Amperometric enzyme biosensors: Past,present and future

Three groups of the amperometric biosensors such as unmediated, mediated and based on direct transfer of electrons have been thoroughly described, and their advantages and disadvantages were shown. The amperometric biosensors are mostly utilized in commercial devices since they are studied to a greater extent and have some advantages. The modern commercial systems based on amperometric biosensors and its applications have been presented. The major field of employing biosensors is medical diagnostics where numerous commercial devices are currently functioning.     

Biosensors

Two decades of research into biosensors has been accelerated recently by the commercial potential offered by biotechnology. New developments in biosensor technology in which a biologically sensitive material is immobilized in intimate contact with a suitable potentiometric, amperometric, optical or other transducer are described. It is expected that some of these devices will be commercialized in 1984.     

Quantitative analysis of calibration dependence of biosensors

Three-parameter Hill's equation, which is used in enzyme kinetics, was shown to applicable to calibration curves of both potentiometric (glucose, pesticides, urea, etc.) and amperometric (surfactants, biphenyl, etc.) biosensors. Possible causes of errors of analyte concentration measurements are discussed.     

Quantitative analysis of the calibration dependences for biosensors

The three-parameter Hill’s equation, which is used in enzyme kinetics, was shown to be applicable to calibration curves of both potentiometric (glucose, pesticides, urea, etc.) and amperometric (surfactants, biphenyl, etc.) biosensors. Possible causes of errors of analyte concentration measurements are discussed.     

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers

Abstract

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen–antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.     

Metabolically engineered methylotrophic yeast cells and enzymes as sensor biorecognition elements

An extended definition of the term metabolic engineering is given and its successful use in the construction of biorecognition elements of sensors is demonstrated. It is shown that genetic and chemical modifications of methylotrophic yeast cells provide directed changes in their physiological responses towards methanol, ethanol and formaldehyde resulting in enhanced selectivity and shorter time response of the corresponding potentiometric and amperometric biosensors.     

Biosensors for process control

Biosensors have been extensively studied during the last 20 years, and a myriad of laboratory biosensors have been developed. Improvements are required in biosensor design and performance before they become widely accepted in industrial process monitoring. However, as the biotechnology industry expands, biosensors may become more acceptable because, despite their limitations, they are the only devices capable of delivering the information required.     

Chiral analysis of amino acids using electrochemical composite bienzyme biosensors.

The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes.     

Biosensors for direct determination of organophosphate pesticides

Direct, selective, rapid and simple determination of organophosphate pesticides has been achieved by integrating organophosphorus hydrolase with electrochemical and opitical transducers. Organophosphorus hydrolase catalyzes the hydrolysis of a wide range of organophosphate compounds, releasing an acid and an alcohol that can be detected directly. This article reviews development, characterization and applications of organophosphorus hydrolase-based potentiometric, amperometric and optical biosensors.     

Clinical uses of enzyme electrode probes

Clinical applications of enzyme electrochemical sensors are reported; they are based on the coupling of enzymes with potentiometric membrane electrodes (pH, iodide) or potentiometric probes (ammonia, carbon dioxide) or amperometric devices (oxygen, hydrogen peroxide). The most popular and successful immobilization procedures for enzymes are reviewed, namely physical entrapments and chemical methods for binding enzymes to solid support like collagen and nylon net; procedures specifically developed for clinical uses of enzyme probes.The simplicity of the apparatus is evidenced, and it is explained how a single instrument can be useful for all kind of measurements. Practical suggestions for constructing a typical probe are given. Single paragraphs are devoted to the determination of urea, cholesterol, creatinine, amino acids, glucose, lactate, protein, choline and acetylcholine to clarify the sequence of enzymatic and electrochemical reactions in order to elucidate the application range the sensitivity and the selectivity as well as the relevant interferences for each metabolite either in the enzymatic or in the electrochemical step. The applications performed in vitro, in vivo and ex vivo and the commercial availability of some instruments are reported.     

Comparisons of platinum, gold, palladium and glassy carbon as electrode materials in the design of biosensors for glutamate

Four electrode materials: Pt, Au, Pd and glassy carbon (GC), were studied to investigate their suitability as substrates in the development of two different classes of glutamate biosensor. Glutamate oxidase cross-linked onto poly(o-phenylenediamine) was chosen as the type 1 biosensor (PPD/GluOx), incorporating PPD as the permselective element to detect H(2)O(2) directly on the electrode surface at relatively high applied potentials. GluOx and horseradish peroxidase/redox polymer modified electrodes (Os(2+)PVP/HRP/GluOx) that relied on enzyme-catalysed H(2)O(2) detection at lower applied potentials were used as type 2 biosensors. The voltammetric and amperometric responses to the enzyme signal transduction molecule, H(2)O(2), and the archetypal interference species in biological applications, ascorbic acid, were determined on the bare and PPD/GluOx-modified surfaces. The amperometric responses of these electrodes were stable over several days of continuous recording in phosphate buffered saline (pH 7.4). The sensitivity of the type 1 biosensors to H(2)O(2) and glutamate showed parallel trends with low limits of detection and good linearity at low concentrations: Pt>Au approximately Pd>GC. Type 2 biosensors out-performed the type 1 design for all electrode substrates, except Pt. However, the presence of the permselective PPD membrane in the type 1 biosensors, not feasible in the type 2 design, suggests that Pt/PPD/GluOx might have the best all-round characteristics for glutamate detection in biological media containing interference species such as ascorbic acid. Other points affecting a final choice of substrate should include factors such as mass production issues.     

Biosensors based on acrylic microgels: a comparative study of immobilized glucose oxidase and tyrosinase

Acrylic microgels are proposed as enzyme immobilizing support in amperometric biosensors. Two enzymes, glucose oxidase and tyrosinase, were entrapped in this matrix and their behaviour is compared. The optimum cross-linking of the polymeric matrix required to retain the enzyme, and to allow the diffusion of the substrate is different for each enzyme, 3.2% for glucose oxidase and 4.5% for tyrosinase. The effect of pH and temperature on the biosensor responses has been studied by experimental design methodology and predictions have been compared with independently performed experimental measurements. A quadratic effect of the variables studied (pH and T) on the biosensor response and the small or null interaction between them was confirmed. The pH results obtained with both methods are coincident revealing an reversible effect on the enzyme. However, the temperature optimum value obtained by experimental design was 10 degrees C lower as a result of an activity decay due to irreversible thermal denaturation of both enzymes.     

Field-effect devices for detecting cellular signals

The integration of living cells together with silicon field-effect devices challenges a new generation of biosensors and bioelectronic devices. Cells are representing highly organised complex systems, optimised by millions of years of evolution and offering a broad spectrum of bioanalytical receptor “tools” such as enzymes, nucleic acids proteins, etc. Their combination with semiconductor-based electronic chips allows the construction of functional hybrid systems with unique functional and electronic properties for both fundamental studies and biosensoric applications. This review article summarises recent advances and trends in research and development of cell/transistor hybrids (cell-based field-effect transistors) as well as light-addressable potentiometric sensors.     

Bio-smart hydrogels: co-joined molecular recognition and signal transduction in biosensor fabrication and drug delivery

Two classes of polymers that are currently receiving widespread attention in biosensor development are hydrogels and conducting electroactive polymers. The present study reports on the integration of these two materials to produce electroactive hydrogel composites that physically entrap enzymes within their matrices for biosensor construction and chemically stimulated controlled release. Enhanced biosensing capabilities of these membranes have been demonstrated in the fabrication of glucose, cholesterol and galactose amperometric biosensors. All biosensors displayed extended linear response ranges (10(-5)-10(-2) M), rapid response times (<60 s), retained storage stabilities of up to 1 year, and excellent screening of the physiological interferents ascorbic acid, uric acid, and acetaminophen. When the cross-linked hydrogel components of these composite membranes were prepared with the amine containing dimethylaminoethyl methacrylate monomer the result was polymeric devices that swelled in response to pH changes (neutral to acidic). Entrapment of glucose oxidase within these materials made them glucose-responsive through the formation of gluconic acid. When insulin was co-loaded with glucose oxidase into these "bio-smart" devices, there was a twofold increase in insulin release rate when the devices were immersed in glucose solutions. This demonstrates the potential of such systems to function as a chemically-synthesized artificial pancreas.     

Role of reaction kinetics and mass transport in glucose sensing with nanopillar array electrodes

The use of nanopillar array electrodes (NAEs) for biosensor applications was explored using a combined experimental and simulation approach to characterize the role of reaction kinetics and mass transport in glucose detection with NAEs. Thin gold electrodes with arrays of vertically standing gold nanopillars were fabricated and their amperometric current responses were measured under bare and functionalized conditions. Results show that the sensing performances of both the bare and functionalized NAEs were affected not only by the presence and variation of the nanoscale structures on the electrodes but also by the reaction kinetics and mass transport of the analyte species involved. These results will shed new light for enhancing the performance of nanostructure based biosensors.     

Design and development of opto-electrochemical biosensing devices for diagnosing chronic kidney disease

Chronic kidney disease (CKD) is emerging as one of the major causes of the increase in mortality rate and is expected to become 5th major cause by 2050. Many studies have shown that it is majorly related to various risk factors, and thus becoming one of the major health issues around the globe. Early detection of renal disease lowers the overall burden of disease by preventing individuals from developing kidney impairment. Therefore, diagnosis and prevention of CKD are becoming the major challenges, and in this situation, biosensors have emerged as one of the best possible solutions. Biosensors are becoming one of the preferred choices for various diseases diagnosis as they provide simpler, cost-effective and precise methods for onsite detection. In this review, we have tried to discuss the globally developed biosensors for the detection of CKD, focusing on their design, pattern, and applicability in real samples. Two major classifications of biosensors based on transduction systems, that is, optical and electrochemical, for kidney disease have been discussed in detail. Also, the major focus is given to clinical biomarkers such as albumin, creatinine, and others related to kidney dysfunction. Furthermore, the globally developed sensors for the detection of CKD are discussed in tabulated form comparing their analytical performance, response time, specificity as well as performance in biological fluids.     

Implantable enzyme amperometric biosensors

The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems.     

Evaluation of permselective membranes for optimization of intracerebral amperometric glutamate biosensors

Monitoring of extracellular brain glutamate concentrations by intracerebral biosensors is a promising approach to further investigate the role of this important neurotransmitter. However, amperometric biosensors are typically hampered by Faradaic interference caused by the presence of other electroactive species in the brain, such as ascorbic acid, dopamine, and uric acid. Various permselective membranes are often used on biosensors to prevent this. In this study we evaluated the most commonly used membranes, i.e. nafion, polyphenylenediamine, polypyrrole, polyaniline, and polynaphthol using a novel silica-based platinum electrode. First we selected the membranes with the highest sensitivity for hydrogen peroxide in vitro and an optimal selectivity against electrochemical interferents. Then we evaluated the performances of these membranes in a short lasting (3-4h) in vivo experiment. We found that best in vitro performance was accomplished with biosensors that were protected by a poly(m-phenylenediamine) membrane deposited onto the platinum electrode by cyclic voltammetry. However, post-implantation evaluation of these membranes showed poor selectivity against dopamine. Combination with a previously applied nafion layer did not protect the sensors against acute biofouling; indeed it was even counter effective. Finally, we investigated the ability of our biosensors to monitor the effect of glutamate transport blocker DL-TBOA on modulating glutamate concentrations in the prefrontal cortex of anaesthetized rats. The optimized biosensors recorded a rapid 35-fold increase in extracellular glutamate, and are considered suitable for further exploration in vivo.     

Electrical contacting of redox proteins by nanotechnological means

Redox enzymes in bioelectronic devices usually lack direct electrical contact with electrodes, owing to the spatial separation of their redox centers from the conductive surfaces by the protein shells. The reconstitution of apo-enzymes on cofactor-functionalized nanostructures associated with electrodes provides a means to align the biocatalysts on the conductive surface and to electrically contact redox enzymes with electrodes. The reconstitution of apo-enzymes on cofactor-functionalized gold nanoparticles or carbon nanotubes has led to effective electrical communication between the redox proteins and the electrodes. Alternatively, the reconstitution of redox enzymes on molecular wires that enable electron tunneling or dynamic charge shuttling represent supramolecular biocatalytic nanostructures exhibiting electrical contact. The bioelectrocatalytic activities of the electrically wired reconstituted enzymes on electrodes have allowed the development of amperometric biosensors and biofuel cell elements.     

PNA biosensors for nucleic acid detection

Biosensor devices, based on the conversion of nucleic acid recognition reactions into useful electrical signals, offer considerable promise for DNA diagnostics. The unique hybridization properties of solution-phase PNA can be extrapolated onto transducer surfaces in connection with the design of remarkably specific DNA biosensors. This article reviews the development of PNA biosensors, and discusses common PNA-biosensing protocols along with their prospects in DNA biosensor technology.