Development of closed bipolar electrode based L-lactate sensor employing quasi-direct electron transfer type enzyme with cyclic voltammetry.

Herein, we present a proof-of-concept of an enzyme sensor combining closed bipolar electrode system with quasi-direct electron transfer (DET) type enzyme. The closed bipolar electrode system was tested using cyclic voltammetry, with L-lactate as a model substrate. L-Lactate was detected through measurement of the change in junction potential across the bipolar electrode. This change in junction potential was caused by reduction of amino reactive phenazine ethosulfate conjugated to Aerococcus vilidans derived engineered L-lactate oxidase (AvLOx) which shows a quasi-DET signal. Using the closed bipolar electrode system allowed simultaneous measuring using cyclic voltammetry and open circuit potential (OCP) and achieved a limit of detection of 400 µM and 76.2 µM lactate respectively. The sensor was then demonstrated to perform with equivalent sensitivity using OCP across varying surface areas. To the best of our knowledge this is the first time a closed bipolar electrode system has been used with an enzyme which is capable of quasi-direct or direct electron transfer. This work can be expanded further to other enzymes capable of directly altering the junction potential of an electrode surface.

Development of the BioBattery: A novel enzyme fuel cell using a multicopper oxidase as an anodic enzyme.

This work presents the development of an enzyme fuel cell, termed "BioBattery", that utilizes multicopper oxidases as the anodic enzyme in a non-diffusion limited system. We evaluated various enzyme variants as the anode, including multicopper oxidase from Pyrobaculum aerophilum, laccase from Trametes versicolor, and bilirubin oxidase from Myrothecium verrucaria. Several combinations of cathodes were also examined, focusing on the reduction of oxygen as the primary electron acceptor. The optimal pairing used multicopper oxidase from Pyrobaculum aerophilum as the anode and amine reactive phenazine ethosulfate modified bovine serum albumin as the cathode. BioBattery was integrated with our previously developed BioCapacitor, proving capable of consistently powering a 470 µF capacitor, positioning it as a modular power source for wearable and implantable systems. This research work addresses and overcomes some of the fundamental limitations seen in enzyme fuel cells, where power and current are often limited by substrate accessibility to the active electrode surface. (152 words).

Development of Direct Electron Transfer-Type Extended Gate Field Effect Transistor Enzymatic Sensors for Metabolite Detection.

In this work, direct electron transfer (DET)-type extended gate field effect transistor (EGFET) enzymatic sensors were developed by employing DET-type or quasi-DET-type enzymes to detect glucose or lactate in both 100 mM potassium phosphate buffer and artificial sweat. The system employed either a DET-type glucose dehydrogenase or a quasi-DET-type lactate oxidase, the latter of which was a mutant enzyme with suppressed oxidase activity and modified with amine-reactive phenazine ethosulfate. These enzymes were immobilized on the extended gate electrodes. Changes in the measured transistor drain current (ID) resulting from changes to the working electrode junction potential (φ) were observed as glucose and lactate concentrations were varied. Calibration curves were generated for both absolute measured ID and ΔID (normalized to a blank solution containing no substrate) to account for variations in enzyme immobilization and conjugation to the mediator and variations in reference electrode potential. This work resulted in a limit of detection of 53.9 µM (based on ID) for glucose and 2.12 mM (based on ID) for lactate, respectively. The DET-type and Quasi-DET-type EGFET enzymatic sensor was then modeled using the case of the lactate sensor as an equivalent circuit to validate the principle of sensor operation being driven through OCP changes caused by the substrate-enzyme interaction. The model showed slight deviation from collected empirical data with 7.3% error for the slope and 8.6% error for the y-intercept.

Continuous glucose monitoring systems - Current status and future perspectives of the flagship technologies in biosensor research.

Diabetes mellitus is a chronic illness in the United States affecting nearly 120 million adults, as well as increasing in children under the age of 18. Diabetes was also the 7th leading cause of death in the United States with 270 K deaths in 2017. Diabetes is best managed by tight glycemic control, as achieving near-normal glucose levels is key to reduce the risk of microvascular complications. Currently, continuous glucose monitoring (CGM) systems have been recognized as the ideal monitoring systems for glycemic control of diabetic patients. Briefly, a CGM system measures blood glucose levels in subcutaneous tissue by attaching a CGM sensor to the skin, allowing the users to make appropriate modifications to their medical interventions according to experience or empirically derived algorithms. The principles of the glucose sensing employed in the current commercially available CGM systems are mainly electrochemical, and employ the gold standard enzyme, glucose oxidase, as the glucose sensing molecule with the combination of hydrogen peroxide monitoring or with the combination of redox mediator harboring hydrogel. Recently, by employing an abiotic synthetic receptor harboring a fluorescent probe combined with a fluorescent detection system, a chronic CGM was commercialized. In addition, the development of less or non-invasive monitoring sensors targeting glucose in tears, sweat, saliva and urine have become of great interest although their clinical relevancy is still controversial. This review article introduces current and future technological aspects of CGM systems, the flagship technology in biosensor research, which was initiated, matured and is still growing in North America.

Development Toward a Triple-Marker Biosensor for Diagnosing Cardiovascular Disease.

Cardiovascular disease (CVD) is the leading cause of death in the United States and is responsible for 30% of all deaths globally. The diagnosis and management of CVD requires monitoring of multiple biomarkers, which comprehensively represents the state of the disease. However, many assays for cardiac biomarkers today are complicated and laborious to perform. Rapid and sensitive biosensors capable of giving accurate measurements of vital cardiac biomarkers without complex procedures are thus in high demand. In the work presented below, rapid, label-free biosensor prototypes for three Food and Drug Administration-approved biomarkers are reported: B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), and C-reactive protein (CRP). The sensors were prepared by immobilizing each biomarker's antibody onto gold working electrodes with platinum counter and silver/silver chloride reference electrodes. The sensors were tested using electrochemical impedance spectroscopy (EIS), a femto-molar sensitive technique capable of label-free, multi-marker detection if a biomarker's optimal frequency (OF) can be identified. The OFs of BNP, cTnI, and CRP were found to be 1.74, 37.56, and 253.9 Hz, respectively. The performance of the BNP biosensor was also evaluated in blood and achieved clinically relevant detection limits of 100 pg/mL.

Development of Electrochemical Methods to Enzymatically Detect Lactate and Glucose Using Imaginary Impedance for Enhanced Management of Glycemic Compromised Patients.

Lactate is an important biological marker that can provide valuable information for patients who have experienced a traumatic injury. Additionally, when coupled with glucose, the severity and likely prognosis of a traumatic injury can be determined. Because monitoring various markers proves useful in diagnosis and treatment of trauma patients, monitoring both glucose and lactate simultaneously may be especially useful for diabetic patients who have suffered a traumatic injury. Previously, using electrochemical impedance spectroscopy (EIS), a sensor capable of measuring two affinity-based biomarkers simultaneously was demonstrated using the biomarker's specific optimal frequency to develop a deconvolution algorithm, which allowed for the measurement of two biomarkers from a single signal. Herein, while developing an EIS lactate sensor, dual enzymatic biomarker detection of lactate and glucose via EIS was also attempted. Both biomarkers were validated individually with the lactate sensor being additionally validated on whole blood samples. The EIS lactate biosensor achieved a range of detection from 0 to 32 mM of lactate and the glucose sensor a range of 0-100 mg/dL of glucose, which are representative of the likely physiological ranges that trauma patients experience. However, the preliminary attempt of dual marker detection was unsuccessful due to suspected accumulation of reduced redox probe on the surface of the self-assembled monolayer (SAM). Individually, the optimal frequency of lactate was determined to be 69.75 Hz and that of glucose was determined to be 31.5 Hz. However, when combined onto one sensor, no discernable optimal frequency could be determined which again was suspected to be due to the accumulation of the reduced redox probe at the surface of the SAM.

Facilitating Earlier Diagnosis of Cardiovascular Disease through Point-of-Care Biosensors: A Review.

Cardiovascular disease (CVD) accounts for 30% of all global deaths and is predicted to dominate in the coming years, despite vast improvements in medical technology. Current clinical methods of assessing an individual's cardiovascular health include blood tests to monitor relevant biomarker levels as well as varying imaging modalities such as electrocardiograms, computed tomography, and angiograms to assess vasculature. As informative as these tools are, they each require lengthy scheduling, preparation, and highly trained personnel to interpret the results before any information is accessible to patients, often leading to delayed treatment, which can be fatal. A point-of-care (POC) sensor platform is thus paramount in rapid and early diagnosis of CVD. Among the many POC detection platforms, including established optical and mechanical methods, electrochemical-based detection mechanisms have become increasingly desirable because of their superior sensitivity, low cost, and label-free detection. Specifically, electrochemical impedance spectroscopy (EIS) has demonstrated remarkable abilities in low-level (femtomolar) detection of several clinically useful biomarkers and has been reported in CVD diagnostic applications. In this review, we provide an in-depth overview of prevalent CVD diseases and clinically relevant proteomic biomarkers for assessing them. Subsequently, we discuss the ongoing development of POC sensors for CVD, highlighting the current clinical gold standard, potential alternative modalities, and electrochemical methodologies previously successful in quantifying specific biomarkers approved by the Food and Drug Administration (FDA). A discussion of EIS highlighting the attributes and capabilities of novel analysis algorithms is included to showcase the possibility of simultaneous dual-marker detection.

The electrochemical behavior of a FAD dependent glucose dehydrogenase with direct electron transfer subunit by immobilization on self-assembled monolayers.

Continuous glucose monitoring (CGM) is a vital technology for diabetes patients by providing tight glycemic control. Currently, many commercially available CGM sensors use glucose oxidase (GOD) as sensor element, but this enzyme is not able to transfer electrons directly to the electrode without oxygen or an electronic mediator. We previously reported a mutated FAD dependent glucose dehydrogenase complex (FADGDH) capable of direct electron transfer (DET) via an electron transfer subunit without involving oxygen or a mediator. In this study, we investigated the electrochemical response of DET by controlling the immobilization of DET-FADGDH using 3 types of self-assembled monolayers (SAMs) with varying lengths. With the employment of DET-FADGDH and SAM, high current densities were achieved without being affected by interfering substances such as acetaminophen and ascorbic acid. Additionally, the current generated from DET-FADGDH electrodes decreased with increasing length of SAM, suggesting that the DET ability can be affected by the distance between the enzyme and the electrode. These results indicate the feasibility of controlling the immobilization state of the enzymes on the electrode surface.

Enhancing Glycemic Control via Detection of Insulin Using Electrochemical Impedance Spectroscopy.

BACKGROUND: Currently, glycemic management for individuals with diabetes mellitus involves monitoring glucose only, which is insufficient as glucose metabolism involves other biomarkers such as insulin. Monitoring additional biomarkers alongside glucose has been proposed to improve glycemic control. In this work, the development of a rapid and label-free insulin biosensor with high sensitivity and accuracy is presented. The insulin sensor prototype also serves as a prior study for a multimarker sensing platform technology that can further improve glycemic control in the future. METHODS: Electrochemical impedance spectroscopy was used to identify an optimal frequency specific to insulin detection on a gold disk electrode with insulin antibody immobilized, which was accomplished by conjugating the primary amines of insulin antibody to the carboxylic bond of the self-assembling monolayer on the gold surface. After blocking with ethanolamine, the insulin physiological concentration gradient was tested. The imaginary impedance was correlated to insulin concentration and the results were compared with standard equivalent circuit analysis and correlation of charge transfer resistance to target concentration. RESULTS: The optimal frequency of insulin is 810.5 Hz, which is characterized by having the highest sensitivity and sufficient specificity. The lower limit of detection was 2.26 [Formula: see text] which is comparable to a standard and better than traditional approaches. CONCLUSION: An insulin biosensor prototype capable of detecting insulin in physiological range without complex data normalization was developed. This prototype will be the ground works of a multimarker platform sensor technology for future all-in-one glycemic management sensors.

Feasibility in the development of a multi-marker detection platform.

A feasibility study for a label-free, multi-marker single sensor using electrochemical impedance spectroscopy (EIS), imaginary impedance, and a signal decoupling technique is reported. To our knowledge, this is the first reported attempt of using imaginary impedance for biomarker detection and multi-marker detection. The electrochemical responses of purified low and high density lipoproteins (LDL and HDL, respectively) were first individually characterized through the immobilization of their molecular recognition elements (MREs) onto gold disk electrodes (GDEs). The co-immobilization was performed by immobilizing the MREs of both LDL and HDL on the same GDE, which was then used to detect LDL and HDL simultaneously in mixed solution. Previous individual purified responses were then used to de-convolute the mixed response, when the two biomarkers were detected in mixed solutions. The optimal frequencies of LDL and HDL were found to be 81.38Hz and 5.49Hz, respectively, which shifted to 175.8Hz and 3.74Hz under co-immobilized conditions. After comparing the electrochemical signal in complex and imaginary impedance, imaginary impedance was found to be more suitable for multi-marker detection purposes. Since imaginary impedance is related to capacitance, electric displacement, relative permittivity, and effective capacitance were derived to elucidate the theory of optimal frequency. This work shows that EIS has the potential for multi-marker detection and can be extended to monitor other complex diseases such as diabetes mellitus for better management and diagnostic purposes.