stem cells seeded on highly ordered inverse opals preferentially differentiated toward an osteogenic lineage with markedly higher biomineralization compared to softer salt-leeched sponges. Alternatively, sponges can be stiffened by creating silk-ceramic composites, either by mixing the two components during the fabrication steps or by incorporating biomineralization active sites in the silk to promote mineralization in situ.459 These techniques allow fabricating 3D sponges with compressive modulus above 1 GPa, which matches the range of cancellous bone.490
2. Topographical cues
During differentiation, cells go beyond the sensing of the simple mechanical characteristics of their environment and look for topographical cues to guide further growth and direct development. Cues such as linear grooves, fibers, nano-pillars, and surface gradients can guide cell migration, differentiation, and maturation through the process of contact guidance.491 These defined patterns can be transferred to silk fibroin through various techniques described earlier such as casting, embossing, and photolithography (Sec. II E). Cells patterned on these linear ridges and valleys align themselves to these patterns, which improves the maturation of tissues with a high-degree linear ordering reminiscent of cellular ordering found in native tissues such as muscles471 and tendons.483 Other patterns such as nano-pillar and nano-pit arrays have been shown to direct stem cell differentiation down osteogenic lineages.492
In 3D scaffolds, silk can be formed into topographies with aligned fibrillar, tubular, and hierarchical structures that guide cellular development and aid in mass transport of nutrients and oxygen. Aligned tubes are attractive for peripheral nerve engineering as extending axons and neurites preferentially grow along elongated tubular structures.493 These tubes can be formed by directional freezing of a silk fibroin hydrogel, which creates elongated ice crystals with controllable size, direction, and aspect ratio that act as the templates for pores. These ice crystals can then be removed by lyophilization, leaving a silk sponge with aligned pores readily usable for cell seeding [Figs. 14(a) and 14(b)]. Hippocampal neurons seeded on a fibroin sponge fabricated in this manner became notably polarized and preferentially extended their axons along the tubular structures [Fig. 14(c) and 14(d)].485 Another method for creating tubular structures consists of rolling flat substrates with linear patterns in the orthogonal direction to the imprinted pattern to create a series of long channels bordered on all longitudinal sides; this strategy was demonstrated using an aligned electrospun fibroin mat to produce a structure morphologically similar to nerve bundles.478 Additionally, the electrospun fibers can be functionalized to promote nerve growth by mixing growth factors with the spinning solution, giving the final construct the ability to provide both topographical and chemical cues for tissue growth.
Bioprinting consists of the simultaneous 3D printing of live cells and extracellular support and it is among the most promising techniques for fabricating whole organs. Compared to traditionally employed bioprinting materials, fibroin has several advantages due to the multiple biofriendly gelation methods that allow the printed silk material to solidify without damaging the encapsulated cells. These methods include enzymatic cross-linking184 or incorporation of another polymer such as gelatin494 or alginate183 that gel in response to heat and presence of Ca2+ ions, respectively. Alternatively, pre-sonicating the silk before mixing it with the cells primes the silk to gel in a predictable time; depending on the silk concentration and the energy applied the gelling time can be tuned from minutes to hours.51 This latter gelation process, in conjunction with UV-crosslinkable methacrylated cellulose, was used to make 3D printable silk/hydroxyapatite hydrogels laden with mesenchymal stem cells.185 The resulting printed hydrogels were suitably tough to be handled and tied in knots, and the encapsulated stem cells survived the process and were still alive 10 days after printing.
Other common means of bioprinting are light-mediated techniques such as cross-linking through digital light processing,60 direct laser writing, multiphoton printing, and subtractive means such as femtosecond machining.495 Native fibroin can be selectively cross-linked with photoinitiators such as riboflavin, which creates di-tyrosine linkages in the material bulk and form a stable hydrogel under illumination with visible light.180 The silk can also be modified with reactive moieties, such as methacroyl groups to form silk-methacrylate (Sil-MA), which can then be polymerized with a biofriendly photoinitiator such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) [Fig. 14(f)].60 This methacrylated silk was used to print mechanically robust hydrogels loaded with human chondrocytes. The encapsulated cells readily proliferated within the hydrogel, showing equivalent proliferation to gelatin-methacryloyl (Gel-MA) [Fig. 14(g)]. Furthermore, after 4 weeks, the chondrocytes had significantly degraded the hydrogel material, showing that covalent cross-linking did not prevent biodegradation [Fig. 14(h)].60
Finally, a cell-laden hydrogel may be subtractively patterned by femtosecond machining which exploits silk's multiphoton absorption due to the presence of tryptophan and tyrosine residues. The absorbed photons thermally disrupt sections of the silk fibroin hydrogel while leaving the surrounding areas (i.e., regions unexposed to laser radiation and below/above the focal point) and the cells contained in those areas unharmed.495 Specifically, 3-photon absorption of an 810 nm femtosecond laser was used to produce voids up to 1 cm into the bulk of a transparent silk fibroin hydrogel, with a lateral resolution of 5 μm. The laser was able to write patterns deep within the bulk of a structure with entrapped mesenchymal stem cells, which survived machining and the subsequent implantation into a mouse host.
4. Hierarchical materials
Natural tissues display a hierarchy of structural organization that starts with the shape and size of individual cells and of the extracellular matrix and continues to the collective organization of these cells into tissues and their further assembly into whole organs. Hierarchically structured bioinspired materials seek to mimic this multiscale assembly with controlled and determined structures from the nano to the macroscale, ideally using a single fabrication process. An example of this design philosophy was demonstrated in a porous 3D printed silk matrix:486 the macroscale properties were defined by the viscoelastic properties of the ink and the programmed 3D structure of the printed device; the meso- and nanoscale were, instead, controlled by the addition of various porogens and by post-processing, which determined the scale, density, and morphology of the pores at these scales [Fig. 14(e)]. This strategy allowed the fabrication of low density closed-cell foams, useful for insulation, as well as higher density open-cell sponges, more suitable as engineered tissue scaffolds. A similar level of hierarchical control can be obtained by controlled fabrication of fibroin aerogels.242 In this case, the macrostructure was determined by the shape of the hydrogel mold as well as by the hydrogel density and cross-linking. Once cast, the hydrogel was mechanically stressed by contraction due to recrystallization or by mechanical forces imparted to the molds. This stress caused the alignment of silk fibrils, which were then locked in place by critical point drying, leaving an aerogel structure. The degree of alignment was found to be proportional to the stress applied, also confirmed by the birefringence displayed by the resulting structures.
5. Optical tissues
Many fibroin solid forms are optically transparent across the visible range and thus are ideal for engineering transparent optical tissues, such as the cornea. One method to fabricate completely optically clear hydrogels is by solvent gelation in acetone;167 hydrogels produced using this method were able to transmit >80% of visible light when cast up to 4 mm thick. Interestingly, despite the highly crystalline nature of solvent-gelled silk, surface culture fibroblasts were able to grow up to 1000 μm into the hydrogel, showing that the solvent-gel remained fully biodegradable. Transparent fibroin hydrogels can also be photopolymerized directly onto the cornea by using riboflavin as a photoinitator.180 In this case, the silk hydrogel bonds tightly to the tissue because of riboflavin-induced cross-links between the silk and natural collagen; this technique offers a potentially efficient and biocompatible approach to reshape the cornea by material addition to correct focal aberrations like myopia.
6. Electroactive silk composites
Tissues, such as musculature and neurological tissue, are inherently electroactive, and their maturation has been shown to be improved by or even be dependent on electrical stimulation. For musculature, electrical stimulation is important for alignment and coherence, allowing for masses of tissue to contract synchronously and uniaxially instead of moving at random times and directions. While silk is intrinsically insulating, silk substrates can readily become electrically conductive via integration with electrically conductive materials such as metals,496 graphene,497 and conducting polymers.471 For instance, chemical priming of the silk by the addition of sulfate moieties allows for the formation of an interpenetrating network of electrically conductive polypyrrole inside the solid silk matrix.471 In this case films were cast onto topographically defined masters yielding a topographically patterned and electrically conductive substrate onto which human-embryonic-stem-cell-derived cardiomyocytes were seeded. The surface pattern and the applied stimulation field were found to promote alignment [Figs. 14(i) and 14(j)], and these factors worked synergistically to promote the development of mature cardiac tissue structures, such as sarcomeres [Fig. 14(k)].471 The presence of conductive elements also improves neuron attachment, connectivity and maturation overall,498 as demonstrated by fabricating 3D printable fibroin/reduced graphene oxide composite hydrogel.497 The methacrylated silk itself was able to act as a reducing element for graphene oxide, enabling conductivity up to 6.5 S/m. This conductive hydrogel was sufficiently neurogenic to induce neuroblastoma cells to differentiate preferentially down a neuron lineage without the aid of chemical induction agents.
One of the biggest challenges in tissue engineering and regenerative medicine is the diffusion problem. Simply stated, there is a maximum diffusion distance for oxygen, nutrients, and waste in an un-vascularized tissue, beyond which any cell dies. Any constructs larger than the diffusion distance has live cells in the outer layers but necrosis in the interior. Silk, like other proteinaceous materials, has a maximum diffusion distance of 100–200 μm, beyond which oxygen becomes the prime limiting factor for cell proliferation.499 Several strategies have been adopted to overcome this challenge. For instance, it is possible to circumvent this to some degree by using very small cell clusters or “organoids” that are smaller in radius than the diffusion limit but can mature and exhibit advanced organ phenotypes.500 Another strategy for implanted devices is to recruit the body's vasculature building mechanisms with signaling molecules like VEGF to vascularize the implant.501 Silk sponges can be used to combine the two above-mentioned techniques in the push for functional organ replacement. This strategy was demonstrated by seeding a porous sponge with kidney organoids;469 the organoids were able to further develop a more adult phenotype, characterized by differentiating into organized epithelial layers. When the sponge was augmented with VEGF and implanted in a mouse, it had a threefold increase in endothelial recruitment compared to untreated fibroin sponges.469 Cells entrapped within silk hydrogels have also been shown to benefit from microchannels molded into the hydrogel matrix.502 Incubation of these microchannel hydrogels with human umbilical vein endothelial cells begins capillary-like vascularization before implantation allowing for greater tissue infiltration after implantation. These macropores also act as access points for cells responsible for extracellular matrix remodeling to accelerate the vascularization process.
8. Engineering fibroin for better tissue response
While native fibroin excels at certain biological tasks, it must be engineered to fulfill others. A primary concern for tissue engineering applications is the lack of integrin-binding domains like RGD in B. mori derived fibroin, which make cellular traction difficult for integrated and extrinsic cells alike.503 This issue could be solved either by sourcing fibroin from alternative species with endogenous RGD binding domains, such as Antheraea mylitta,504 or by artificially supplementing the B. mori fibroin with these domains via chemical ligation,505 enzymatic conjugation,506 recombinant507 or transgenic508 expression as well as blending509 or coating461 with a copolymer such as collagen that contains inherent integrin-binding domains. These same recombinant and ligation strategies can be applied to other bindings/recognition domains such as laminin-derived IKVAV510 or YIGSR/GYIGSR.511 This supplementation has consistently shown improvements to cellular traction, attachment, spreading and tissue-specific maturation signals like neurite outgrowth.
VII. FOOD SUPPLY CHAIN
Among the favorable functional attributes of silk, being edible allows for unusual applications which connect technology and food. Over the past few years, there has been growing interest in the use of silk fibroin from B. mori in the food supply chain with a focus on the agriculture, food safety, and food security fields. In particular, this section highlights the use of silk as an edible coating to boost crop production and improve foods' shelf life and as an interface to monitor food spoilage.
A. Silk-based technologies to improve crop production
Given that the world population is expected to reach 9.7 billion people by 2050512 and with roughly 2 billion people worldwide experiencing moderate or severe food insecurity,513 there is increasing interest in the development of sustainable materials and devices which could boost crop yields.
In this context, silk fibroin was used in combination with trehalose to develop a seed coating that can boost seed germination and mitigate abiotic stressors by encapsulating, preserving, and releasing biofertilizers in the soil.514 This study aimed to engineer the microenvironment of seeds in contrast to traditional approaches which, instead, aim to design plants that can adapt to different environments. The technology leverages the film-forming and preservation abilities of silk fibroin, which impart mechanical robustness, adhesiveness, conformability, and controlled biodegradation, and the ability of trehalose to support the survival of the biofertilizer (i.e., plant growth-promoting rhizobacteria) during desiccation and resuscitation. Silk coatings were applied to Phaseolus vulgaris seeds resulting in improved germination rate and seedling growth, leading to taller seedling with longer and more articulated roots compared to uncoated controls [Fig. 15(a)]. Additionally, the effect of the coating was more evident for seeds grown in saline soil (i.e., 8 ds/m).
Together with boosting seed germination, the challenge of increasing food demand can be addressed by enhancing plants' resistance and increasing the sustainability of crop production.515 To achieve this goal, the use of biomaterials and drug delivery principles to engineer the delivery and distribution of payloads in plants has been widely studied.516 So far, the most used delivery methods (i.e., foliar spray, root application, and trunk injection/petiole feeding) either suffer from significant material loss and low efficiency due to the plant's barrier tissues or are not suitable for small plants. To overcome these limitations, silk fibroin was applied to fabricate a microneedle-like device, named phytoinjector, which can deliver a variety of payloads (i.e., from small molecules to large proteins) into specific loci of various plant tissues.516 The phytoinjector was fabricated from silk fibroin blended with hydrophilic peptides (Cs) extracted from silk fibroin, which yielded a more hydrophilic silk material. This device can be used to deliver payloads into the vasculature of tomato plants to study material transport in xylem and phloem and to perform complex biochemical reactions in situ [Fig. 15(b)]. Furthermore, by tuning the silk to Cs ratio, another device, named phytosampler, was obtained with the aim of sampling plant sap for detection of metabolites.
B. Edible coatings to extend food shelf-life
While efforts are underway to increase food production, the Food and Agriculture Organization (FAO) of the United Nations estimated that one-third of the food produced for human consumption is wasted globally.517 Between 194 and 389 kg of food per capita per year are wasted on a global scale across the whole supply chain.518–520 For fruits and vegetables, the FAO estimated that 50% of crops are lost, starting from agricultural production until household consumption, mostly due to the premature decay of perishable products.517,521,522 In this scenario, the development of silk fibroin-based edible coatings has been studied as a method to preserve crop freshness.523,524 Silk fibroin is an ideal candidate for this kind of application since, once applied as a coating, it is edible, tasteless, odorless, transparent, biodegradable, and possesses outstanding mechanical properties as well as low permeability to oxygen and water vapors.78,110 Furthermore, silk fibroin obtained Generally Recognized as Safe (GRAS) food item status by the U.S. Food and Drug Administration.525 Silk fibroin was applied as an edible coating on strawberries and bananas by dipping the fruits in a silk fibroin suspension, repeating this step from 1 to 4 times, and storing them at 22 °C and 38% RH after drying.524 The coatings were able to extend the shelf life of both kinds of fruit, decreasing the respiration rate, weight loss, water vapor and oxygen diffusion, preserving firmness and color, and delaying ripening of bananas compared to uncoated control during 14 and 9 days of storage for strawberries and bananas, respectively [Figs. 15(c) and 15(d)]. The properties of the coatings can be tuned by controlling the silk's polymorphism (β-sheet content) through water annealing by exposing the coated fruit to water vapors under vacuum for several hours; this treatment enables the modulation of the mechanical properties and the diffusion of gases. By increasing the β-sheet content from 36% up to 58%, the permeability of this coating to water vapor and oxygen decreases and makes it more effective in extending the fruits' shelf life. As an alternative to the water annealing process, which can be time consuming and difficult to apply on a large scale, gas permeabilities and mechanical properties of the edible coatings can be modulated by mixing silk fibroin with other polymers, thus leveraging the intrinsic properties of each constituent of the mixture.522,523 For instance, silk fibroin was mixed with poly(vinyl alcohol) (PVOH) at different ratios to develop edible coatings in which the two components, after phase separation, self-assemble and spontaneously form a bilayer structure.523 PVOH, which is GRAS526–528 and has low water vapor and oxygen permeability, as well as good mechanical properties, has been coupled with silk fibroin to reduce the gas diffusion and improve stretchability. The 1:1 silk fibroin:PVOH ratio showed the best performances in achieving these goals and was chosen to conduct preservation tests on fresh-cut apples. The fresh-cut apples were dipped in a silk fibroin and PVOH suspension and were stored at 4 °C for 14 days after drying. Silk fibroin formed a coating layer on the fresh-cut apples' surface and coated the apple fibers by infiltrating in the cut apple flesh, while PVOH settled on silk fibroin and formed a homogeneous outer protective layer. The coating was effective in preserving the apple pieces by reducing both weight loss and browning compared to both uncoated controls and samples coated with pristine either silk fibroin or PVOH523 [Figs. 15(e) and 15(f)].
C. Sensing interfaces for the detection of food spoilage
In combination with the use of edible coatings to extend produce shelf life and delay spoilage, food quality monitoring and the detection of bacterial pathogens are critical in minimizing food loss and ensuring food safety. The World Health Organization (WHO) reported that foodborne contamination globally kills 420000 people per year,529 and the Centers for Disease Control and Prevention (CDC) in the United States estimated that every year 1 in 6 people get sick because of foodborne diseases.530 A viable method to monitor food quality is represented by silk-based microneedle patches that can both sample fluids in food tissues and provide a colorimetric signal to indicate the presence of pathogenic bacteria or food spoilage.531 The silk-based microneedle patches were obtained via replica molding and water annealed for 8 h to form few-microns-large pores. The backsides of the microneedle patches were printed with polydiacetylene (PDA) liposomes, which can be conjugated with antibodies for bacterial sensing or left unfunctionalized to detect changes in samples' pH. After being printed and exposed to specific environments, the PDA liposomes and antibodies are stabilized and preserved by silk fibroin, which acts as protective carriers. Silk microneedles undergo poroelastic reswelling as soon as they come into contact with food; this causes the absorption of fluids, which reach the printed backside of the patches where the PDA liposomes develop a pathogen-specific colorimetric response.531 This technology was effective in detecting the contamination of fresh hake and haddock fillets by E. coli and Salmonella typhimurium within 16 h from the microneedle application; the presence of contamination was displayed by the color change of the printed patterns from blue to red [Fig. 15(g)]. The silk microneedle patches are also strong enough to penetrate packaging films allowing the monitoring of packaged food quality without requiring opening of the package.531
Another study reported the possibility to monitor food quality by using silk fibroin as a substrate for wireless gold antennas that can easily adhere to food (e.g., apples, egg, tomatoes, cheese, and bananas) and monitor changes in quality.280 The devices were tested by monitoring their resonant responses continuously during the spoilage of food. The efficacy of this type of approach is demonstrated by monitoring fruit ripening with a conformally attached radio frequency identification (RFID)-like silk sensor applied onto the fruit skin, while spoilage of dairy products was monitored through surface contact with solid food or immersion in liquid goods.
VIII. ART AND SCIENCE
In the last ten years, a growing interest in scientific research from the field of art and design has fueled experimental projects and productive collaborations resulting in promising fibroin-based products. These products are not subjected to the biocompatibility constraints of biomedical applications and can combine fibroin with a larger range of materials (such as natural plasticizers, food-grade colorants, or sensing molecules) as well as make use of existing fabrication machinery (such as 3D printing extrusion nozzles, CNC-milling platforms, and screen-printing ateliers). Consequently, art and science collaborations output silk-based constructs at larger scales and with immediate potential for human interaction as, for instance, silk domes co-fabricated by robots and silkworms, larger-than-human pH sensors, and alternative fashion materials that could substitute traditional polluting ones.
A. Digital-natural co-construction
The silk pavilion532 [Fig. 16(a)] is the result of a collaboration between scientists and architects who devised a 3-m wide silk dome made by 6000 spinning silkworms on top of 26 panels of robotically-woven industrial silk thread. The project investigated the potential for biological and industrial co-fabrication of large-scale fibrous structures. In particular, physiological parameters of the silkworm's behavior determined the geometric angles at which the panels would be placed so that flat patches were spun by the silkworms instead of 3D traditional cocoons. The maximum size of the panel scaffolds woven with industrial silk thread was, instead, determined by mechanical constraints of the robotic weaving platform. The environmental forces acting on the site were calculated for solar radiation to steer silkworms toward warmer areas of the dome and achieve a homogeneous density of spun silk. The process successfully generated a silk dome with superior material density and structural stability compared to biologically spun silk or industrial woven silk alone, demonstrating promising pathways toward digital-natural co-manufacturing.
B. Large-scale distributed sensing
Silk fibroin can be effectively combined with other biomaterials and thickeners to display comparable properties to commercial screen-printing inks.350 Moreover, it can encapsulate active molecules such as pH dyes, including bromocresol green (BG), nitrazine yellow (NY), and phenol red (PR), which are pH-responsive in the ranges 3–5, 4–7, and 6–8, respectively, and are able to sense and change color to display pH levels in fluids such as sweat. Silk-based distributed screen-printed colorimetric pH sensors were demonstrated first on T-shirts (Sec. V D) and then extended to large scale (3 × 1 m2) fabric tapestries [Fig. 16(b)]. Their robustness was tested by subjecting the tapestries to the action of more than 10 000 visitors with hand-held spray bottles emulating human sweat, harsh chemical solutions, and acid rain (Lachesis exhibit533) Not only did the tapestries display consistent colorimetric responses, but they also withstood dry cleaning and reversible processing to their original colorimetric state, allowing them to be re-exhibited and sprayed by new audiences. The project demonstrated scalability in ink synthesis, adaptability to existing fabrication methods, endurance of intended use cycles, public outreach of scientific discovery, and a great potential for large-scale distributed sensing mapping to human and environmental wellbeing.
C. 2.5D printed leather-like materials
The versatility of silk fibroin inks can be harnessed in yet another existing fabrication method. Numeric control pneumatic extrusion systems operating under ambient conditions, and pioneered in the water-based fabrication platform,534 can digitally distribute silk fibroin inks. This technique was employed in forming meter-scale 2.5-dimension surfaces with a wide range of patterns printed with or without a homogeneous base layer and generating regular square, honeycomb, or triangular grids as well as differential density diamond grids or reptile skin inspired lattices.535 On top of geometric control, these surfaces can be generated with variations in feature-size, flexibility, opacity, responsiveness, and assembly [Fig. 16(c)]. Feature sizes of the top layers range from 0.25 to 5 mm, which confer differential strength to constructs. Maximum rigidity is displayed by sheets with coarse regular grid structures that tend to stay flat while maximum flexibility is based on single-layer non-regular lattice designs that drape like fabric. Opacity is controlled by tuning layer thicknesses and food-grade pigments within the blends. Responsiveness can be programmed by adding thermochromic powder dyes that respond to human body temperature ranges, for instance, 34 °C activated black-to-yellow or 28 °C activated gray-to-orange dye. Assembled structures withstand the folding, bending, piercing, stretching, and sewing typically used to create leather goods. These surfaces offer new routes for tough and strong, environmentally friendly, leather-like materials as promising alternatives to heavily polluting animal leathers.
IX. FUTURE DIRECTIONS
Due to its properties, regenerated silk fibroin has demonstrated enormous potential in a broad variety of fields. Its biocompatibility first seeded its use in the biomedical field, while properties such as optical transparency and ease of functionalization promoted its growth in disciplines such as optics, electronics, and sensing, while also branching to architectural fields as a re-engineered smart material.
Silk's optical and material properties allowed for the development of silk-based photonic crystals, waveguides, and resonators for advanced color filters, sensors, and free-standing laser particles among others.221,222,234 Notably for a naturally derived material, silk also exhibits unique third-order nonlinear optical behaviors unlike other conventional materials such as silicon and silicon dioxide;536 as a novel nonlinear optical material, it is expected that the combination of silk with microresonators will open new opportunities for nonlinear light-matter interaction studies. In addition, silk's tunable crystallinity and biocompatibility enable the development of optical platforms that could be directly integrated within the human body and have a programmable functional lifetime, which can be exploited for sensors and drug-delivery systems with an optical feedback.
Within the sensing field, silk fibroin can be used both as an adhesive layer and as the active layer of free-standing sensing devices. Silk's variable formats, biocompatibility, and biodegradability enable the design and fabrication of reactive interfaces that can be easily integrated into flexible platforms as wearable and implantable devices. These silk-based sensors can be used within health monitors and human-machine interfaces, establishing safe contact with tissues and organs. There are, however, critical challenges that still need to be addressed to enable the widespread use of silk fibroin based sensing devices. For instance, the design and implementation of smart sensors that can self-heal after damage would provide promising solutions by exploiting biologically inspired and stimuli-responsive composites with controllable shape-changing properties. Moreover, the sensing performance is affected by operational stress caused by cyclical mechanical deformation, heating during prolonged use, UV exposure through natural light, environmental temperature, and humidity fluctuations. These issues can be tackled by taking inspiration from silk fibroin's naturally occurring outstanding mechanical properties, which can be recapitulated and exploited by controlling its conformational state, degree of crystallinity, and functionalization, to tune the degradation rate, the release and preservation of encapsulated sensing molecules, and the mechanical robustness of the sensing interface.
Fibroin has many prospects for the future of biomedical devices and tissue constructs. Its biocompatibility and resorbability have been extensively demonstrated, as have the potential for adapting the fibroin constructs to match the mechanical properties of tissues as diverse as brain matter, musculature, and bone. However, silk fibroin suffers from being biologically inert and poorly permeable to oxygen. Future bioactive silks should not merely be limited to preventing the foreign body response but seek to actively induce a regenerative response. The next generation of silk biomaterials will need to build on the foundations of biofunctionalized silk to improve tissue interface and promote cell adhesion, proliferation, and stem cell differentiation, and induce local anti-inflammatory responses in immune cells. Further strategies will also need to be developed to facilitate oxygen and nutrient transport across multiple dimensional scales, from the nano- to the macroscale, within bulk silk materials for large scale tissue constructs to be grown and maintained.
Silk fibroin has been proven to be successful in extending food shelf life by developing colorless and tasteless edible coatings on the strength of its edibility, transparency, and tunable gas barrier and mechanical properties. The performance of silk fibroin coatings could be further enhanced with the formulation of active edible coatings through the incorporation of antimicrobial (e.g., essential oils) and antioxidant (e.g., ascorbic and citric acids) agents, which would prevent molding and oxidative rancidity. Silk fibroin could also be applied in the fabrication of self-standing biodegradable food packaging. An open challenge remains to adapt and optimize silk fibroin films fabrication processes to match the properties of widely adopted synthetic packaging materials (e.g., PET, polypropylene, polyethylene) both from the mechanical and gas permeability standpoint. Possible strategies of interest from a sustainable standpoint include combining silk fibroin with other biomaterials to form composites (e.g., fiber or laminar composites) to improve its barrier and/or mechanical properties.
For the above-mentioned fields, a few challenges remain unsolved and partially hinder silk's use as a ubiquitous material both at the laboratory scale and at the industrial level. As with all natural materials, the manufacturing process that relies on the use of B. mori cocoons renders silk fibroin production dependent on environmental factors, which will require the development of dedicated, monitored, and automated large-scale production approaches to mitigate batch to batch variability, external contaminants, and environmental influence. Moreover, genetic engineering techniques can help us to achieve higher protein yields, via sericulture employing B. mori or recombinantly expression in single cell organisms, ultimately leading to silk proteins with new capabilities and, in the future, more economically competitive products. Genome editing, indeed, could enable the advancement of silk-based devices with finely tuned physicochemical properties along with advanced functionalities, while broadening the manufacturing capacity as a result of a robust control on genomic sequence, protein size, and homogeneity, as well as degradation rate, thereby generating responsive materials.537–541
Due to the complex mechanisms involved in the spinning process of the B. mori silkworm taking place at the microscopic scale of its silk glands, full replication of the natural silk properties is difficult to achieve via the silk fibroin regeneration process. A better understanding of the natural silkworm spinning process may inspire better designs for artificial systems that can closely mimic the silkworm gland native environment and improve properties of regenerated silk fibroin. Additionally, further insight into mechanisms of the directed assembly of the protein, whether in the native spinning process or from regenerated solutions, could allow abundant new opportunities for silk-based applications by providing another method of controlling properties across multiple length-scales. Beyond the molecular level, high throughput technologies, such as 3D printing, thermal molding, and screen-printing, have the potential to boost industrial applications of regenerated silk fibroin on a broader scale in several fields, including pharmaceutical screening and delivery, bioresponsive textiles, novel sensing geometries, functional membranes and filters, on to solid objects and architectural components.
Throughout history and through the modern times, silk is a material with exceptional properties that keep fascinating and inspiring researchers around the globe. The possibility to integrate different capabilities on flexible, naturally derived technological and biomedical platforms, inspires the exploration of new research branches and the development of next-generation devices beyond the familiar lustrous fiber and the generation of new ideas at the intersection of materials science, healthcare, and technology.
The authors acknowledge Gregory Callahan for providing SEM images of silk formats. The authors would like to gratefully acknowledge support from the Office of Naval Research (ONR) and from the Stavros Niarchos Foundation (SNF).
Conflict of Interest
Tufts University has filed patents on several silk-based technologies and has licensed the technologies to startup companies. F.G.O. is a scientific founder of Mori LLC, Vaxess LLC, and Sofregen LLC.
Data sharing is not applicable to this article as no new data were created or analyzed in this study.