Flexible Biosensors for the Impedimetric Detection of Protein Targets Using Silk-Conductive Polymer Biocomposites

To expand the applications of flexible biosensors in point-of-care healthcare applications beyond monitoring of biophysical parameters, it is important to devise strategies for the detection of various proteins and biomarkers. Here, we demonstrate a flexible, fully organic, biodegradable, label-free impedimetric biosensor for the critical biomarker, vascular endothelial growth factor (VEGF). This biosensor was constructed by photolithographically patterning a conducting ink consisting of a photoreactive silk sericin coupled with a conducting polymer. These functional electrodes are printed on flexible fibroin substrates that are controllably thick and can be free-standing, or conform to soft surfaces. Detection was accomplished via the antibody to VEGF which was immobilized within the conducting matrix. The results indicated that the developed flexible biosensor was highly sensitive and selective to the target protein, even in challenging biofluids such as human serum. The biosensors themselves are biocompatible and degradable. Through this work, the developed flexible biosensor based on a simple and label-free strategy can find practical applications in the monitoring of wound healing or early disease diagnosis

Nanomechanics of Full-Length Nebulin: An Elastic Strain Gauge in the Skeletal Muscle Sarcomere

Nebulin, a family of giant modular proteins (MW 700−800 kDa), acts as a F-actin thin filament ruler and calcium-linked regulator of actomyosin interaction. The nanomechanics of full length, native rabbit nebulin was investigated with an atomic force microscope by tethering, bracketing, and stretching full-length molecules via pairs of site-specific antibodies that were attached covalently, one to a protein resistant self-assembled monolayer of oligoethylene glycol and the other to the cantilever. Using this new nanomechanics platform that enables the identification of single molecule events via an unbiased analysis of detachment force and distance of all force curves, we showed that nebulin is elastic and extends to ∼1 μm by external force up to an antibody detachment force of ∼300−400 pN. Upon stretching, nebulin unravels and yields force spectra with craggy mountain range profiles with variable numbers and heights of force peaks. The peak spacings, analyzed by the model-independent, empirical Hilbert−Huang transform method, displayed underlying periodicities at ∼15 and ∼22 nm that may result from the unfolding of one or more nebulin modules between force peaks. Nebulin may act as an elastic strain gauge that interacts optimally with actin only under appropriate strain and stress. This stretch to match protein ruler may also exert a compressive force that stabilizes thin filaments against stress during contraction. We propose that the elasticity of nebulin is integral and essential in the muscle sarcomere

Degradable Elastomeric Silk Biomaterial for Flexible Bioelectronics

The integration of degradable and biomimetic approaches in material and device development can facilitate the next generation of sustainable (bio) electronics. The use of functional degradable materials presents exciting opportunities for applications in healthcare, soft robotics, energy, and electronics. These include conformability to curved surfaces, matching of stiffness of tissue, and the ability to withstand mechanical deformations. Nature-derived materials such as silk fibroin (SF) provide excellent biocompatibility, resorbability, and tunable properties toward such goals. However, fibroin alone lacks the required mechanical properties and durability for processing in biointegrated electronics and dry conditions. To overcome these limitations, we report on an elastomeric photocurable composite of silk fibroin and poly­(dimethylsiloxane) (PDMS). Photofibroin (containing methacryl functionalities) is doped with photoPDMS (methacryloxypropyl-terminated poly­(dimethylsiloxane)) to form an elastomeric photofibroin (ePF) composite. The elastomeric silk is photocurable, allowing for microfabrication using UV photolithography. It is suitable for circuits, strain-sensing devices, and biointegrated systems. The ePF exhibits flexibility in both wet and dry conditions, enhanced mechanical strength and long-term durability, and optical transparency. It is stable at high temperatures, compatible with electronic materials, and cytocompatible while being enzymatically degradable. This work therefore highlights a path toward combining natural and synthetic materials to achieve versatile properties and demonstrates the potential of silk fibroin composites in (bio) electronics, encapsulation, and packaging

Table_1_Use of Silk Proteins to Form Organic, Flexible, Degradable Biosensors for Metabolite Monitoring.DOCX

The development of sustainable and degradable biosensors and bioelectronics has implications for implantable systems, as well in addressing issues of electronic waste. Mechanically flexible and bioresorbable sensors can find applications at soft biological interfaces. While devices typically use metallic and synthetic components and interconnects that are non-degradable or have the potential to cause adverse tissue reactions, the use of nature-derived materials and conducting polymers can provide distinct advantages. In particular, silk fibroin and sericin can provide a unique palette of properties, providing both structural and functional elements. Here, a fully organic, mechanically flexible biosensor in an integrated 3-electrode configuration is demonstrated. Silk sericin conducting ink is micropatterned on a silk fibroin substrate using a facile photolithographic process. Next, using a conducting polymer wire sheathed in silk fibroin, organic interconnects are used to form the electrical connections. This fully organic electrochemical system has competitive performance metrics for sensing in comparison to conventional systems, as verified by detection of a model analyte—ascorbic acid. The stability of the silk biosensor through biodegradation was observed, showing that the sensors can function for several days prior to failure. Such protein-based systems can provide a useful tool for biomonitoring of analytes in the body or environment for controlled periods of time, followed by complete degradation, as transient systems for various applications.</p

Tunable Light-Actuated Interpenetrating Networks of Silk Fibroin and Gelatin for Tissue Engineering and Flexible Biodevices

Soft materials with tunable properties are valuable for applications such as tissue engineering, electronic skins, and human–machine interfaces. Materials that are nature-derived offer additional advantages such as biocompatibility, biodegradability, low-cost sourcing, and sustainability. However, these materials often have contrasting properties that limit their use. For example, silk fibroin (SF) has high mechanical strength but lacks processability and cell-adhesive domains. Gelatin, derived from collagen, has excellent biological properties, but is fragile and lacks stability. To overcome these limitations, composites of gelatin and SF have been explored. However, mechanically robust self-supported matrices and electrochemically active or micropatterned substrates were not demonstrated. In this study, we present a composite of photopolymerizable SF and photogelatin, termed photofibrogel (PFG). By incorporating photoreactive properties in both SF and gelatin, control over material properties can be achieved. The PFG composite can be easily and rapidly formed into free-standing, high-resolution architectures with tunable properties. By optimizing the ratio of SF to gelatin, properties such as swelling, mechanical behavior, enzymatic degradation, and patternability are tailored. The PFG composite allows for macroscale and microscale patterning without significant swelling, enabling the fabrication of structures using photolithography and laser cutting techniques. PFG can be patterned with electrically conductive materials, making it suitable for cell guidance and stimulation. The versatility, mechanical robustness, bioactivity, and electrochemical properties of PFG are shown for skeletal muscle tissue engineering using C2C12 cells as a model. Overall, such composite biomaterials with tunable properties have broad potential in flexible bioelectronics, wound healing, regenerative medicine, and food systems

Backbone-Branched DNA Building Blocks for Facile Angular Control in Nanostructures

Nanotechnology based on the highly specific pairing of nucleobases in DNA has been used to generate a wide variety of well-defined two- and three-dimensional assemblies, both static and dynamic. However, control over the junction angles to achieve them has been limited. To achieve higher order assemblies, the strands of the DNA duplex are typically made to deviate at junctions with configurations based on crossovers or non-DNA moieties. Such strand crossovers tend to be intrinsically unstructured with the overall structural rigidity determined by the architecture of the nanoassembly, rather than the junction itself. Specific approaches to define nanoassembly junction angles are based either on the cooperative twist- and strain-promoted tuning of DNA persistence length leading to bent DNA rods for fairly large nano-objects, or <i>de novo</i> synthesis of individual junction inserts that are typically non-DNA and based on small organic molecules or metal-coordinating ligand moieties. Here, we describe a general strategy for direct control of junction angles in DNA nanostructures that are completely tunable about the DNA helix. This approach is used to define angular vertices through readily accessible backbone-branched DNAs (bbDNAs). We demonstrate how such bbDNAs can be used as a new building block in DNA nanoconstruction to obtain well-defined nanostructures. Angular control through readily accessible bbDNA building block provides a general and versatile approach for incorporating well-defined junctions in nanoconstructs and expands the toolkit toward achieving strain free, highly size- and shape-tunable DNA based architectures

Investigations of Chemical Modifications of Amino-Terminated Organic Films on Silicon Substrates and Controlled Protein Immobilization

Fourier transform infrared spectroscopy by grazing−angle attenuated total reflection (FTIR-GATR), ellipsometry, atomic force microscopy (AFM), UV−visible spectroscopy, and fluorescence microscopy were employed to investigate chemical modifications of amino-terminated organic thin films on silicon substrates, protein immobilization, and the biological activity and hydrolytic stability of immobilized proteins. Amino-terminated organic films were prepared on silicon wafers by self-assembling 3-aminopropyltriethoxysilane (APTES) in anhydrous toluene. Surface amino groups were derivatized into three different linkers: N-hydroxysuccinimide (NHS) ester, hydrazide, and maleimide ester groups. UV−visible absorption measurements and fluorescence microscopy revealed that more than 40% of surface amino groups were chemically modified. Protein immobilization was carried out on modified APTES films containing these linkers via coupling with primary amines (−NH2) in intact monoclonal rabbit immunoglobulin G (IgG), the aldehyde (−CHO) of an oxidized carbohydrate residue in IgG, or the sulfhydryl (−SH) of fragmented half-IgG, respectively. FTIR spectra contain vibrational signatures of these functional groups present in modified APTES films and immobilized IgGs. Changes in the APTES film thickness after chemical modifications and protein immobilization were also observed by ellipsometric measurements. The biological activity and long-term hydrolytic stability of immobilized IgGs on modified APTES films were estimated by fluorescence measurements of an adsorbed antigen, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (FITC-Ab). Our results indicate that the FITC-Ab binding capacity of half-IgG immobilized via maleimide groups is greater than that of the oxidized IgG and the intact IgG immobilized via hydrazide and NHS ester groups, respectively. In addition, IgGs immobilized using all coupling chemistries were hydrolytically stable in phosphate-buffered saline (PBS)

Integrated Compositional and Nanomechanical Analysis of a Polyurethane Surface Modified with a Fluorous Oxetane Siliceous-Network Hybrid

Investigating the surface characteristics of heterogeneous polymer systems is important for understanding how to better tailor surfaces and engineering specific reactions and desirable properties. Here we report on the surface properties for a blend consisting of a major component, a linear polyurethane or thermoplastic elastomer (TPU), and a minor component that is a hybrid network. The hybrid network consists of a fluorous polyoxetane soft block and a hydrolysis/condensation inorganic (HyCoin) network. Phase separation during coating formation results in surface concentration of the minor fluorous hybrid domain. The TPU is H₁₂MDI/BD­(50)-PTMO-1000 derived from bis­(cyclohexylmethylene)-diisocyanate and butane diol (50 wt %) and poly­(tetramethylene oxide). Surface modification results from a novel network-forming hybrid composed of poly­(trifluoroethoxymethyl–methyl oxetane) diol) (3F) as the fluorous moiety end-capped with 3-isocyanatopropylriethoxysilane and bis­(triethoxysilyl)­ethane (BTESE) as a siliceous stabilizer. We use an integrated approach that combines elemental analysis of the near surface via X-ray photoelectron microscopy with surface mapping using atomic force microscopy that presents topographical and phase imaging along with nanomechanical properties. Overall, this versatile, high-resolution approach enabled unique insight into surface composition and morphology that led to a model of heterogeneous surfaces containing a range of constituents and properties

Biomimetic Designing of Functional Silk Nanotopography Using Self-assembly

In nature inorganic–organic building units create multifunctional hierarchical architectures. Organic silk protein is particularly attractive in this respect because of its micro-nanoscale structural blocks that are attributed to sophisticated hierarchical assembly imparting flexibility and compressibility to designed biohybrid materials. In the present study, aqueous silk fibroin is assembled to form nano/microtopography on inorganic silica surface via a facile diffusion-limited aggregation process. This process is driven by electrostatic interaction and only possible at a specified aminated surface chemistry. The self-assembled topography depends on the age and concentration of protein solution as well as on the surface charge distribution of the template. The self-assembled silk trails closely resemble natural cypress leaf architecture, which is considered a structural analogue of neuronal cortex. This assembled surface significantly enhances anchorage of neuronal cell and cytoskeletal extensions, providing an effective nano/microtopographical cue for cellular recognition and guidance

Stretchable and Electroactive Crosslinked Gelatin for Biodevice and Cell Culture Applications

Biomimetic substrates that incorporate functionality such as electroactivity and mechanical flexibility, find utility in a variety of biomedical applications. Toward these uses, nature-derived materials such as gelatin offer inherent biocompatibility and sustainable sourcing. However, issues such as high swelling, poor mechanical properties, and lack of stability at biological temperatures limit their use. The enzymatic crosslinking of gelatin via microbial transglutaminase (mTG) yields flexible and robust large area substrates that are stable under physiological conditions. Here, we demonstrate the fabrication and characterization of strong, stretchable, conductive mTG crosslinked gelatin thin films. Incorporation of the conductive polymer poly­(3,4-ethylenedioxythiophene) polystyrene sulfonate in the gel matrix with a bioinspired polydopamine surface coating is used to enable conductivity with enhanced mechanical properties such as extensibility and flexibility, in comparison to plain gelatin or crosslinked gelatin films. The electroconductive substrates are conducive to cell growth, supporting myoblast cell adhesion, viability, and proliferation and could find use in creating active cell culture systems incorporating electrical stimulation. The substrates are responsive to motion such as stretching and bending while being extremely handleable and elastic, making them useful for applications such as electronic skin and flexible bioelectronics. Overall, this work presents facile, yet effective development of bioinspired conductive composites as substrates for bio-integrated devices and functional tissue engineering