The curing duration of crosslinked redox hydrogels is a critical yet often overlooked parameter influencing the fabrication reproducibility, signal output and signal precision of wired enzymatic biosensors. This study investigates the effect of curing duration on the performance of glucose sensing enzyme electrodes based on polyethylene glycol diglycidyl ether (PEGDGE) crosslinked with an osmium redox polymer mediator (Os(bpy)PVI) and glucose oxidizing enzymes for development of amperometric glucose sensors. The results demonstrate that a curing duration of at least 7 days achieves consistent and precise electrochemical glucose responses for enzyme electrodes based on both glucose oxidase (GOx) and an engineered ascomycetous Class II cellobiose dehydrogenase variant (CDH) (WTChCDH) capable of glucose oxidation. Enzyme electrodes produced using GOx/PEGDGE/Os(bpy)PVI exhibited an initial decline in sensitivity when cured for 1 to 4 days, followed by stabilization in sensitivity after approximately 7 days. The intra-electrode relative standard deviation (%RSD) of sensitivity improved significantly from 53 % (over day 1–7) to 16 % (days 7 – 21), indicating a transition toward precise sensing response. The improved precision is attributed to progressive crosslinking within the redox hydrogel matrix over an extended (up to 7 day) period. These findings identify curing time as a critical design parameter governing signal reproducibility and long-term electrode integrity, providing fundamental insight into the scalable fabrication and industrial translation of wired enzymatic biosensor platforms.

Pyrolytic carbon derived from thermosetting polymer precursors is a promising electrode material for bioelectrochemical applications due to its structural robustness, chemical stability, biocompatibility, and amenability to surface modification. Crucially, it allows the integration of diverse nano- and microstructures on the electrode surface. In this study, we introduce pyrolytic carbon nanograss (CNG) electrodes fabricated from SU-8 photoresist as a novel and scalable platform for biophotovoltaic (BPV) applications. Using the cyanobacterium Synechocystis sp. PCC 6803 as a photobiocatalyst, we evaluate biophotocurrent generation both in the absence and presence of an osmium redox polymer mediator. The CNG structures, with tunable lengths, significantly increase the electroactive surface area compared to planar carbon electrodes. This enhancement, combined with the redox polymer, leads to a remarkable 40-fold increase in biophotocurrent output for the longest nanograss structures. Additionally, the nanostructured surfaces improve bacterial cell retention and promote stable electron transfer. Importantly, the bioanodes exhibit excellent operational stability under prolonged high-intensity illumination, highlighting their robustness. The simple and scalable microfabrication of CNG electrodes positions pyrolytic carbon as a compelling candidate for next-generation microbial solar cells.

A glucose/O2-based biofuel cell employing nanoporous gold electrodes (NPG) supported by Kapton® was prepared. The anode was prepared by drop-casting a solution of Crassicarpon hotsonii cellobiose dehydrogenase (ChCDH) and an Os complex-based polymer and the cathodes by covalent immobilization of Magnaporthe oryzae bilirubin oxidase (MoBOD) on 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM). Both electrodes were coated with poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) (MPC) to reduce biofouling. The anode had a Jmax of 172 ± 10 μA cm-2 and a KMapp of 19 ± 3 mM in phosphate buffer saline, with a linear detection range from 1 to 5 mM and a sensitivity of 9.4 ± 0.3 μA cm-2 mM-1. In artificial plasma, the response was saturated at 3 mM, with Jmax of 6.8 ± 10 μA cm-2, KMapp of 1 mM and a linear detection range from 1 to 5 mM. The cathode had a Jmax of 103 μA cm2 and retained 80 % of its response after 18 h of continuous measurement in phosphate buffered saline, while in artificial plasma, the stability was significantly reduced with a half-life of 1 h under continuous operation. The power outputs in PBS and artificial serum were 4.4 and 1.0 μWcm-2, respectively.

Conductive inks are used for the development of disposable electrochemical sensors. They trigger the possibility of building screen- or stencil-printed electrodes with similar efficiency with respect to solid electrodes. [1,2] In particular, biocompatible inks are formulated and stencil-printed on a flexible support that could be easily integrated within smart-devices for the continuous and minimally invasive monitoring of lactate and glucose. First, the conductive ink formulation has been optimized based on electrochemical and rheological measurements implemented within a multivariate analysis model. Afterwards, the active carbon electrode was modified with osmium redox polymers (ORPs) to establish an electronic connection with enzymes, since neither glucose oxidase (GOx) nor lactate oxidase (LOx) are able to directly transfer electrons. [3,4] Finally, both biosensors have been tested in model solution and sweat to determine the analytical figures of merit. The latest evolution of enzyme-based amperometric biosensors is represented by edible biosensors that can now detect a variety of parameters, ranging from basic physiological measurements such as temperature and pH to complex analyzes of organic and biological gases, and provide the data in real time. They can monitor a wide range of biomarkers, including those related to gastrointestinal health, enzymes, hormones, glucose levels, and even drug concentrations. [5] References Bollella, P. Enzyme-based amperometric biosensors: 60 years later...Quo Vadis? Anal. Chim. Acta , 340517 (2022). Tricase, A., et al. Water‐Based Conductive Ink Formulations for Enzyme‐Based Wearable Biosensors. Adv. Sensor Res., 2300036 (2023). Marchianò, V., et al., High Voltage Flexible Glucose/O2 Fully Printed Hydrogel-based Enzymatic Fuel Cell, J. Phys. D , accepted. Marchianò, V., et al., Tailoring Water-based Graphite Conductive Ink Formulation for Enzyme Stencil-Printing: Experimental Design to Enhance Wearable Biosensors Performance, Chem. Mat., accepted. V. Marchianò et al., Inside Out: Exploring Edible Biosensors for Health Monitoring, Bioelectrochemistry, accepted.

The development of implantable glucose sensors is of significant interest in the management of diabetes. This work focuses on developing an implantable, biocompatible nanoporous gold electrode prototype based on Kapton® for the subcutaneous detection of glucose. The electrodes were first modified with a layer containing glucose oxidase and Os(2,2'-bipyridine)2Cl·PVI (Os(bpy)2Cl PVI). An additional polymeric layer containing poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) was then added to reduce biofouling and foreign body reaction effects. The modified electrode had a VMAX of 211 ± 13 μA cm-2 and a KMapp of 6.1 ± 0.8 mM in pseudo physiological conditions, with a linear detection range from 1 to 4 mM and a sensitivity of 28.6 ± 2.1 μA cm-2 mM-1. In artificial plasma, the response of the sensor was saturated at 3 mM, with a VMAX of 113 ± 10 μA cm-2 and a KMapp of 2.1 ± 0.4 mM with a linear detection range from 1 to 2.5 mM and a sensitivity of 14.6 ± 3.3 μA cm-2 mM-1. Mechanical stress testing demonstrated that there was a 40 % reduction of the redox polymer coverage after 320 deformation events, however the catalytic activity was still detectable after 160 events. Minimal cytotoxicity effects of the electrodes were observed. When subcutaneously implanted the electrodes showed fairly good mechanical stability after one week and detachment of the metallic layer on some electrodes after 21 days, probably due to electrode bending. A limited foreign body reaction was observed. These results indicated that the electrodes could be implanted for a period of up to 1 week.

This study presents the development of second-generation wearable biosensor arrays using printed electrode technology with a homemade water-based graphite ink. The electrodes were prepared by stencil-printing, then modified with [Os(bpy)2(Cl)(PVI) 10 ] to facilitate electron transfer from lactate oxidase (LOx) and glucose oxidase (GOx). These biosensor arrays exhibit a low limit of detection, high sensitivities, and selectivity when tested in both buffer solution and artificial sweat. They also demonstrate good operational/storage stability, retaining about 80% of the initial signal after 20 days. Integrated into a wristband, the biosensors were successfully tested for continuous monitoring of L-lactate and D-glucose in a healthy volunteer during daily activities. This technology holds potential for real-time wearable applications in different fields, ranging from sports to medicine and healthcare.

Abstract Herein, this work reports the first example of second‐generation wearable biosensor arrays based on a printed electrode technology involving a water‐based graphite ink, for the simultaneous detection of l‐lactate and d‐glucose. The water‐based graphite ink is deposited onto a flexible polyethylene terephthalate sheet, namely stencil‐printed graphite (SPG) electrodes, and further modified with [Os(bpy)2(Cl)(PVI)10] as an osmium redox polymer to shuttle the electrons from the redox center of lactate oxidase from Aerococcus viridans (LOx) and gluocose oxidase from Aspergillus niger (GOx). The proposed biosensor array exhibits a limit of detection as low as (9.0 ± 1.0) × 10−6 m for LOx/SPG‐[Os(bpy)2(Cl)(PVI)10] and (3.0 ± 0.5) × 10−6 m for GOx/SPG‐[Os(bpy)2(Cl)(PVI)10], a sensitivity as high as 1.32 μA mm−1 for LOx/SPG‐[Os(bpy)2(Cl)(PVI)10] and 28.4 μA mm−1 for GOx/SPG‐[Os(bpy)2(Cl)(PVI)10]. The technology is also selective when tested in buffer and artificial sweat and is endowed with an operational/storage stability of ≈80% of the initial signal retained after 20 days. Finally, the proposed array is integrated in a wristband and successfully tested for the continuous monitoring of l‐lactate and d‐glucose in a healthy volunteer during daily activity. This is foreseen as a real‐time wearable device for sport‐medicine and healthcare applications.

Galactose monitoring in individuals allows the prevention of harsh health conditions related to hereditary metabolic diseases like galactosemia. Current methods of galactose detection need development to obtain cheaper, more reliable, and more specific sensors. Enzyme-containing amperometric sensors based on galactose oxidase activity are a promising approach, which can be enhanced by means of their inclusion in a redox polymer coating. This strategy simultaneously allows the immobilization of the biocatalyst to the electroactive surface and hosts the electron shuttling units. An additional deposition of capping polymers prevents external interferences like ascorbic or uric acid as well as biofouling when measuring in physiological fuels. This work studies the protection effect of poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate (MPC) and polyvinylimidazole-polysulfostyrene (P(VI-SS)) when incorporated in the biosensor design for the detection of galactose in human plasma.

Abstract Research on implantable glucose biosensors is driven by the need for innovative medical devices for continuous glucose monitoring in patients with diabetes mellitus. However, biosensor sterilization is a step that is widely omitted during the process of innovation. To compare the effects of gamma irradiation and chemical treatment with ethylene oxide (carbon microarray electrodes are fabricated, functionalized with glucose oxidizing enzymes (cellobiose dehydrogenase CDH or glucose oxidase GOx), and coated with a specifically designed zwitterionic polymer prior to the sterilization step. Cyclic voltammetry in the presence of 100 mm glucose of the biosensors before and after sterilization shows that gamma irradiation with a low radiation rate (25 kGy, 260 Gy h−1) does not induce a sensor performance loss, unlike the EtO treatment. In addition, no cytotoxic by‐products are released after gamma sterilization. Based on these results obtained with both glucose oxidizing enzymes (CDH and GOx), gamma irradiation of the glucose biosensors with a low dose rate is preferable to exposure to EtO for biosensor terminal sterilization.

In this study, we introduce a novel enzymatic fuel cell (EFC) utilizing stencil printed electrodes modified with specific enzymes. These electrodes are assembled in two configurations: one with a liquid electrolyte and another with six EFCs connected in series, resembling a Voltaic pile. The second configuration significantly improves performance, achieving an open circuit voltage (OCV) of 2.36 +/- 0.22 V and a maximum power output of 22.9 +/- 0.9 mu W at a cell voltage of 1.95 V (using 10 mM D-glucose). This approach showcases potential for renewable power sources and could be crucial for powering implantable bioelectronics like pacemakers.

Herein, we report for the first time an experimental design-based approach to develop water-based graphite conductive ink containing enzymes and redox mediators to obtain fully printed wearable biosensors for lactate and glucose monitoring. The experimental design encompasses both electrochemical parameters, such as electroactive area and electron transfer rate constant, and rheological parameters, including elastic (G ') and viscous (G '') moduli where G ''/G ' is expressed as tan delta. Notably, the printed electrodes exhibited an electroactive area A(EA) of 3.95 +/- 0.31 cm(2) and a roughness factor, rho, of 43.8, which is 50 times higher than those of commercially available screen-printed electrodes. Furthermore, lactate oxidase and glucose oxidase are integrated within water-based graphite conductive ink to obtain enzyme-based inks: enzyme-ink (E-INK), to detect lactate, and enzyme mediator-ink (EM-INK), to detect glucose. The resulting biosensors demonstrated high sensitivity and low limit of detection 3.3 mu A mM(-1) and 0.3 +/- 0.1 mu M (ferricyanide as electron mediator), and 4.3 mu A mM(-1) and 3 +/- 1 mu M, for E-INK and EM-INK, respectively. The biosensors also exhibited excellent selectivity, maintaining their storage stability, with approximately 80-90% of the initial signal retained after 90 days. Overall, this promising system holds potential to be utilized as a flexible and wearable biosensor. Its use of biocompatible water-based inks makes it suitable for applications in sports medicine and remote clinical care.

Managing blood glucose can affect important clinical outcomes during the intraoperative phase of surgery. However, currently available instruments for glucose monitoring during surgery are few and not optimized for the specific application. Here we report an attempt to exploit an enzymatic sensor in a vein replica that could continuously monitor glucose level in an authentic human bloodstream. First, detailed investigations of the superficial venous systems of volunteers were carried out using ocular and palpating examinations, as well as advanced ultrasound measurements. Second, a tubular glucose-sensitive biosensor mimicking a venous system was designed and tested. Almost ideal linear dependence of current output on glucose concentration in phosphate buffer saline was obtained in the range 2.2-22.0 mM, whereas the dependence in human plasma was less linear. Finally, the developed biosensor was investigated in whole blood under homeostatic conditions. A specific correlation was found between the current output and glucose concentration at the initial stage of the biodevice operation. However, with time, blood coagulation during measurements negatively affected the performance of the biodevice. When the experimental results were remodeled to predict the response without the influence of blood coagulation, the sensor output closely followed the blood glucose level.

Monitoring galactose levels in dairy products can help to prevent severe complications with a hereditary metabolic disease such as galactosemia, a life-threatening disease. The current state of the art requires the development of less expensive, more reliable and specific methods to determine galactose levels in food in a practical way. We report the development and optimization of an amperometric biosensor for determination of galactose in dairy products based on galactose oxidase (GaOx) co-immobilized with an osmium-complex modified redox polymer on glassy carbon electrodes. To attain the maximum catalytic currents based on mediated electron transfer, two Os-complex based polymers with different redox potentials and different enzyme:redox polymer ratios were studied. The optimized GaOx-modified electrode gave a maximum electrocatalytic response of galactose oxidation that was not affected by the presence of O2, indicating fast wiring of the enzyme by the Os-complex modified redox polymer. The biosensor that gave the best analytical parameters for galactose detection was further tested for measuring galactose concentration in lactose-containing and lactose-free milk and yogurt samples under aerobic conditions. The results obtained with the amperometric biosensor were validated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

The lifetime of implantable electrochemical glucose monitoring devices is limited due to the foreign body response and detrimental effects from ascorbic acid (AA) and uric acid (UA) interferents that are components of physiological media. Polymer coatings can be used to shield biosensors from these interferences and prolong their functional lifetime. This work explored several approaches to protect redox polymer-based glucose biosensors against such interferences by designing six targeted multi-layer sensor architectures. Biological interferents, like cells and proteins, and UA and AA interferents were found to have individual effects on the current density and operational stability of glucose biosensors, requiring individual protection and treatment. Protection against biofouling can be achieved using a poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) (MPC) zwitterionic polymer coating. An enzyme-scavenging approach was compared to electrostatic repulsion by negatively charged polymers for protection against AA and UA interferences. A multi-layer novel polymer design (PD) system consisting of a cross-linkable negatively charged polyvinylimidazole-polysulfostyrene co-polymer inner layer and a cross-linkable MPC zwitterionic polymer outer layer showed the best protection against AA, UA, and biological interferences. The sensor protected using the novel PD shield displayed the lowest mean absolute relative difference between the glucose reading without the interferent and the reading value with the interferent present and also displayed the lowest variability in sensor readings in complex media. For sensor measurements in artificial plasma, the novel PD extends the linear range (R2 = 0.99) of the sensor from 0-10 mM for the control to 0-20 mM, shows a smaller decrease in sensitivity, and retains high current densities. The application of PD multi-target coating improves sensor performance in complex media and shows promise for use in sensors operating in real conditions.

Cellobiose dehydrogenase (CDH) is capable of oxidizing cellobiose and related carbohydrates and generating electrical current at carbon-based electrodes through direct electron transfer (DET) or mediated electron transfer (MET) mechanisms. As a result, CDHs have been utilized as biocatalysts in biosensors and biofuel cell anodes. A novel engineered ascomycetous Class II CDH with enhanced glucose activity was tested as a bioelectrocatalyst for application to DET or MET-based glucose biosensors with the electrode component amount selection optimized for maximum current in 5 mM glucose solutions. The optimised DET biosensor showed a similar sensitivity and 3-fold lower K-M,K-app when compared to non-optimised DET sensor based on the same engineered CDH. The optimized MET biosensor had a similar K-M,K-app to non-optimized MET biosensor. However, it showed 15-fold improvement in j(max) and 17-fold improvement in sensitivity over the DET biosensor. The sensor signals are not affected by the presence of oxygen, although operation in artificial serum results in 43 % and 28 % lower sensitivity for the DET and MET sensors, respectively. While no individually tested potential interferent breaches a mean absolute relative difference of 20 % of the current, the cumulative co-operative effect in complex media, such as artificial serum, decreases the glucose oxidation current signal.

NADH is a cofactor used by a wide range of dehydrogenases. Measurement of the concentration of NADH is widely used to measure the activity of enzymes. Extensive efforts have been made to develop electrochemical sensors for NADH determination at low overpotentials to avoid interferences. The development of an enzymatic electrochemical biosensor for the detection of NADH is described. Nanoporous gold electrodes were utilised for the immobilization of diaphorase and osmium-based polymer Os(bpy)(2)(PVI). Nanoporous gold electrodes of different pore sizes were manufactured by varying the dealloying temperature. To optimise the enzymatic response, the concentrations of polymer and enzyme, average pore size and the operating temperature were examined with the optimal performance observed using 10 mu L of Os(bpy)(2)(PVI) and 5 mu L of diaphorase at concentrations of 6 and 10 mg/mL, respectively, on nanoporous gold electrodes with an average pore size of 5.9 nm. The biosensor showed a high sensitivity of 89.6 mu A/cm(2) mM, a low LOD of 0.8 mu M and a linear range from 5 to 100 mu M at a potential of 0.35 V vs Ag/AgCl at a temperature of 40 ?C.

Prostate cancer (PCa) is the most common cancer among men diagnosed worldwide. The conventional non-invasive test for detection and staging of prostate cancer include prostate specific antigen (PSA) test which often can give false positives and is unable to distinguish between aggressive and indolent stages of PCa. This results in doing unnecessary invasive tests like digital rectal examination (DRE), biopsy etc., which only gives further anxiety to patients. Here we describe a rapid and oxygen-insensitive microfluidic immunoassay for on-line capture and precise detection of potential prostate cancer protein biomarkers. Measurement of a panel of biomarkers will help in not only diagnosing the PCa precisely but may also help in staging the cancer. The microfluidic system includes protein capture from serum using a sandwich ELISA arrangement and signal-transducing poly-horseradish peroxidase (poly-HRP) enzyme labels on an 8-electrode screen-printed carbon array. A major challenge in using these screen-printed electrode arrays is the estimation of the electrochemical surface area (ECSA) of electrodes because the current signal is proportional to ECSA and variability in the electrode surface areas generate variability in the corresponding currents. We show that normalization of electrode responses having different surface areas can be achieved using in-situ experimental methods and that the normalization methods can improve the precision of the immunoassay. Another challenge in electrochemical immunoassay systems is the interference of oxygen using the mediator hydroquinone1. A range of osmium polypyridyl complexes were studied to find the best mediator that is not affected by oxygen. Using an optimized mediator and a simple method to estimate carbon electrode surface area, immunoassays can be designed with improved detection characteristics. (1) Dhanapala, L.; Jones, A. L.; Czarnecki, P.; Rusling, J. F. Sub-Zeptomole Detection of Biomarker Proteins Using a Microfluidic Immunoarray with Nanostructured Sensors. Anal. Chem. 2020, 92 (12), 8021–8025. ttps://doi.org/10.1021/acs.analchem.0c01507.

Foreign body response (FBR) is a major challenge that affects implantable biosensors and medical devices, including glucose biosensors, leading to a deterioration in device response over time. Polymer shields are often used to mitigate this issue. Zwitterionic polymers (ZPs) are a promising class of materials that reduce biofouling of implanted devices. A series of ZPs each containing tetherable epoxide functional groups was synthesised for application as a polymer shield for eventual application as implantable glucose biosensors. The polymer shields were initially tested for the ability to resist fibrinogen adsorption and fibroblast adhesion. All synthesised ZPs showed comparable behaviour to a commercial Lipidure ZP in resisting fibrinogen adsorption. Nafion, a common anionic shield used against electrochemical interferents, showed higher protein adsorption and comparable cell adhesion resistance as uncoated control surfaces. However, a poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate) (MPC)-type ZP showed similar behaviour to Lipidure, with approximately 50% reduced fibrinogen adsorption and 80% decrease in fibroblast adhesion compared to uncoated controls. An MPC-coated amperometric glucose biosensor showed comparable current density and a 1.5-fold increase in sensitivity over an uncoated control biosensor, whereas all other polymer shields tested, including Lipidure, Nafion and a poly(ethyleneglycol) polymer, resulted in lower sensitivity and current density. Collectively, these characteristics make MPC-polymer shield coatings an appealing possibility for use in implantable glucose sensors and other implanted devices with the aim of reducing FBR while maintaining sensor performance.

Enzymatic glucose electrodes based on mediated electron transfer have potential for application as semi implantable or implantable sensors. Enzyme electrodes consisting of adsorbed osmium-based redox polymer crosslinked with a glucose oxidising enzyme are promising systems for continuous glucose monitoring, but suffer from signal output magnitude and long-term stability issues. The inclusion of carbon nanosupports such as multiwalled carbon nanotubes (MWCNTs) into these sensors tends to increase characteristics such as current density and surface coverage of enzyme or mediator. However, large quantities of nanomaterials are often necessary to see significant effects. Grafting of the enzyme to the surface of the MWCNTs improves dispersibility of the nanosupport aiding enzyme electrode fabrication, and increases enzyme activity. Here we report on a design of experiments (DoE) approach to determine the optimum amount of each component in enzyme electrodes, using glucose oxidase grafted to carbon nanotube support, to maximise current density and stability for application to continuous use glucose biosensing. Using the DoE approach while considering current density and stability responses delivers a set of component amounts where both responses are optimised. Thus far stability has not been investigated as a response to be optimised using a DoE approach. The optimised enzyme electrodes show a current density of 3.18 +/- 0.30 mA cm(-2), representing a 146% increase in current density in 50 mM phosphate-buffered saline at 37 degrees C containing 5 mM glucose when compared to similar systems where enzyme and nanosupport are not grafted to each other. Using the predictive DoE model, component amounts were then modified to minimise the quantity of the nanoconjugate while showing similar electrochemical behaviour and current density to the optimised system, using 93% less of the nanoconjugate. However, the operational stability under continuous use was moderate with only approximate to 50% amperometric current retained after 12 hr use. Overcoating with a Nafion protective layer improved stability to 72-75% over the same period. The coupling of adsorbed films to the electrode surface, use of additional perm-selective membranes, and/or use of pulsed potentials to implement intermittent sampling of glucose levels, rather than continuous amperometry, is proposed to improve operational stability. (C) 2021 The Authors. Published by Elsevier Ltd.