Creatinine sensitive biosensor based on ISFETs and creatinine deiminase immobilised in BSA membrane

A creatinine sensitive biosensor based on ion sensitive field-effect transistors (ISFETs) with immobilised creatinine deiminase (CD) is developed. CD is immobilised on the transducer surface by classical cross-linking with bovine serum albumin (BSA) in a glutaraldehyde (GA) vapour. The linear dynamic ranges of biosensors are between 0 and 5 mM creatinine concentration, and the sensor sensitivity depends on the sample buffer concentration. Minimal detection limit for creatinine determination in model solution with 144 mM NaCl and 5% BSA, pH 7.4, is about 10 muM. Biosensor responses are reproducible and stable during continuous work at least for 8 h, and the relative standard deviation of sensor response is approximately 3% (n=48, for creatinine concentrations of 0.2 and 0.6 mM). An investigation about storage stability of creatinine sensitive ENFETs kept in dry at 4-6 degrees C shows that biosensors demonstrate an excellent storage stability for at least 6 months and more. Moreover, creatinine sensitive enzymatic field-effect transistors (ENFETs), demonstrating very good performances, are very selective and specific and well suitable for hemodialysis monitoring.     

Multibiosensor based on enzyme inhibition analysis for determination of different toxic substances

An original concept of an enzyme multibiosensor for determination of toxic substances based on enzyme inhibition analysis has been proposed and its main performances have been analysed. For the development of this multibiosensor, two types of transducers such as potentiometric pH-sensitive field-effect transistors and conductometric thin-films interdigitated electrodes, and three enzymes, namely urease, acetylcholinesterase and butyrylcholinesterase have been used. The experimental data have been treated by multivariate correspondence analysis. A complete procedure for a simultaneous determination of some heavy metal ions and pesticides has been proposed and its advantages have been discussed.     

Potentiometric Biosensors Based on ISFETs and Immobilized Cholinesterases

This paper describes the authors' achievements in the development, investigation, and application of cholinesterase biosensors based on ISFETs. Various biosensors for determination of concentrations of different toxic substances (organophosphorous and carbamate pesticides, hypochlorite, glycoalkaloids) were designed on the basis of reversible and/or irreversible enzyme inhibition effects. The main analytical characteristics of the biosensors developed have been studied under different conditions and optimal experimental protocols for toxic substances determination have been proposed. Most of these biosensors show a high reproducibility and a good operational and storage stability. A quite good correlation with results obtained through routinely used standard methods as HPLC has been shown.     

Enzyme-modified field effect transistors based on surface-conductive single-crystalline diamond

Enzyme-modified field effect transistors (ENFETs) were realized using surface-conductive single-crystalline diamond films. The enzymes penicillinase and acetylcholinesterase were immobilized onto the active area of diamond-based electrolytic solution gated FETs, using different organic linker molecules and cross-linking chemistries. The active area of the devices was patterned to generate enzyme-modified regions next to surface-conductive regions. Penicillinase was chosen as a robust model system, but the main focus of the present paper is on acetylcholinesterase, an enzyme essential for many neuronal signal transduction processes. All the different ENFETs show a clear and specific response to the corresponding substrate, penicillin and acetylcholine. The device response is based on the pH sensitivity of the surface-conductive active area and is enabled by the local pH change induced during the enzymatic reaction. The devices demonstrate promising stability and characteristic variations of the enzymatic activity with measurement conditions. Furthermore, the results from the ENFET measurements were compared with the results of spectrophotometric experiments, carried out with enzymes immobilized on diamond substrates and also with free enzymes in solution. This allows an analysis of the enzyme kinetics, as well as qualitative comparison of the different functionalization methods employed in this study.     

场效应晶体管生物传感器

综述了场效应晶体管生物传感器的研究进展。介绍了场效应晶体管生物传感器的原理、分类及其生物功能物质的固定化技术,并探讨了其存在的问题和发展趋势。     

Enzyme biosensors based on ion-selective field-effect transistors

The key theoretical principles of the work on ion-selective field-effect transistor connected with their application in bioanalytical practice, some specifics of modern microtechnologies for their creation, and measurement schemes with set-ups are discussed. The achievements in the creation of enzyme biosensors based on ion-selective field-effect transistors and prospects for their application are described in detail.     

Piezoquartz biosensors for the analysis of environmental objects, foodstuff and for clinical diagnostic

It is reviewed opportunities and practical application features of gravimetric biosensors for determination of high-and low-molecular biologically active substances and microorganisms in environments, food stuffs and clinical diagnostics. Characteristics of piezosensors based on different nature receptor molecules (an antibodies, antigens, hapten-protein conjugates etc.) are given. It’s reflected the modern view on the wide range of questions, connected with their function in liquids. The special attention is directed towards immunosensors, the most perspective for quantification of trace concentrations in complex matrixes. Possible immunoassay formats in liquid (direct and competitive detection) are given. The questions of formation and regeneration of sensitive layer on the base of biomolecules of different nature are discussed.     

Modelling of the Potentiometric Response of ENFETs Based on Enzymatic Multilayer Membranes

Based on the modified site‐binding model, the response of urea‐sensitive enzymatic field effect transistors (ENFETs) is fitted. The effect of two types of surface sites (silanol and amine sites) and of additional polymeric permselective membrane on the sensitivity of the biosensor is discussed. It is found that the dependence of the slope of the sensor response versus pUrea, on the buffer concentration and on the diffusion of the urea in the enzymatic membrane can be translated by a parameter α that depends on the membrane composition. Moreover, the enhancement of the sensor sensitivity by deposition of additional permselective membranes can be illustrated by a parameter δ that depends on the association of additional permselective membranes and a buffer; the higher values of parameter δ are obtained with a Nafion membrane associated with a phosphate buffer.     

Micro-enzyme field effect transistor sensor using direct covalent bonding of urease

Enzyme field effect transistors (ENFETs), which have been previously proposed for the detection of urea, consist of a pH ion-sensitive FET with urease enzyme immobilized in a polymeric membrane. A new means of preparing the enzymatic sensing layer is proposed in which urease is directly covalently bonded onto the silica insulator. The sensitivity, lifetime and response time of the ENFETs obtained are fairly good.     

Glucose sensor using a phospholipid polymer-based enzyme immobilization method

An electroenzymatic glucose sensor based on a simple enzyme immobilization technique was constructed and tested. The glucose sensor measures glucose concentrations as changes of oxygen concentrations induced by enzymatic reactions. The immobilizing procedure was developed with the purpose of producing wearable biosensors for clinical use. Two types of biocompatible polymers, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with dodecyl methacrylate (PMD) and MPC copolymerized with 2-ethylhexyl methacrylate, were compared as a sensitive membrane of biosensors. The PMD enzyme membrane had a better response time. Linearity, reproducibility, effect of the concentrations of immobilized enzyme and drifts of sensor characteristics in long-term tests were also investigated. The linear characteristics were confirmed with glucose concentration from 0.01 to 2.00 mmol/l, with a coefficient of determination of 0.9999. The average output current for 1 mmol/l and the standard deviation were 0.992 and 0.0283 muA. Significant changes in the sensor's characteristics were not observed for 2 weeks when it was kept in a refrigerator at 4 degrees C. Because of the simple procedure, the enzyme immobilization method is not only useful for wearable devices but also other devices such as micro total analysis systems.     

Formaldehyde-sensitive conductometric sensors based on commercial and recombinant formaldehyde dehydrogenase

Novel formaldehyde-sensitive conductometric biosensors have been developed that are based on commercial bacterial formaldehyde dehydrogenase (FDH) from Pseudomonas putida and recombinant formaldehyde dehydrogenase (rFDH) from the yeast Hansenula polymorpha as the bio-recognition elements. The bio-recognition membranes have mono-layer architecture and consist of enzyme cross-linked with albumin and of the cofactors NAD (for FDH-based sensor) or NAD and glutathione (for rFDH-based sensor). This architecture of the biosensor allows the determination of formaldehyde without adding NAD and glutathione to the analyzed sample at every analysis and conducting measurements on the same transducer without cofactors regeneration since the bio-membrane contains it at high concentration (100 mM for NAD and 20 mM for glutathione). The response is linear in the range from 10 to 200 mM of formaldehyde concentration depending on the enzyme used. The dependence of the biosensor output signals on pH and buffer concentration as well as operational/storage stability and selectivity/specificity of the developed conductometric biosensors have been investigated. The relative standard deviation of the intra-sensor response did not exceed 4% and 10% for rFDH- and FDH-based sensors, respectively. The relative standard deviation of the inter-sensor response constituted 20% for both dehydrogenases used. The biosensors have been validated for formaldehyde detection in some real samples of pharmaceutical (Formidron), disinfectant (Descoton forte) and an industrial product (Formalin). A good correlation does exist between the concentration values measured by the conductometric biosensor developed in this work, an enzymatic method, amperometric biosensors developed earlier, and standard analytical methods of formaldehyde determination.     

Bio-field-effect transistors as detectors in flow-injection analysis

The development of enzyme-modified bio-field-effect transistors (BioFETs) for the determination of glucose, urea, penicillin G, penicillin V and cephalosporin C is reported. BioFETs are produced by covering the pH-sensitive gate areas of ion-selective field-effect transistors with enzyme membranes. The characteristics of the resulting BioFETs and the influence of several parameters, e.g., pH and buffer capacity, are described. The measuring range covers 1–2 orders of magnitude of substrate concentration, and the BioFETs are applicable for 3–12 weeks, depending on the enzyme. They show a short response time and are well suited for detection in flow systems. The frequency of determination with BioFETs in flow systems is high (15–20 measurements per hour). The application of a BioFET in on-line bioprocess control is described. A glucose oxidase FET monitors the glucose concentration during cultivation of Escherichia coli. The results correspond well with off-line liquid chromatographic determinations.     

A Neural Network Model in the Calibration of Glucose Sensor Based on the Immobilization of Glucose Oxidase into Polypyrrole Matrix

The immobilization of enzymes on an electrode surface is of great importance in bioelectrochemistry. The entrapment of enzymes into a polymer matrix is simple and a speedy technique for the production of biosensors. This procedure of enzyme immobilization by electropolymerization has a great significance in fabrication of micro sensors in the preparation of multiplayer devices. In current study, glucose oxidase enzyme that is specific for the glucose determination was entrapped into polypyrrole matrix containing p‐benzoquinone in PIPES buffer and glucose sensitivity of the biosensor was investigated. Then, artificial neural network analysis was done for the nonlinear calibration plot. This implementation can be used for the sensor failure detection, as well. The estimation power of the neural network used in the direct and inverse calibration modelling was examined by statistical methods. It presented the good performance for the estimation power.     

Recent trends in potentiometric sensor arrays--a review

Nowadays there exists a large variety of ion sensors based on polymeric or solid-state membranes that can be used in a sensor array format in many analytical applications. This review aims at providing a critical overview of the distinct approaches that were developed to build and use potentiometric sensor arrays based on different transduction principles, such as classical ion-selective electrodes (ISEs) with polymer or solid-state membranes, solid-contact electrodes (SCE) including coated wire electrodes (CWE), ion-sensitive field-effect transistors (ISFETs) and light addressable potentiometric sensors (LAPS). Analysing latest publications on potentiometric sensor arrays development and applications certain problems are outlined and trends are discussed.     

Evaluation of redox mediators for amperometric biosensors: Ru-complex modified carbon-paste/enzyme electrodes

The properties of reagentless amperometric biosensors are mainly governed by the interaction of the used redox enzyme and the redox mediators used to facilitate the electron-transfer reaction. Both the used redox mediators and the redox enzymes differ concerning their hydrophilicity and their properties within the matrix of a carbon-paste electrode. Since there is no general procedure which is applicable for any enzyme in combination with any redox mediator, optimisation is necessary for each possible combination. Three approaches for the development of biosensors were investigated using carbon-paste electrodes enriched with redox mediator as a base in all sensor architectures. A class of redox mediators with the common formula Ru(LL)(2)(X)(2) (where LL are 1,10-phenantroline or 2,2'-bipyridine type ligands, and X is an acido ligand) was investigated. In the first approach, enzymes were integrated into the carbon paste; in the second, the enzymes were adsorbed on the surface of the mediator-containing carbon-paste electrode and held in place by a Nafion film; and in the third approach, enzymes were entrapped in polymer films, which were electrochemically deposited onto the electrode's surface. The properties of the obtained biosensors strongly depend on the sensor architecture and the specific features of the used enzyme. Thus, our investigation using three different sensor architectures can provide valuable information about the possible interaction between a specific enzyme and a redox mediators with specific properties.     

Urea Biosensors Based on PVC Membrane Ion-Selective Electrodes

Abstract

This paper presents a general method of enzyme immobilization at the surface of ion selective membranes. Covalent binding of enzymes directly on the electrode surface is a very effective method that results in stable enzymatic membranes. As an example the construction of enzymatic sensors for urea determination based on ammonium and hydrogen carbonate ion selective electrodes is presented. The optimum working conditions for these biosensors were found. Bioelectrodes based on an ammonium sensor show very good analytical parameters: dynamic stability - over 2 months without decrease of sensitivity, response time - shorter then 20 s. high sensitivity, determination range from 0.3 to 70 mM. In the contrast to the ammonium ion based biosensors, those constructed on the basis of anion selective electrodes have worse analytical parameters. It is mainly due to poor selectivity and instability of an applied ion selective electrode. In spite of this, both types of urea biosensors were used for measurements in the differential potentiometry mode. The application of such system increased the sensitivity of urea determination.     

Bi-enzyme sensor based on thick-film carbon electrode modified with electropolymerized tyramine

Bi-enzyme sensor based on thick-film epoxy-carbon electrode modified with polytyramine has been developed and examined for the determination of peroxidase substrates and cholinesterase inhibitors. Polytyramine was obtained on the electrode surface by repeated scanning of the potential from +600 to +1800 mV vs. Ag/AgCl in tyramine solution. The enzymes were immobilized in the polytyramine matrix by cross-linking with glutaraldehyde. The biosensor developed provides a reliable and inexpensive way for preliminary testing of common environmental pollutants with a single sensor in accordance with assumed toxic effect by the choice of appropriate substrate and measurement conditions. The bi-enzyme sensor makes it possible to determine substituted phenols and aromatic amines in the micromolar range of their concentrations and anticholinesterase pesticides with detection limits of 0.1 (Coumaphos) and 0.03 micromol l(-1) (Chloropyrifos-methyl).     

Use of the clark oxygen sensor with immobilized enzymes for determinations in flow systems

A small-volume cell has been constructed for amperometric flow measurements with a Clark oxygen sensor and its performance was tested. The Clark sensor can be combined with immobilized enzymes for determination of substances after enzymatic conversion during which oxygen is consumed or released. Two enzymes, glucose oxidase and tyrosinase, were used and two measuring techniques, employing the enzyme immobilized on the Clark sensor membrane and with the enzyme bound on a support in a preceding reactor, were tested and compared. It was found that, in the given system, measurement with the enzyme immobilized on the sensor membrane has better sensitivity, precision and response rate.     

Label-free electrical determination of trypsin activity by a silicon-on-insulator based thin film resistor.

A silicon-on-insulator (SOI) based thin film resistor is employed for the label-free determination of enzymatic activity. We demonstrate that enzymes, which cleave biological polyelectrolyte substrates, can be detected by the sensor. As an application, we consider the serine endopeptidase trypsin, which cleaves poly-L-lysine (PLL). We show that PLL adsorbs quasi-irreversibly to the sensor and is digested by trypsin directly at the sensor surface. The created PLL fragments are released into the bulk solution due to kinetic reasons. This results in a measurable change of the surface potential allowing for the determination of trypsin concentrations down to 50 ng mL(-1). Chymotrypsin is a similar endopeptidase with a different specificity, which cleaves PLL with a lower efficiency as compared to trypsin. The activity of trypsin is analyzed quantitatively employing a kinetic model for enzyme-catalyzed surface reactions. Moreover, we have demonstrated the specific inactivation of trypsin by a serine protease inhibitor, which covalently binds to the active site of the enzyme.     

Design of an Interference‐Free Cholesterol Amperometric Biosensor Based on the Electrosynthesis of Polymeric Films of Diaminonaphthalene Isomers

Different cholesterol amperometric biosensors were developed based on entrapment of cholesterol esterase and/or cholesterol oxidase in polymer films of diaminonaphthalene isomers, electrochemically synthesised from aqueous solutions of the monomers and enzymes in phosphate buffer at neutral pH. These conditions permit the growth of films with extraordinary selective properties which allow the preparation of interference‐free biosensors for application in biological media without the response being affected by the presence of either endogenous species (ascorbic and uric acid) or exogenous species like 4‐acetamidophenol. These selective properties were evaluated for the different monolayer and bilayer configurations proposed in function of the film permeation factor. All the steps involved in the preparation of the biosensors and determination of free or total cholesterol were carried out in a flow system. A comparative study was made of the analytical properties of each of the configurations developed and their application to the flow‐injection determination of cholesterol in a synthetic serum.