Ultrasensitive tapered optical fiber refractive index glucose sensor.

Refractive index (RI) sensors are of great interest for label-free optical biosensing. A tapered optical fiber (TOF) RI sensor with micron-sized waist diameters can dramatically enhance sensor sensitivity by reducing the mode volume over a long distance. Here, a simple and fast method is used to fabricate highly sensitive refractive index sensors based on localized surface plasmon resonance (LSPR). Two TOFs (l = 5 mm) with waist diameters of 5 µm and 12 µm demonstrated sensitivity enhancement at λ = 1559 nm for glucose sensing (5-45 wt%) at room temperature. The optical power transmission decreased with increasing glucose concentration due to the interaction of the propagating light in the evanescent field with glucose molecules. The coating of the TOF with gold nanoparticles (AuNPs) as an active layer for glucose sensing generated LSPR through the interaction of the evanescent wave with AuNPs deposited at the tapered waist. The results indicated that the TOF (Ø = 5 µm) exhibited improved sensing performance with a sensitivity of 1265%/RIU compared to the TOF (Ø = 12 µm) at 560%/RIU towards glucose. The AuNPs were characterized using scanning electron microscopy and ultraviolent-visible spectroscopy. The AuNPs-decorated TOF (Ø = 12 µm) demonstrated a high sensitivity of 2032%/RIU toward glucose. The AuNPs-decorated TOF sensor showed a sensitivity enhancement of nearly 4 times over TOF (Ø = 12 µm) with RI ranging from 1.328 to 1.393. The fabricated TOF enabled ultrasensitive glucose detection with good stability and fast response that may lead to next-generation ultrasensitive biosensors for real-world applications, such as disease diagnosis.

Advances in tissue engineering and biofabrication for in vitro skin modeling.

The global prevalence of skin disease and injury is continually increasing, yet conventional cell-based models used to study these conditions do not accurately reflect the complexity of human skin. The lack of inadequate in vitro modeling has resulted in reliance on animal-based models to test pharmaceuticals, biomedical devices, and industrial and environmental toxins to address clinical needs. These in vivo models are monetarily and morally expensive and are poor predictors of human tissue responses and clinical trial outcomes. The onset of three-dimensional (3D) culture techniques, such as cell-embedded and decellularized approaches, has offered accessible in vitro alternatives, using innovative scaffolds to improve cell-based models' structural and histological authenticity. However, these models lack adequate organizational control and complexity, resulting in variations between structures and the exclusion of physiologically relevant vascular and immunological features. Recently, biofabrication strategies, which combine biology, engineering, and manufacturing capabilities, have emerged as instrumental tools to recreate the heterogeneity of human skin precisely. Bioprinting uses computer-aided design (CAD) to yield robust and reproducible skin prototypes with unprecedented control over tissue design and assembly. As the interdisciplinary nature of biofabrication grows, we look to the promise of next-generation biofabrication technologies, such as organ-on-a-chip (OOAC) and 4D modeling, to simulate human tissue behaviors more reliably for research, pharmaceutical, and regenerative medicine purposes. This review aims to discuss the barriers to developing clinically relevant skin models, describe the evolution of skin-inspired in vitro structures, analyze the current approaches to biofabricating 3D human skin mimetics, and define the opportunities and challenges in biofabricating skin tissue for preclinical and clinical uses.

Flexible battery-less wireless glucose monitoring system.

In this work, a low power microcontroller-based near field communication (NFC) interfaced with a flexible abiotic glucose hybrid fuel cell is designed to function as a battery-less glucose sensor. The abiotic glucose fuel cell is fabricated by depositing colloidal platinum (co-Pt) on the anodic region and silver oxide nanoparticles-multiwalled carbon nanotubes (Ag2O-MWCNTs) composite on the cathodic region. The electrochemical behavior is characterized using cyclic voltammetry and chronoamperometry. This glucose hybrid fuel cell generated an open circuit voltage of 0.46 V, short circuit current density of 0.444 mA/cm2, and maximum power density of 0.062 mW/cm2 at 0.26 V in the presence of 7 mM physiologic glucose. Upon device integration of the abiotic glucose hybrid fuel cell with the NFC module, the data from the glucose monitoring system is successfully transmitted to an android application for visualization at the user interface. The cell voltage correlated (r2 = 0.989) with glucose concentration (up to 19 mM) with a sensitivity of 13.9 mV/mM•cm2.

Cu and Ni Co-sputtered heteroatomic thin film for enhanced nonenzymatic glucose detection.

In this work, we report a wafer-scale and chemical-free fabrication of nickel (Ni) and copper (Cu) heteroatomic Cu-Ni thin films using RF magnetron sputtering technique for non-enzymatic glucose sensing application. The as-prepared wafer-scale Cu-Ni thin films exhibits excellent electrocatalytic activity toward glucose oxidation with a 1.86 µM detection limit in the range of 0.01 mM to 20 mM range. The Cu-Ni film shows 1.3- and 5.4-times higher glucose oxidation activity in comparison to the Cu and Ni electrodes, respectively. The improved electrocatalytic activity is attributed to the synergistic effect of the bimetallic catalyst and high density of grain boundaries. The Cu-Ni electrodes also possessed excellent anti-interference characteristics. These results indicate that Cu-Ni heteroatomic thin film can be a potential candidate for the development of non-enzymatic glucose biosensor because of its chemical free synthesis, excellent reproducibility, reusability, and long-term stability.

Plasmonic-Based Biosensor for the Early Diagnosis of Prostate Cancer.

A tapered optical fiber (TOF) plasmonic biosensor was fabricated and used for the sensitive detection of a panel of microRNAs (miRNAs) in human serum obtained from noncancer and prostate cancer (PCa) patients. Oncogenic and tumor suppressor miRNAs let-7a, let-7c, miR-200b, miR-141, and miR-21 were tested as predictive cancer biomarkers since multianalyte detection minimizes false-positive and false-negative rates and establishes a strong foundation for early PCa diagnosis. The biosensing platform integrates metallic gold triangular nanoprisms (AuTNPs) laminated on the TOF to excite surface plasmon waves in the supporting metallic layer and enhance the evanescent mode of the fiber surface. This sensitive TOF plasmonic biosensor as a point-of-care (POC) cancer diagnostic tool enabled the detection of the panel of miRNAs in seven patient serums without any RNA extraction or sample amplification. The TOF plasmonic biosensor could detect miRNAs in human serum with a limit of detection between 179 and 580 aM and excellent selectivity. Statistical studies were obtained to differentiate cancerous from noncancerous samples with a p-value <0.0001. This high-throughput TOF plasmonic biosensor has the potential to expand and advance POC diagnostics for the early diagnosis of cancer.

Label-free tapered optical fiber plasmonic biosensor.

We designed and fabricated a novel label-free ultrasensitive tapered optical fiber (TOF) plasmonic biosensor that successfully detected a five panel of microRNAs with good selectivity. The biosensing platform integrates three different metallic nanoparticles: gold spherical nanoparticles (AuNPs), gold nanorods (AuNRs), and gold triangular nanoprisms (AuTNPs) laminated TOF to enhance the evanescent mode. The dip in the intensity profile of the transmission spectrum corresponded to the specific wavelength of the nanoparticle. The AuTNPs laminated TOF was found to exhibit the highest refractive index sensitivity and was therefore used to assay the panel of microRNAs. Single stranded DNA probes were self-assembled on the AuTNPs TOF plasmonic biosensors to achieve the highest sensitivity from the formation of hydrogen bonds between the ssDNAs and the target microRNAs. Experimentally, we observed that by measuring the spectral shifts, a limit of detection (LOD) between 103 aM and 261 aM for the panel of microRNAs can be achieved. Additionally, the ssDNA layer immobilized on the TOF plasmonic biosensor resulted in an extended dynamic range of 1 fM - 100 nM. In human serum solution, clinically relevant concentration of the panel of microRNAs were successfully detected with a LOD between 1.097 fM to 1.220 fM. This is the first report to demonstrate the applicability of our TOF plasmonic biosensor approach to detect a panel of microRNAs. This simple yet highly sensitive approach can provide a high-throughput and scalable sensor for detecting and quantifying large arrays of microRNAs, thereby expanding the applications of biosensors.

Flexible electrochemical uric acid and glucose biosensor.

Fully integrated uric acid (UA) and glucose biosensors were fabricated on polydimethylsiloxane/polyimide platform by facile one step laser scribed technique. The laser scribed graphene (LSG) on the thin polyimide film was functionalized using pyrenebutanoic acid, succinimide ester (PBSE) to improve the electrochemical activity of the biosensors. The LSG was further decorated with platinum nanoparticles (PtNPs) to promote the electrocatalytic activity towards the oxidation of UA. Glucose oxidase was immobilized on the PtNPs modified surface for selective detection of glucose. The fabricated biosensors were characterized via scanning electron microscopy (SEM), Energy dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS), and electrochemical methods (cyclic voltammetry and amperometry measurements). Outstanding electrocatalytic activities toward oxidation of UA and glucose were demonstrated. A wide detection range of 5 µM to 480 µM UA with a high sensitivity of 156.56 µA/mMcm2 and a calculated detection limit (LOD) of 0.018 µM (S/N = 3) were achieved for the UA biosensor. The glucose biosensor exhibited a detection range of 5 µM to 3200 µM with a sensitivity of 12.64 µA/mMcm2 and an LOD of 2.57 µM (S/N = 3). These integrated biosensors offer great promise for potential applications in wearable UA and glucose sensing due to their good sensitivity, selectivity, and stability properties.

A Glucose Monitoring System With Remote Data Access.

A cost-effective portable glucose monitoring system with remote data access based on a novel e-oscilloscope was developed using a glucose biofuel cell and a capacitor circuit interfaced to an ESP8266 microcontroller programmed to convert the charge/discharge rates of the capacitor functioning as a transducer. The capacitor charge/discharge rates were converted into glucose concentration readings that is monitored remotely. The glucose monitoring system comprise a glucose biofuel cell, a charge pump circuit, a capacitor and an ESP microcontroller. The anode was fabricated by modifying a gold microwire with nanoporous colloidal platinum (Au-co-Pt) and the cathode was constructed using a mesh dense network of multiwalled carbon nanotubes modified with bilirubin oxidase, respectively. The glucose monitoring system showed sensitivity of 1.18 Hz/mM · cm2 with a correlation coefficient of 0.9939 with increasing glucose concentration from 1 mM to 25 mM. In addition, the glucose monitoring system exhibited optimal operation at a pH of 7.4 and 37 °C, which is ideal for physiological glucose monitoring.

Electrochemical Detection of Neurotransmitters.

Neurotransmitters are important chemical messengers in the nervous system that play a crucial role in physiological and physical health. Abnormal levels of neurotransmitters have been correlated with physical, psychotic, and neurodegenerative diseases such as Alzheimer's, Parkinson's, dementia, addiction, depression, and schizophrenia. Although multiple neurotechnological approaches have been reported in the literature, the detection and monitoring of neurotransmitters in the brain remains a challenge and continues to garner significant attention. Neurotechnology that provides high-throughput, as well as fast and specific quantification of target analytes in the brain, without negatively impacting the implanted region is highly desired for the monitoring of the complex intercommunication of neurotransmitters. Therefore, it is crucial to develop clinical assessment techniques that are sensitive and reliable to monitor and modulate these chemical messengers and screen diseases. This review focuses on summarizing the current electrochemical measurement techniques that are capable of sensing neurotransmitters with high temporal resolution in real time. Advanced neurotransmitter sensing platforms that integrate nanomaterials and biorecognition elements are explored.

Label-Free MicroRNA Optical Biosensors.

MicroRNAs (miRNAs) play crucial roles in regulating gene expression. Many studies show that miRNAs have been linked to almost all kinds of disease. In addition, miRNAs are well preserved in a variety of specimens, thereby making them ideal biomarkers for biosensing applications when compared to traditional protein biomarkers. Conventional biosensors for miRNA require fluorescent labeling, which is complicated, time-consuming, laborious, costly, and exhibits low sensitivity. The detection of miRNA remains a big challenge due to their intrinsic properties such as small sizes, low abundance, and high sequence similarity. A label-free biosensor can simplify the assay and enable the direct detection of miRNA. The optical approach for a label-free miRNA sensor is very promising and many assays have demonstrated ultra-sensitivity (aM) with a fast response time. Here, we review the most relevant label-free microRNA optical biosensors and the nanomaterials used to enhance the performance of the optical biosensors.

A Fully-Flexible Solution-Processed Autonomous Glucose Indicator.

We present the first demonstration of a fully-flexible, self-powered glucose indicator system that synergizes two flexible electronic technologies: a flexible self-powering unit in the form of a biofuel cell, with a flexible electronic device - a circuit-board decal fabricated with biocompatible microbial nanocellulose. Our proof-of-concept device, comprising an enzymatic glucose fuel cell, glucose sensor and a LED indicator, does not require additional electronic equipment for detection or verification; and the entire structure collapses into a microns-thin, self-adhering, single-centimeter-square decal, weighing less than 40 mg. The flexible glucose indicator system continuously operates a light emitting diode (LED) through a capacitive charge/discharge cycle, which is directly correlated to the glucose concentration. Our indicator was shown to operate at high sensitivity within a linear glucose concentration range of 1 mM-45 mM glucose continuously, achieving a 1.8 VDC output from a flexible indicator system that deliver sufficient power to drive an LED circuit. Importantly, the results presented provide a basis upon which further development of indicator systems with biocompatible diffusing polymers to act as buffering diffusion barriers, thereby allowing them to be potentially useful for low-cost, direct-line-of-sight applications in medicine, husbandry, agriculture, and the food and beverage industries.

Towards a dual in-line electrochemical biosensor for the determination of glucose and hydrogen peroxide.

Herein, we report the development of a sensitive and selective dual mode electrochemical platform for the detection of glucose and H2O2. The platform is based on tungsten and gold microwire electrodes decorated with gold nanoparticles (AuNPs) and colloidal platinum (colloidal-Pt), respectively. The nanostructured AuNPs electrode was used as a support matrix for the immobilization of horseradish peroxidase (HRP) and the colloidal-Pt served as the non-enzymatic glucose biosensor in the construction of a dual in-line electrochemical biosensor that provides a microenvironment for HRP and a pathway for analyte diffusion via the high surface area. The dual-mode approach allows for the simultaneous detection of glucose and H2O2. The glucose biosensor exhibited a dynamic linear range of 0.5 mM to 8 mM glucose. Linearity for H2O2 was up to 70 µM. Operational stability resulted in 75% of the initial biosensor response after 27 days. The dual in-line biosensor showed good sensitivity of 0.403 mA mM-1 cm-2 for glucose and 0.193 mA mM-1 cm-2 for H2O2 in addition to good anti-interference ability. Thus, the low-cost and simple dual in-line biosensor can be used for the real-time detection of glucose and H2O2 in the clinical, biological and environmental fields.

A hybrid glucose fuel cell based on electrodeposited carbon nanotubes and platinized carbon.

Herein, we report on a hybrid fuel cell using electrodeposited multi-walled carbon nanotubes (MWCNTs) as a bioanode template for the immobilization of pyrolloquinoline quinone glucose dehydrogenase (PQQ-GDH) and electrodeposited platinized screen printed carbon nanotubes as the cathode. By depositing these nanostructures, high surface area is realized, wherein efficient direct electron transfer and excellent bioelectrocatalytic performance is achieved. The hybrid fuel cell comprised Nafion/PQQ-GDH/MWCNTs as the bioanode and a platinized carbon as the cathode to oxidize the glucose fuel and reduce oxygen, respectively. The hybrid fuel cell generated an open circuit voltage and a short circuit current density of 345 mV and 352.48 µA/cm2, respectively. The maximum power density of 58.08 µW/cm2 at a cell voltage of 198.5 mV is achieved at physiological conditions. This hybrid glucose fuel cell may be helpful for exploiting novel nanostructure carbon and platinum derived electrode substrate framework that incorporates the advantages of both enzymatic and non-enzymatic glucose fuel cells. The method employed herein further shows promise in the development of biomedical power source to drive bio-implantable devices without the use of batteries.

Wireless bipotentiostat circuit for glucose and H2O2 interrogation.

Here we present a cost-effective point-of-use wireless platform for the electrochemical detection of low concentrations of glucose and hydrogen peroxide (H2O2), simultaneously. The electrochemical system utilizes a dual sensor integrated with a portable bipotentiostat. The bipotentiostat hardware implements a basic designed that reduces the cost of construction and increase the affordability of the instrument, while providing similar functionality as the more expensive bench-top potentiostats. The bipotentiostat utilizes inexpensive components and common Ag/AgCl reference and platinum counter electrodes and two working electrodes, and it is designed to detect currents within the range of 20 uA - 7 mA. Additionally, the bipotentiostat is integrate with wireless module ESP8266 that interfaces with a smartphone to enable real-time monitoring and visualization of the analyte concentration levels. The results show that the selfdesigned bipotentiostat is capable of performing chronoamperometry and demonstrate an electrochemical detection system that is a portable alternative system for laboratory and point-of-use testing.

Operation stability of chitosan and nafion-chitosan coatings on bioelectrodes in enzymatic glucose biofuel cells.

The performance of bioelectrodes in enzymatic glucose biofuel cell is not only dependent on the enzyme immobilization schemes but it is greatly influenced by the ability of the enzyme to exhibit favorable orientation for a direct electron transfer (DET) between the enzyme and the current collector. The electrochemical investigation of chitosan and nafion-chitosan coatings on multi-walled carbon nanotubes (MWCNTs) immobilized with pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BOD) at the bioanode and biocathode, respectively revealed interesting operational stability performance for the enzymatic biofuel cells. The bioelectrodes operated in DET mode and the chitosan coated biofuel cell system overall demonstrated higher power (156 µW) output. The stability of PQQ-GDH bioanodes varied based on the enzyme concentrations, wherein a concentration of 2.5 mg/ml resulted in a significant enhancement in stability and the maximum power density of 1.6 mW/cm2 compared to enzyme concentrations of 5 mg/ml PQQ-GDH or higher.

Fabrication of highly effective hybrid biofuel cell based on integral colloidal platinum and bilirubin oxidase on gold support.

A hybrid biofuel cell (HBFC) is explored as a low-cost alternative to abiotic and enzymatic biofuel cells. Here the HBFC provides an enzymeless approach for the fabrication of the anodic electrode while employing an enzymatic approach for the fabrication of the cathodic electrode to develop energy harvesting platform to power bioelectronic devices. The anode employed 250 µm braided gold wire modified with colloidal platinum (Au-co-Pt) and bilirubin oxidase (BODx) modified gold coated Buckypaper (BP-Au-BODx) cathode. The functionalization of the gold coated multi-walled carbon nanotube (MWCNT) structures of the BP electrodes is achieved by 3-mercaptopropionic acid surface modification to possess negatively charged carboxylic groups and subsequently followed by EDC/Sulfo-NHS (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-Hydroxysulfosuccinimide) crosslinking with BODx. The integration of the BODx and gold coated MWCNTs is evaluated for bioelectrocatalytic activity. The Au-co-Pt and BP-Au-BODx exhibited excellent electrocatalytic activity towards glucose oxidation with a linear dynamic range up to 20 mM glucose and molecular oxygen reduction, respectively. The HBFC demonstrated excellent performance with the largest open circuit voltages of 0.735 V and power density of 46.31 µW/cm2 in 3 mM glucose. In addition, the HBFC operating on 3 mM glucose exhibited excellent uninterrupted operational stability while continuously powering a small electronic device. These results provide great opportunities for implementing this simple but efficient HBFC to harvest the biochemical energy of target fuel(s) in diverse medical and environmental applications.

Gold foil-based biosensor for the determination of hydrogen peroxide.

Hydrogen peroxide ($\text{H}_{\mathbf {2}} \mathbf {O} _{\mathbf {2}}$) plays a critical role in the regulation of multifarious physiological processes. We developed a sensor containing a mercaptopropionic acid (MPA) monolayer covalently immobilized with Horseradish peroxidase (HRP) enzyme for the electrochemical detection of hydrogen peroxide ($\textbf{H}_{\mathbf {2}} \mathbf {O} _{\mathbf {2}}$). A gold foil substrate was chemically treated with nitric acid and were used as working electrode. Platinum wire and Ag-Ag/Cl were used as counter and reference electrodes, respectively. The acid treated gold electrode with the immobilized enzyme shown to have improved catalytic activity in the reduction of $\textbf{H}_{\mathbf {2}} \mathbf {O} _{\mathbf {2}}$. The steady-state current response increases linearly with $\textbf{H}_{\mathbf {2}} \mathbf {O} _{\mathbf {2}}$ concentration from 10 $\mu \textbf{M}$ to 9 mM with a low detection limit of 60 $\mu \textbf{M}$ and showed a sensitivity of 0.4 mA/ mM cm$^{\mathbf {2}}$. This electrochemical sensor is demonstrated to be highly selective and sensitive in the presence of interfering analytes. The improved activity and simple preparation method of the electrode makes the MPAHRP modified gold electrode promising for being developed as an attractive robust material for electrochemical $\textbf{H}_{\mathbf {2}} \mathbf {O} _{\mathbf {2}}$ sensing.

Cross-linked electrospun gelatin nanofibers for cell-based assays.

The present study evaluates the crosslinking of electrospun gelatin nanofibers by physical and chemical methods to further elucidate the importance of the application of gelatin scaffold platforms for cell-based assays. The dehydrothermally cross-linked electrospun gelatin scaffolds were unable to retained their structure morphology and integrity upon exposure to 1X PBS or cell-culture media. The DHT and EDC/Sulfo-NHS cross-linked gelatin scaffolds exhibited fiber diameter on average in the nanometer range. Subsequently, we utilized 1X PBS and cell culture media to evaluate the stability of the nanofibers in solution. The immersion evaluation indicated that the chemically crosslinked gelatin nanofibers maintained their random nanofiber distribution and morphology. However, a high degree of swelling was observed in the presence of cell culture media. Overall, the gelatin scaffold demonstrated good performance in PBS and cell culture media. Hence, EDC/Sulfo-NHS crosslinked electrospun gelatin nanofibrous scaffolds have good biocompatibility and are promising bio-scaffolds for cell-based assays.

Carbon Nanotube-Cellulose Pellicle for Glucose Biofuel Cell.

Carbon nanotube (CNT)-cellulose pellicle was developed to create a conductive CNT network on 20 µm nanostructured cellulose film. The flexible and electrically conductive film was prepared by the modification of bacterial nanocellulose pellicle with multi-walled carbon nanotubes (MWCNTs). The composite film was further modified with redox enzymes including pyroquinoline quinone glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BODx) functioning as the anodic and cathodic catalyst, respectively with glucose as the biofuel source. The enzyme functionalized MWCNT-cellulose based glucose/O2 biofuel cell system harnessed the biochemical energy of glucose via the oxidation of glucose and reduction of molecular oxygen to generate electrical power in the microwatt range. The biofuel cell system exhibited an open circuit voltage and power density of 470 mV and 46.25 µW/cm2, respectively, with a current density of 381 µA/cm2 in the presence of 25 mM glucose. At physiological glucose concentration, the biofuel cell exhibited an open circuit voltage and power density of 418 mV and 24.975 µW/cm2 respectively, with a current density of 293.75µA/cm2. As a result, we expect that this facile strategy to prepare flexible conductive bioelectrodes for the development of glucose biofuel cell system using synthesized bacterial nanocellulose crosslinked with MWCNTs and enzyme can be readily extended to diverse applications in enzymatic biofuel cell and biosensor technology.

Storage stability of electrospun pure gelatin stabilized with EDC/Sulfo-NHS.

With the rapid development of biomimetic polymers for cell-based assays and tissue engineering, crosslinking electrospun nanofibrous biopolymer constructs is of great importance for achieving sustainable and efficient three-dimensional scaffold constructs. Uncrosslinked electrospun gelatin nanofibrous constructs immediately and completely dissolved in aqueous solutions due to their aqueous solubility and poor storage stability. Here, a novel and versatile approach for the fabrication and crosslinking of electrospun gelatin construct with tunable porosity and high aspect ratio nanofibers is presented. Uncrosslinked electrospun gelatin/genipin nanofibrous and pure gelatin nanofibrous constructs exhibited smooth surfaces that were well-defined, with a diameter in the range of 448 ± 364 nm and 257 ± 57 nm, respectively. Dehydrothermal, genipin-EDC/Sulfo-NHS, and EDC/Sulfo-NHS crosslinking approaches were examined to achieve insoluble gelatin nanofibrous constructs that were suitable for cell-based assays. Mechanical characterization demonstrated that the pure gelatin nanofibrous construct crosslinked via EDC/Sulfo-NHS exhibited an increased mechanical strength and stiffness and showed no dissolution in aqueous solutions and retained its fiber morphology. An excellent 1 month storage stability was demonstrated at 22, 4, -20, and -80°C (dehydrated) and at 4°C (hydrated). The as-crosslinked gelatin nanofibrous construct was highly biocompatible (90% cell viability), as demonstrated by the promoted proliferation of PC12 cells.