rus
Issue #1/2025
E.V.Panfilova, K.V.Mozer, A.A.Maltsev
TECHNOLOGY FOR PRODUCING SILK FIBROIN AND STRUCTURES BASED ON IT FOR WEARABLE ELECTRONICS PRODUCTS
TECHNOLOGY FOR PRODUCING SILK FIBROIN AND STRUCTURES BASED ON IT FOR WEARABLE ELECTRONICS PRODUCTS
DOI: https://doi.org/10.22184/1993-8578.2025.18.1.16.29
Silk fibroin biopolymer is one of the promising materials for organic electronics. It is characterized by optical transparency, thermal stability sufficient for proteins, biocompatibility and high tensile strength. Silk fibroin-based structures can be used to manufacture sensor elements of wearable electronics. Their properties are determined by the conformation of the protein structure, which depends on the methods and modes of formation of regenerated fibroin from its native form. In this project, a process for the formation of silk fibroin solution, films and photonic crystal structures based on them was developed.
Silk fibroin biopolymer is one of the promising materials for organic electronics. It is characterized by optical transparency, thermal stability sufficient for proteins, biocompatibility and high tensile strength. Silk fibroin-based structures can be used to manufacture sensor elements of wearable electronics. Their properties are determined by the conformation of the protein structure, which depends on the methods and modes of formation of regenerated fibroin from its native form. In this project, a process for the formation of silk fibroin solution, films and photonic crystal structures based on them was developed.
Теги: biopolymers fibroin photonic crystal structures protein structures silk белковые структуры биополимеры фиброин фотонно-кристаллические структуры шелк
INTRODUCTION
Wearable flexible electronics is an actively developing field of electronics [1]. Its distinctive feature is the small size of devices, their flexibility and lightness, as well as the use of organic materials as both substrate material and functional layers. In this regard, wearable electronic devices can be used to measure human body parameters such as temperature, chemical analysis of biological fluids, pressure, and pulse. The demand for this type of electronic devices is constantly growing [2], which imposes requirements for their production and utilisation to reduce waste and harm to the environment. Functional structures should be biocompatible, mechanically robust, environmentally friendly in production and easily recyclable.
Biopolymers, in particular silk fibroin, fulfil these requirements. Fibroin is a fibrillar protein. It is most often obtained from the cocoons of the mulberry silkworm Bombyx mori (Fig.1) by purifying the cocoons from the fibre-binding protein sericin.
Natural silk is produced on an industrial scale, usually for textile industry, but it is also interesting for medical wearable electronics production. Fibroin consists of two chains, a heavy chain (350–370 kDa) and a light chain (25 kDa). The heavy chain is composed of 90% of fibroin’s fibrophobic amino acid residues (Gly ∼43–46%, Ala ∼25–30%, Ser ∼12%) [3] (Fig.2). The friable regions of fibroin consist mainly of polar amino acid residues [4].
This material has unique properties that can be varied for three main types of conformations: α-helixes, β-folds, and random turns and twists. In the native state, 50–60% of fibroin consists of β phase [5], which characterises the crystalline regions of the polymer and determines its strength and stiffness. Spirals (α conformation – amorphous peptide chain) provide silk elasticity. Fibroin fibres have a tensile strength of 610–690 MPa and an elastic modulus of 15–17 MPa [6]. This protein is thermostable: its denaturation temperature is higher than 127 °C. Films from regenerated fibroin are optically transparent: they have 90–95% transmittance of the visible spectrum of radiation, refractive index in the visible spectrum nf = 1.54 [7]. Fibroin is biocompatible and biodegradable [8]. The presence of active functional sites in fibroin, such as primary amino group, facilitates incorporation of various conductive elements such as conductive polymers, carbon-based fillers, and metal interfaces [9]. Thus, fibroin, which is inherently an insulator, can be endowed with electrically conductive properties. In addition, by acting on the polar groups of fibroin macromolecules through external stimuli such as water vapour, methyl/ethyl alcohol, temperature, ultrasound, pH, and UV radiation, the protein structure can be modified and controlled [10].
In electronic devices, fibroin can be used in various forms: hydrogels, nanofibres, films, particles, and sponges [11] (Fig.3). In [12] application of a hydrogel based on fibroin, polyvinyl alcohol and borax for manufacturing of artificial leather and triboelectric generators is considered. The addition of silk to polyvinyl alcohol hydrogels improved their stability and also significantly increased the moisture absorption.
The options for using silk fibroin as a functional material are varied and numerous. In [13] a variant of fibroin application as an optical waveguide on CR-39 polymer substrate is presented. The obtained fibroin waveguide had a transparency of 85% in the visible light region and low optical losses both in the visible spectrum and in the IR region. Chemical analysis of biological fluids requires high sensitivity to biomarkers. In [14], an electrochemical sensor based on carbon cloth and nitrogen-doped silk (SilkNCT) was proposed. Compared with conventional electrode materials, SilkNCT, exhibits an internal hierarchical and porous mesh woven structure, which provides good contact with reagents and efficient electron transfer. A fibroin-based moisture sensor is described in [15]. In [16] it is shown that fibroin films variability under external factors influence can be used in information coding devices. In connection with biocompatibility and biodegradation of silk fibroin, application and creation of "smart" sensor structures based on it, for example, for measuring intraocular pressure or analysing composition of biological fluid, is being actively studied. Impregnation of colloidal photonic crystal with fibroin solution and subsequent obtaining of inverse photonic-crystalline structure expands the possibilities of adjusting the photonic forbidden zone under changing humidity, pressure [17] and mechanical force. In [18], we analysed a spectrophotometric method for measuring the longitudinal strain of the flexible photonic crystal structure of silk fibroin (Fig.4). It was shown that acceptable values of uncertainty of such measurements (less than 10%) open prospects for their use in electronics and medicine.
In the presented project the technology of obtaining silk fibroin solution and its solid phase with predominance of β conformation has been worked out, and possibility of protein photonic crystal structure formation has been shown.
MATERIALS AND METHODS
The process of preparating an aqueous solution of fibroin consists of the steps of cocoons preparation, degumming followed by washing and drying, dissolution, dialysis, centrifugation and filtration to remove the undissolved precipitate (Fig.5). The choice of methods and modes of their implementation in laboratory conditions was carried out on the basis of analysing information from literature sources and studying the nature of the processes.
The degumming step is necessary to purify cocoons from sericin. The process can be realised by physical, chemical, physicochemical and biological methods [19]. During the degumming process, it is important not only to remove the fibre-binding protein, but also not to destroy the long fibroin chains. Degumming leads to degradation of the heavy chain of fibroin from a molecular mass of 350–390 kDa to a wide mass distribution from an average molecular mass of about 150 kDa to small fragments of 40–50 kDa [20], which further affects the mechanical properties of the resulting structures and materials. The most common method is to clean the cocoons in an alkaline or neutral medium at elevated temperature. The medium can be soda ash (Na2CO3), laundry soap or neutral soap.
Since fibroin is 3/4 composed of non-polar hydrophobic amino acids, it is resistant to most solvents. To dissolve fibroin, it is necessary to break the strong hydrogen bonds, thereby converting it from a hydrophobic β-structure to a hydrophilic α-structure. To break the hydrogen bonds between macromolecules, solvent ions must interact with polar and charged groups of fibroin side chains. For this purpose, it is necessary to choose a solvent that can effectively penetrate into a protein molecule. Table 1 shows the liquids used in practice for fibroin dissolution.
The main disadvantage of saline-containing aqueous, aqueous-organic and organic solutions of fibroin is the long preparation time: fibroin has to be pre-activated by means of a saline solution, once obtained, the solutions have to be dialysed to remove solvents, considerable time cost is also required to regenerate the solvents in order to reuse them [4]. When ionic liquids are used, solubility of fibroin depends more on the anion nature. Ions with a greater ability to form hydrogen bonds dissolve fibroin better. The disadvantage of these solvents is their property of sorbing water from the atmosphere, which negatively affects the process [28].
A dialysis step is necessary to remove salt if an aqueous or aqueous-organic solvent is used. A cellulose dialysis bag with a pore diameter of 12–14 kDa is typically used as the membrane. The buffer solution is distilled or bidistilled water.
Fibroin-based films are prepared by drop casting on a substrate, centrifugation, and printing methods [32]. The protein molecule of fibroin is prone to the self-organisation process and can be subjected to controlled functionalisation in the film [33]. Fig.6 shows external influences both stimulating the growth of the crystalline phase (water vapour, temperature, etc.) and destroying the long chains of fibroin (UV).
When temperature rises above 40 °C, stability of many polypeptides decreases, resulting in the unfolding of coiled globules or spirals of molecules. The fibroin macromolecule is characterised by an increase in conformational mobility and destabilisation of the helical conformation with increasing temperature. As a result of this process, active groups compensate their potential to form H-bonds by interacting with neighbouring molecules. Thus, intramolecular bonds switch to intermolecular bonds and hydrophobic groups fold into a β-structure, which is more energetically favourable [5]. For temperatures just above the glass transition region, the crystal growth rate tends to be low and their final stable crystallinity at equilibrium is also small. When moving to the region of higher temperatures, the kinetics of crystal growth changes, and a higher degree of crystallinity can be achieved in a shorter time [34]. When treated with alcohols (methanol, ethanol), the change in conformation is due to the peculiarity of the polypeptide structure – the double character of the C-N bond in fibroin strictly restricts rotation around it. The interaction of ions or polar molecules with CO or NH groups favours electron density transfer, a decrease in the strength of the double bond and an increase in mobility [5].
As crystallinity increases, flexibility of the film decreases. Increased plasticity can be achieved by steam treatment and by adding plasticisers to the film. When treated with water vapour, water molecules penetrate the network of macromolecules and plasticise the system. The main effect of introducing both water vapour and plasticiser is to lower the glass transition temperature (Тс). With increasing plasticiser content, the glass transition temperature decreases uniformly up to some value of plasticiser concentration in the polymer. In presence of plasticiser, the polymer retains highly elastic properties at lower temperature [35]. Glycerol, calcium chloride, glucose (dextrose) can act as a plasticiser for fibroin. Table 2 shows the mechanical characteristics of plasticised fibroin films depending on the plasticiser and its content in the film.
RESULTS AND DISCUSSION
Obtaining silk fibroin solution in laboratory conditions was carried out in several steps (Fig.7). To transfer the protein from the native state with β confirmations to the water-soluble state, a three-component solution of calcium chloride, ethyl alcohol, and water with a molar ratio of 1 : 2 : 8 was used as a medium capable of penetrating into the structure and destroying it to a helical state.
In order to work out the formation technology of homogeneous solid-phase structures with β conformation, 7 film samples of fibroin were formed by drop method. The peculiarities of the process of their obtaining are described in Table 3. The fold growth was controlled qualitatively by wetting angle and quantitatively by IR spectrometry by bands of amide I, which is responsible for the secondary structure of the protein. The ratio of α and β conformations in the structures was estimated by the ratio of the areas of the corresponding peaks (Fig.8). The studies showed that the protein is represented predominantly by the b sheet structure, the ratio of conformations for the tested samples takes values from 0.17 to 0.57. Sample No.2 has the most ordered structure (the content of the disordered part of the film is 0.11), which appeared due to strong interchain interactions, as evidenced by the shift of the main bands to the region of lower wave numbers. And it also has one of the highest contents of β conformations: 0.73. This sample was heated at 40 оС in the presence of water vapour and after drying was further stretched over a water bath to orient the crystallites along the film. In contrast, sample No. 7 is characterised by a high content of a-helicals, indicating a greater plasticity of the film resulting from the interaction of Ca2 ⁺ with the protein.
The introduction of fibroin solution into three-dimensional colloidal photonic crystals obtained by the method described in [40] was carried out by impregnation followed by drying at 55 °C and treatment with ethyl alcohol for 1 hour. As a result, structures with clearly visualisable PVZ were obtained (Fig.9). Filling the interspherical voids of the photonic crystal with fibroin expectedly led to a shift of the forbidden zone λb towards the IR region.
Studies have shown that composite structure is most successfully formed at a fibroin solution concentration of 3.5%. In this case, viscosity of the solution allows it, on the one hand, to penetrate into the voids of the colloidal lattice and, on the other hand, to form a solid-phase framework inside the matrix of the photonic crystal.
CONCLUSIONS
"Smart" structures based on silk fibroin represent a promising class of materials for fabrication of sensor structures and MEMS for their use in electronics, biophotonics and medicine. The technological solutions obtained in this work can be applied to obtain solutions and solid-phase protein structures in such products prototypes production. Continuation of the work will be devoted to refining technology of inverting and obtaining inverse photonic crystals from silk fibroin.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Wearable flexible electronics is an actively developing field of electronics [1]. Its distinctive feature is the small size of devices, their flexibility and lightness, as well as the use of organic materials as both substrate material and functional layers. In this regard, wearable electronic devices can be used to measure human body parameters such as temperature, chemical analysis of biological fluids, pressure, and pulse. The demand for this type of electronic devices is constantly growing [2], which imposes requirements for their production and utilisation to reduce waste and harm to the environment. Functional structures should be biocompatible, mechanically robust, environmentally friendly in production and easily recyclable.
Biopolymers, in particular silk fibroin, fulfil these requirements. Fibroin is a fibrillar protein. It is most often obtained from the cocoons of the mulberry silkworm Bombyx mori (Fig.1) by purifying the cocoons from the fibre-binding protein sericin.
Natural silk is produced on an industrial scale, usually for textile industry, but it is also interesting for medical wearable electronics production. Fibroin consists of two chains, a heavy chain (350–370 kDa) and a light chain (25 kDa). The heavy chain is composed of 90% of fibroin’s fibrophobic amino acid residues (Gly ∼43–46%, Ala ∼25–30%, Ser ∼12%) [3] (Fig.2). The friable regions of fibroin consist mainly of polar amino acid residues [4].
This material has unique properties that can be varied for three main types of conformations: α-helixes, β-folds, and random turns and twists. In the native state, 50–60% of fibroin consists of β phase [5], which characterises the crystalline regions of the polymer and determines its strength and stiffness. Spirals (α conformation – amorphous peptide chain) provide silk elasticity. Fibroin fibres have a tensile strength of 610–690 MPa and an elastic modulus of 15–17 MPa [6]. This protein is thermostable: its denaturation temperature is higher than 127 °C. Films from regenerated fibroin are optically transparent: they have 90–95% transmittance of the visible spectrum of radiation, refractive index in the visible spectrum nf = 1.54 [7]. Fibroin is biocompatible and biodegradable [8]. The presence of active functional sites in fibroin, such as primary amino group, facilitates incorporation of various conductive elements such as conductive polymers, carbon-based fillers, and metal interfaces [9]. Thus, fibroin, which is inherently an insulator, can be endowed with electrically conductive properties. In addition, by acting on the polar groups of fibroin macromolecules through external stimuli such as water vapour, methyl/ethyl alcohol, temperature, ultrasound, pH, and UV radiation, the protein structure can be modified and controlled [10].
In electronic devices, fibroin can be used in various forms: hydrogels, nanofibres, films, particles, and sponges [11] (Fig.3). In [12] application of a hydrogel based on fibroin, polyvinyl alcohol and borax for manufacturing of artificial leather and triboelectric generators is considered. The addition of silk to polyvinyl alcohol hydrogels improved their stability and also significantly increased the moisture absorption.
The options for using silk fibroin as a functional material are varied and numerous. In [13] a variant of fibroin application as an optical waveguide on CR-39 polymer substrate is presented. The obtained fibroin waveguide had a transparency of 85% in the visible light region and low optical losses both in the visible spectrum and in the IR region. Chemical analysis of biological fluids requires high sensitivity to biomarkers. In [14], an electrochemical sensor based on carbon cloth and nitrogen-doped silk (SilkNCT) was proposed. Compared with conventional electrode materials, SilkNCT, exhibits an internal hierarchical and porous mesh woven structure, which provides good contact with reagents and efficient electron transfer. A fibroin-based moisture sensor is described in [15]. In [16] it is shown that fibroin films variability under external factors influence can be used in information coding devices. In connection with biocompatibility and biodegradation of silk fibroin, application and creation of "smart" sensor structures based on it, for example, for measuring intraocular pressure or analysing composition of biological fluid, is being actively studied. Impregnation of colloidal photonic crystal with fibroin solution and subsequent obtaining of inverse photonic-crystalline structure expands the possibilities of adjusting the photonic forbidden zone under changing humidity, pressure [17] and mechanical force. In [18], we analysed a spectrophotometric method for measuring the longitudinal strain of the flexible photonic crystal structure of silk fibroin (Fig.4). It was shown that acceptable values of uncertainty of such measurements (less than 10%) open prospects for their use in electronics and medicine.
In the presented project the technology of obtaining silk fibroin solution and its solid phase with predominance of β conformation has been worked out, and possibility of protein photonic crystal structure formation has been shown.
MATERIALS AND METHODS
The process of preparating an aqueous solution of fibroin consists of the steps of cocoons preparation, degumming followed by washing and drying, dissolution, dialysis, centrifugation and filtration to remove the undissolved precipitate (Fig.5). The choice of methods and modes of their implementation in laboratory conditions was carried out on the basis of analysing information from literature sources and studying the nature of the processes.
The degumming step is necessary to purify cocoons from sericin. The process can be realised by physical, chemical, physicochemical and biological methods [19]. During the degumming process, it is important not only to remove the fibre-binding protein, but also not to destroy the long fibroin chains. Degumming leads to degradation of the heavy chain of fibroin from a molecular mass of 350–390 kDa to a wide mass distribution from an average molecular mass of about 150 kDa to small fragments of 40–50 kDa [20], which further affects the mechanical properties of the resulting structures and materials. The most common method is to clean the cocoons in an alkaline or neutral medium at elevated temperature. The medium can be soda ash (Na2CO3), laundry soap or neutral soap.
Since fibroin is 3/4 composed of non-polar hydrophobic amino acids, it is resistant to most solvents. To dissolve fibroin, it is necessary to break the strong hydrogen bonds, thereby converting it from a hydrophobic β-structure to a hydrophilic α-structure. To break the hydrogen bonds between macromolecules, solvent ions must interact with polar and charged groups of fibroin side chains. For this purpose, it is necessary to choose a solvent that can effectively penetrate into a protein molecule. Table 1 shows the liquids used in practice for fibroin dissolution.
The main disadvantage of saline-containing aqueous, aqueous-organic and organic solutions of fibroin is the long preparation time: fibroin has to be pre-activated by means of a saline solution, once obtained, the solutions have to be dialysed to remove solvents, considerable time cost is also required to regenerate the solvents in order to reuse them [4]. When ionic liquids are used, solubility of fibroin depends more on the anion nature. Ions with a greater ability to form hydrogen bonds dissolve fibroin better. The disadvantage of these solvents is their property of sorbing water from the atmosphere, which negatively affects the process [28].
A dialysis step is necessary to remove salt if an aqueous or aqueous-organic solvent is used. A cellulose dialysis bag with a pore diameter of 12–14 kDa is typically used as the membrane. The buffer solution is distilled or bidistilled water.
Fibroin-based films are prepared by drop casting on a substrate, centrifugation, and printing methods [32]. The protein molecule of fibroin is prone to the self-organisation process and can be subjected to controlled functionalisation in the film [33]. Fig.6 shows external influences both stimulating the growth of the crystalline phase (water vapour, temperature, etc.) and destroying the long chains of fibroin (UV).
When temperature rises above 40 °C, stability of many polypeptides decreases, resulting in the unfolding of coiled globules or spirals of molecules. The fibroin macromolecule is characterised by an increase in conformational mobility and destabilisation of the helical conformation with increasing temperature. As a result of this process, active groups compensate their potential to form H-bonds by interacting with neighbouring molecules. Thus, intramolecular bonds switch to intermolecular bonds and hydrophobic groups fold into a β-structure, which is more energetically favourable [5]. For temperatures just above the glass transition region, the crystal growth rate tends to be low and their final stable crystallinity at equilibrium is also small. When moving to the region of higher temperatures, the kinetics of crystal growth changes, and a higher degree of crystallinity can be achieved in a shorter time [34]. When treated with alcohols (methanol, ethanol), the change in conformation is due to the peculiarity of the polypeptide structure – the double character of the C-N bond in fibroin strictly restricts rotation around it. The interaction of ions or polar molecules with CO or NH groups favours electron density transfer, a decrease in the strength of the double bond and an increase in mobility [5].
As crystallinity increases, flexibility of the film decreases. Increased plasticity can be achieved by steam treatment and by adding plasticisers to the film. When treated with water vapour, water molecules penetrate the network of macromolecules and plasticise the system. The main effect of introducing both water vapour and plasticiser is to lower the glass transition temperature (Тс). With increasing plasticiser content, the glass transition temperature decreases uniformly up to some value of plasticiser concentration in the polymer. In presence of plasticiser, the polymer retains highly elastic properties at lower temperature [35]. Glycerol, calcium chloride, glucose (dextrose) can act as a plasticiser for fibroin. Table 2 shows the mechanical characteristics of plasticised fibroin films depending on the plasticiser and its content in the film.
RESULTS AND DISCUSSION
Obtaining silk fibroin solution in laboratory conditions was carried out in several steps (Fig.7). To transfer the protein from the native state with β confirmations to the water-soluble state, a three-component solution of calcium chloride, ethyl alcohol, and water with a molar ratio of 1 : 2 : 8 was used as a medium capable of penetrating into the structure and destroying it to a helical state.
In order to work out the formation technology of homogeneous solid-phase structures with β conformation, 7 film samples of fibroin were formed by drop method. The peculiarities of the process of their obtaining are described in Table 3. The fold growth was controlled qualitatively by wetting angle and quantitatively by IR spectrometry by bands of amide I, which is responsible for the secondary structure of the protein. The ratio of α and β conformations in the structures was estimated by the ratio of the areas of the corresponding peaks (Fig.8). The studies showed that the protein is represented predominantly by the b sheet structure, the ratio of conformations for the tested samples takes values from 0.17 to 0.57. Sample No.2 has the most ordered structure (the content of the disordered part of the film is 0.11), which appeared due to strong interchain interactions, as evidenced by the shift of the main bands to the region of lower wave numbers. And it also has one of the highest contents of β conformations: 0.73. This sample was heated at 40 оС in the presence of water vapour and after drying was further stretched over a water bath to orient the crystallites along the film. In contrast, sample No. 7 is characterised by a high content of a-helicals, indicating a greater plasticity of the film resulting from the interaction of Ca2 ⁺ with the protein.
The introduction of fibroin solution into three-dimensional colloidal photonic crystals obtained by the method described in [40] was carried out by impregnation followed by drying at 55 °C and treatment with ethyl alcohol for 1 hour. As a result, structures with clearly visualisable PVZ were obtained (Fig.9). Filling the interspherical voids of the photonic crystal with fibroin expectedly led to a shift of the forbidden zone λb towards the IR region.
Studies have shown that composite structure is most successfully formed at a fibroin solution concentration of 3.5%. In this case, viscosity of the solution allows it, on the one hand, to penetrate into the voids of the colloidal lattice and, on the other hand, to form a solid-phase framework inside the matrix of the photonic crystal.
CONCLUSIONS
"Smart" structures based on silk fibroin represent a promising class of materials for fabrication of sensor structures and MEMS for their use in electronics, biophotonics and medicine. The technological solutions obtained in this work can be applied to obtain solutions and solid-phase protein structures in such products prototypes production. Continuation of the work will be devoted to refining technology of inverting and obtaining inverse photonic crystals from silk fibroin.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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