Label-Free Neuropeptide Detection beyond the Debye Length Limit.

Biosensors with high selectivity, high sensitivity, and real-time detection capabilities are of significant interest for diagnostic applications as well as human health and performance monitoring. Graphene field-effect transistor (GFET) based biosensors are suitable for integration into wearable sensor technology and can potentially demonstrate the sensitivity and selectivity necessary for real-time detection and monitoring of biomarkers. Previously reported DC-mode GFET biosensors showed a high sensitivity for sensing biomarkers in solutions with a low salt concentration. However, due to Debye length screening, the sensitivity of the DC-mode GFET biosensors decreases significantly during operation in a physiological fluid such as sweat or interstitial fluid. To overcome the Debye screening length limitation, we report here alternating current (AC) mode heterodyne-based GFET biosensors for sensing neuropeptide-Y (NPY), a key stress biomarker, in artificial sweat at physiologically relevant ionic concentrations. Our AC-mode GFET biosensors show a record ultralow detection limit of 2 × 10-18 M with an extensive dynamic range of 10 orders of magnitude in sensor response to target NPY concentration. The sensors were characterized for various carrier frequencies (ranging from 30 kHz to 2 MHz) of the applied AC voltages and various salt concentrations (10, 50, and 100 mM). Contrary to DC-mode sensing, the AC-mode sensor response increases with an increase in salt concentration in the electrolyte. The sensor response can be further enhanced by tuning the carrier frequency of the applied AC voltage. The optimum response frequency of our sensor is approximately 400-600 kHz for salt concentrations of 50 and 100 mM, respectively. The salt-concentration- and frequency-dependent sensor response can be explained by an electrolyte-gated capacitance model.

Molecular Dynamics Investigation into pH Dependent Metal Binding of the Intrinsically Disordered Worm Jaw Protein, Nvjp-1.

Sclerotization of the Nereis virens jaw is mediated by metal binding to the histidine-rich jaw protein, Nvjp-1. Previous studies showed that the mechanical properties of Nvjp-1 hydrogels could be modulated with zinc binding as well as the associated anion. Here, we show that the mechanical properties of Nvjp-1 hydrogels can be modulated by pH and that zinc binding to Nvjp-1 is stable at both acidic and alkaline pH conditions. To probe the mechanism of Zn2+ binding to Nvjp-1 at different pH conditions, we utilized all atom molecular dynamics simulations employing a polarizable force field. At low pH conditions, polar residues predominantly interacted with Zn2+, with at most two residues interacting with a given zinc ion. Surprisingly, little to no Zn2+ binding was observed with the abundant Nvjp-1 acidic residues, which form salt-bridges with the protonated histidines to effectively block their binding to Zn2+ ions. As the pH was shifted to alkaline conditions, Zn2+ binding residues reconfigured to form additional coordination bonds with histidine, resulting in a reduction in the radius of gyration that correlated with hydrogel sclerotization. Furthermore, acetate ions were shown to facilitate the capture of zinc ions through association with protonated histidines at low pH, freeing acidic residues to interact with Zn2+ ions and increasing the number of Zn2+ ions that diffuse into the Nvjp-1 interior. Thus, these studies provide valuable molecular insights into how amino acid residues in Nvjp-1 manage metal salt binding and coordination in hydrogels as a function of the pH and ionic environments.

Gamma estimator of Jarzynski equality for recovering binding energies from noisy dynamic data sets.

A fundamental problem in thermodynamics is the recovery of macroscopic equilibrated interaction energies from experimentally measured single-molecular interactions. The Jarzynski equality forms a theoretical basis in recovering the free energy difference between two states from exponentially averaged work performed to switch the states. In practice, the exponentially averaged work value is estimated as the mean of finite samples. Numerical simulations have shown that samples having thousands of measurements are not large enough for the mean to converge when the fluctuation of external work is above 4 kBT, which is easily observable in biomolecular interactions. We report the first example of a statistical gamma work distribution applied to single molecule pulling experiments. The Gibbs free energy of surface adsorption can be accurately evaluated even for a small sample size. The values obtained are comparable to those derived from multi-parametric surface plasmon resonance measurements and molecular dynamics simulations.

3D Printing of Regenerated Silk Fibroin and Antibody-Containing Microstructures via Multiphoton Lithography.

Regenerated silk fibroin, a biopolymer derived from silkworm cocoons, is a versatile material that has been widely explored for a number of applications (e.g., drug delivery, tissue repair, biocompatible electronics substrates, and optics) due to its attractive biochemical properties and processability. Here, we report on the free-form printing of silk-based, 3D microstructures through multiphoton lithography. Utilizing multiphoton lithography in conjunction with specific photoinitiator chemistry and postprint cross-linking, a number of microarchitectures were achieved including self-supporting fibroin arches. Further, the straightforward production of high fidelity and biofunctional protein architectures was enabled through the printing of aqueous fibroin/immunoglobulin solutions.

Vertically aligned peptide nanostructures using plasma-enhanced chemical vapor deposition.

In this study, we utilize plasma-enhanced chemical vapor deposition (PECVD) for the deposition of nanostructures composed of diphenylalanine. PECVD is a solvent-free approach and allows sublimation of the peptide to form dense, uniform arrays of peptide nanostructures on a variety of substrates. The PECVD deposited d-diphenylalanine nanostructures have a range of chemical and physical properties depending on the specific discharge parameters used during the deposition process.

Dielectric breakdown strength of regenerated silk fibroin films as a function of protein conformation.

Derived from Bombyx mori cocoons, regenerated silk fibroin (RSF) exhibits excellent biocompatibility, high toughness, and tailorable biodegradability. Additionally, RSF materials are flexible, optically clear, easily patterned with nanoscale features, and may be doped with a variety bioactive species. This unique combination of properties has led to increased interest in the use of RSF in sustainable and biocompatible electronic devices. In order to explore the applicability of this biopolymer to the development of future bioelectronics, the dielectric breakdown strength (Ebd) of RSF thin films was quantified as a function of protein conformation. The application of processing conditions that increased ß-sheet content (as determined by FTIR analysis) and produced films in the silk II structure resulted in RSF materials with improved Ebd with values reaching up to 400 V/µm.

The impact of culture medium on the development and physiology of biofilms of Pseudomonas fluorescens formed on polyurethane paint.

Microbial biofilms cause the deterioration of polymeric coatings such as polyurethanes (PUs). In many cases, microbes have been shown to use the PU as a nutrient source. The interaction between biofilms and nutritive substrata is complex, since both the medium and the substratum can provide nutrients that affect biofilm formation and biodeterioration. Historically, studies of PU biodeterioration have monitored the planktonic cells in the medium surrounding the material, not the biofilm. This study monitored planktonic and biofilm cell counts, and biofilm morphology, in long-term growth experiments conducted with Pseudomonas fluorescens under different nutrient conditions. Nutrients affected planktonic and biofilm cell numbers differently, and neither was representative of the system as a whole. Microscopic examination of the biofilm revealed the presence of intracellular storage granules in biofilms grown in M9 but not yeast extract salts medium. These granules are indicative of nutrient limitation and/or entry into stationary phase, which may impact the biodegradative capability of the biofilm.

Epidermal growth factor: layered silicate nanocomposites for tissue regeneration.

Wound healing is a complex, multistep process that can be summarized into three stages, namely, hemostasis and inflammation, proliferation, and finally, tissue remodeling. Battlefield wound healing demands rapid hemostasis using clotting or cauterizing agents to immediately limit blood loss, but this occurs at the expense of proper tissue repair beyond hemostasis. Layered silicate clays such as kaolin and montmorillonite (MMT) have been previously shown to induce blood clotting due to their ability to form charged interactions with clotting factors. The charge characteristics of sodium MMT (Na-MMT) also enable functionalization with active biomolecules. Herein we functionalized Na-MMT with epidermal growth factor (EGF) via ion exchange reaction to create a nanocomposite (MMT-EGF) with approximately 0.004 EGF molecules per Na(+) exchange site and conduct biochemical analyses of keratinocytes after treatment with MMT-EGF. Our results demonstrate that EGF immobilized on MMT retains the ability to activate the epidermal growth factor receptor (EGRF), causing phosphorylation of the AKT and MEK1 pathways, as well as upregulation of its downstream target gene expression involved in cell growth and migration. This study also shows that like EGF, MMT-EGF treatment can stimulate cell migration in vitro, which is dependent on ERK1/2 phosphorylation.