Mechanisms controlling the sensitivity of amperometric biosensors in flow injection analysis systems

This paper numerically investigates the sensitivity of an amperometric biosensor acting in the flow injection mode when the biosensor contacts an analyte for a short time. The analytical system is modelled by non-stationary reaction-diffusion equations containing a non-linear term related to the Michaelis-Menten kinetics of an enzymatic reaction. The mathematical model involves three regions: the enzyme layer where enzymatic reaction as well as the mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region. The biosensor operation is analysed with a special emphasis to the conditions at which the biosensor sensitivity can be increased and the calibration curve can be prolonged by changing the injection duration, the permeability of the external diffusion layer, the thickness of the enzyme layer and the catalytic activity of the enzyme. The apparent Michaelis constant is used as a main characteristic of the sensitivity and the calibration curve of the biosensor. The numerical simulation was carried out using the finite difference technique.     

Mathematical modeling of biosensor action in the region between diffusion and kinetic modes

We describe the action of electrochemical enzyme-based biosensor by applying mathematical modeling. We consider two types of biosensors: a biosensor containing a single heterogeneous enzyme layer and biosensor containing an additional protecting polymer-based layer. The initial parameters of the biosensor were selected on the basis of typical immobilized glucose oxidase-based electrochemical biosensor. A phenomenon of the accumulation of the electrochemically active product inside the biocatalytic layer was evaluated. It was shown that accumulation of the product can increase sensitivity of the biosensor no more than 2.6 times. Due to the asymmetric distribution of the electrochemically active product inside the enzyme-containing membrane and asymmetric diffusion of the substrate, it was shown that the thickness of the membrane possesses an optimal value. For the selected set of initial parameters, the optimal thickness of the enzyme-containing layer was 2.9–4.5  $\upmu $ m. Real profiles of the impact of the thickness of the membranes were evaluated. A method for the evaluation of acceptable fluctuations of the membrane diffusion parameters on biosensor response was created, and the profiles of the dependence were calculated. These dependencies can be used for development of the software for biosensor monitoring.     

Mathematical modeling of the action of biosensor possessing variable parameters

Electrochemical biosensor containing flat semi-permeable membrane covering enzyme-containing layer has been investigated. Mathematical modeling of the action modes of electrochemical biosensors with outer diffusion membrane was performed. Operation of the biosensor under the conditions when the permeability of the membrane and the activity of the biocatalytic layer depend on the parameters of the probe has been examined. The pH and temperature were selected as the main parameters which often affect the action of biosensors. A set of parameters was selected when the biosensor operates in kinetic and diffusion modes of action. The response time of the biosensor was shown to be sensitive to the mode of the biosensor action especially in the boundary region of the biosensor action. The linearity of the biosensor (the linear dependence of the biosensor response on the substrate concentration) in the deep diffusion mode can be increased by several magnitudes, whereas the response time increases only several times.     

Mathematical Model and Numerical Simulation of Conductometric Biosensor of Urea

In this article, a mathematical model was developed to describe and optimize the configuration of the urea biosensor. The biosensor is based on interdigitated gold microelectrodes modified with a urease enzyme membrane. The model presented here focuses on the enzymatic reaction and/or diffusion phenomena that occur in the enzyme membrane and in the diffusion layer. Numerical resolution of differential equations was performed using the finite difference technique. The mathematical model was validated using experimental biosensor data. The responses of the biosensor to various conditions were simulated to guide experiments, improve analytical performance, and reduce development costs.     

The Effect of Diffusion Limitations on the Response of Amperometric Biosensors with Substrate Cyclic Conversion

A mathematical model of amperometric biosensors in which chemical amplification by cyclic substrate conversion takes place in a single enzyme membrane has been developed. The model involves three regions: the enzyme layer where enzyme reaction as well as mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region where the analyte concentration is maintained constant. Using computer simulation the influence of the thicknesses of the enzyme layer and the diffusion region on the biosensor response was investigated. This paper deals with conditions when the mass transport in the exterior region may be neglected to simulate the biosensor response in a well-stirred solution. The digital simulation was carried out using the finite difference technique.     

Amperometric Determination of Hydrogen Peroxide and its Mathematical Simulation for Horseradish Peroxidase Immobilized on a Sonogel Carbon Electrode

A mathematical model of a horseradish peroxidase biosensor was applied to simulate the amperometric response for the detection of hydrogen peroxide. The development of the mathematical model was based on the Michaelis–Menten equation and Fick’s Second Law. The theoretical study is based on the determination of physico-chemical and geometric parameters of a horseradish peroxidase biosensor as well as the kinetic parameters of reaction mechanism such as diffusion coefficients of hydrogen peroxide, the thickness of enzymatic layer, and the Michaelis–Menten kinetic constant. The theoretical analysis provides an accurate estimate of parameters affecting the biosensor performance such as the diffusion coefficient of hydrogen peroxide in the biomembrane that was estimated to be 56?×?10^?12 m²/s. The thickness of diffusion layer was estimated to be 80–100?µm and the biomembrane 7.5?µm. The experimental and numerical values of kinetic parameters were 0.92 and 0.98?µM for the Michaelis–Menten constants and 0.010 and 0.012?µM/s for the catalytic activity rates. The model was validated for hydrogen peroxide detection and exhibited a good agreement with the experimental measurements.     

Mathematical Modelling of a Non‐enzymatic Amperometric Electrochemical Biosensor for Cholesterol

Thickness of the electro‐polymerized layer grown on a substrate and used as the recognition element for the analyte is critical to measuring the response of a biosensor, with high sensitivity and accuracy. However, it is difficult to control the thickness during synthesis. A mathematical model is developed in this study that considers thickness of the electro‐polymerized layer in simulating the electrochemical response of a non‐enzymatic biosensor for cholesterol in blood. The model includes transient kinetics and one‐dimensional diffusion of the analyte in the poly‐methyl orange (PMO) recognition layer electrochemically grown on the electrode. The governing partial differential equations resulting from the species conservation balances in the PMO layer are numerically solved. Time and spatial concentration profiles of the analyte in the PMO layer are determined. Model predictions are calibrated with the experimental data for different PMO thicknesses. Interestingly, model predictions show a linear response over the calibrated concentration range of cholesterol for all PMO layer thicknesses. Based on the chronoamperometry measurements, the model predictions for the cholesterol concentrations measured in the laboratory samples were also found to be remarkably accurate. This is the first mathematical model developed to understand the transport and kinetics of an analyte in the electro‐polymerized layer used as the recognition element of a non‐enzymatic biosensor.     

Biosensor for determination of carboxylic acids in wines based on the inhibition of sarcosine oxidase

The flow-through amperometric biosensor is presented for determination of carboxylic acids. It is based on two sensor layers that are deposited on a platinum electrode. The inner layer serves to eliminate interferences by limiting diffusion of electrochemically active substances such as ascorbic acid. This layer is electro-polymerized using an equimolar mixture of o-phenylenediamine and resorcinol. The outer layer is prepared by cross-linking the enzyme sarcosine oxidase and bovine serum albumin using glutaraldehyde. The formation of enzymatically produced hydrogen peroxide is monitored at 600 mV vs. an Ag/AgCl reference electrode. The addition of carboxylic acids causes competitive inhibition of the enzyme and a decrease in signal. The assay was optimized for determination of carboxylic acids in wine samples. Following 10-fold dilution, most samples contain 1–10 mM individual carboxylic acids and thus a 5 mM concentration of sarcosine was chosen as being optimal for competition. In case of real samples, the biosensor measures the sum of all carboxylic acids, which serves as a parameter describing the quality of wines. Results from testing several wine samples are reported.     

Computational modelling of amperometric biosensors in the case of substrate and product inhibition

In this paper the response of an amperometric biosensor at mixed enzyme kinetics and diffusion limitations is modelled in the case of the substrate and the product inhibition. The model is based on non-stationary reaction–diffusion equations containing a non-linear term related to non-Michaelis–Menten kinetics of an enzymatic reaction. A numerical simulation was carried out using a finite difference technique. The complex enzyme kinetics produced different calibration curves for the response at the transition and the steady-state. The biosensor operation is analysed with a special emphasis to the conditions at which the biosensor response change shows a maximal value. The dependence of the biosensor sensitivity on the biosensor configuration is also investigated. Results of the simulation are compared with known analytical results and with previously conducted researches on the biosensors.     

An Analysis of Mixtures Using Amperometric Biosensors and Artificial Neural Networks

This paper presents a sensor system based on a combination of an amperometric biosensor acting in batch as well as flow injection analysis with the chemometric data analysis of biosensor outputs. The multivariate calibration of the biosensor signal was performed using artificial neural networks. Large amounts of biosensor calibration as well as test data were synthesized using computer simulation. Mathematical and corresponding numerical models of amperometric biosensors have been built to simulate the biosensor response to mixtures of compounds. The mathematical model is based on diffusion equations containing a non-linear term related to Michaelis–Menten kinetics of the enzymatic reaction. The principal component analysis was applied for an optimization of calibration data. Artificial neural networks were used to discriminate compounds of mixtures and to estimate the concentration of each compound. The proposed approach showed prediction of each component with recoveries greater that 99% in flow injection as well as in batch analysis when the biosensor response is under diffusion control.     

Gas Diffusion Electrodes for Use in an Amperometric Enzyme Biosensor

The preparation of gas diffusion electrodes and their use in an amperometric enzyme biosensor for the direct detection of a gaseous analyte is described. The gas diffusion electrodes are prepared by covering a PTFE membrane (thickness 250 μm, pore size 2 μm, porosity 35%) with gold, platinum, or a graphite/PTFE mixture. Gold and platinum are deposited by e‐beam sputtering, whereas the graphite/PTFE layer is prepared by vacuum filtration of a respective aqueous suspension. These gas diffusion electrodes are exemplarily implemented as working electrodes in an amperometric biosensor for gaseous formaldehyde containing NAD‐dependent formaldehyde dehydrogenase from P. putida [EC. 1.2.1.46] as enzyme and 1,2‐naphthoquinone‐4‐sulfonic acid as electrochemical mediator. The resulting sensors are compared with regard to background current, signal noise, linear range, sensitivity, and detection limit. In this respect, sensors with gold or graphite/PTFE covered membranes outclass ones with platinum for this particular analyte and sensor configuration.     

Effects of nonionic surfactants on electrochemical behavior of ubiquinone and menaquinone incorporated in a carbon paste electrode

A carbon paste electrode, in which the carbon particles were coated with a thin layer of a nonionic surfactant (NIS), was constructed with a pasting liquid containing ubiquinone (UQ) or menaquinone (MQ). It has revealed that the layer acts not only as a diffusion barrier but also as a matrix for the redox reaction of quinones at electrode surface, and its effects on the electrochemical behavior of quinones depend on both the physico-chemical structure of a surfactant and the kind of quinones. Further, such a modification was applied to the preparation of an enzyme electrode in which the quinone molecule act as a redox mediator and the influences on the sensitivity of the glucose biosensor was demonstrated.     

流动注入式乳酸生物传感器

研制了一种测定L－乳酸的生物传感器,将乳酸氧化酶（LOD）通过共价键固定在尼龙钢上制备乳酸氧化酶膜,将制得的酶膜固定在氧电极上构成乳酸生物传感器;将透析膜放在氧化酶膜上产生对L－乳酸扩散高度限制来改变该生物传感器的响应;酶膜机械强度高,在氧电极上反复装卸而不损坏,所构成的乳酸生物传感器的校正曲线的乳酸定量上限达5mmol/L,响应时间小于30s;初步血样测试的结果显示该乳酸生物传感器用于临床血乳酸的测定具有可行性。     

Optical Urea Biosensor Based On Ammonium Ion Selective Membrane

ABSTRACT

An optical urea biosensor was developed by immobilizing an urease enzyme layer on a thin ammonium-selective polymer membrane. The ammonium optical membrane utilized dichlorofluorescein octadecyl ester (DCFOE) as anionic chromophore and nonactin as neutral ionophore. The urease layer was coated on the top of the ammonium layer by gelatin entrapment combined with glutaradehyde cross-linking. Hydrolysis of urea catalyzed by urease produced ammonium ion, which was extracted into the-polymer film to form complexes with nonactin. A proton was released which resulted in a color change of the optical membrane due to charge neutrality principle. The biosensor     

Fabrication of a bilayer potentiometric phosphate biosensor by cross-link immobilization with bovine serum albumin and glutaraldehyde

Chemical cross-linking of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) with glutaraldehyde (GLA) and bovine serum albumin (BSA) has been used to fabricate a stable and reliable bilayer potentiometric phosphate biosensor. The bilayer arrangement consists of an inner BSA-GLA layer and an outer BSA-GLA-PNP-XOD layer. The inclusion of the inner BSA-GLA layer improves the adhesion of the outer BSA-GLA-PNP-XOD layer and ensures stability of the phosphate biosensor. Established optimum conditions for immobilization of the enzymes in the outer layer and for reliable potentiometric measurement were 4.5% v/v GLA, 6.8% w/v BSA, XOD:PNP mole ratio of 1:8, and a film drying time of 30 min. As little as 20 μM of phosphate can be detected with the BSA-GLA/BSA-GLA-XOD-PNP bilayer biosensor with a linear concentration range between 40 and 120 μM. The biosensor was very stable for 21 days, achieving a good reproducibility with a rsd of only 5.7% and, even after more than a month, the change in the initial potential value was only 10%.     

纳米复合物修饰电极的电化学传感器检测芦丁

研制了纳米复合物修饰电极,碳纳米管与表面含有大量氨基的壳聚糖在玻碳电极表面首先形成碳纳米管/壳聚糖膜,通过膜表面丰富的氨基与纳米Au的强静电吸附,在玻碳电极表面获得均匀致密的纳米金修饰层.这种基于纳米复合材料制备的新型电化学传感器对芦丁具有很好的响应,可以快速地实现电极与芦丁之间的直接电子转移,有良好的稳定性.芦丁的测定线性范围为4.00×10-7～1.77×10-5 mol/L,最低检测限为1.29×10-7 mol/L.由于抗坏血酸在该修饰电极上的氧化电位出现显著负移,因此可避免抗坏血酸对芦丁测定的干扰.该方法可以不经预分离直接检测药物中的芦丁含量.     

Fabrication of multiwalled carbon nanotubes/polyaniline modified Au electrode for ascorbic acid determination

An ascorbate oxidase (AsOx) (E.C.1.10.3.3) purified from Lagenaria siceraria fruit was immobilized covalently onto a carboxylated multiwalled carbon nanotubes and polyaniline (c-MWCNT/PANI) layer electrochemically deposited on the surface of an Au electrode. The diffusion coefficient of ascorbic acid was determined as 3.05 × 10(-4) cm(2) s(-1). The behavior of different electrolytes on electro-deposition was also studied. An ascorbate biosensor was fabricated using a AsOx/c-MWCNT/PANI/Au electrode as a working electrode, Ag/AgCl (3 M/saturated KCl) as standard and Pt wire as an auxiliary electrode connected through a potentiostat. Linear range, response time and detection limit were 2-206 μM, 2 s and 0.9 μM respectively. The biosensor showed optimum response at pH 5.8 and in a broader temperature range (30-45 °C), when polarized at +0.6 V. The biosensor was employed for determination of ascorbic acid level in sera, fruit juices and vitamin C tablets. The sensor was evaluated with 91% recovery of added ascorbic acid in sera and 6.5% and 11.4% within and between batch coefficients of variation respectively for five serum samples. There was a good correlation (r = 0.98) between fruit juice ascorbic acid values by the standard 2,6-dichlorophenolindophenol (DCPIP) method and the present method. The enzyme electrode was used 200 times over a period of two months, when stored at 4 °C. The biosensor has advantages over earlier enzyme sensors in that it has no leakage of enzyme, due to the covalent coupling of enzyme with the support, lower response time, wider working range, higher storage stability and no interference by serum substances.     

Development of a diamine biosensor

An amperometric diamine sensor is developed for clinical applications in diagnosis of bacterial vaginosis (BV). The sensor is based on crosslinked putrescine oxidase (PUO) which catalyzes the conversion of diamines (mainly putrescine and cadaverine) to products including hydrogen peroxide. The hydrogen peroxide is detected anodically at platinum electrode polarized at 0.5 V versus Ag/AgCl. Platinum-plated gold electrodes used as a substrate for the sensor construction, are batch-fabricated on a flexible polyimide foil (Kapton(R), DuPont). A three-electrode cell configuration is used in all amperometric measurements. The sensor construction is based on three layers: an inner layer to reject the interference effect of oxidizable molecules, an outer diffusion controlling layer, and in addition, an enzyme middle layer. The enzyme layer was immobilized by crosslinking PUO with bovine serum albumin (BSA) using glutaraldehyde (GA). An optimization study of the enzyme solution composition was carried out. With the optimized enzyme layer, the biosensor showed a very high sensitivity and fast response time of ca. 20 s. The sensor has a linear dynamic range from (0.5-300 muM) for putrescine that covers the expected biological levels of the analyte. Details on sensor fabrication and characterization are given in the present work.     

Evaluation of DNA damage and antioxidant capacity of sericin by a DNA electrochemical biosensor based on dendrimer-encapsulated Au-Pd/chitosan composite

Au-Pd bimetallic nanoparticles were prepared in the presence of an amine-terminated dendrimer of a fourth generation poly(amidoamine) type. A biosensor was fabricated by immobilizing dsDNA on a thin layer of a dendrimer-encapsulated bimetallic nanoparticles (Au-Pd) in a chitosan composite on a glassy carbon electrode. The biosensor was evaluated by square wave voltammetry for the determination of the oxidative damage of immobilized DNA and the antioxidant capacity of sericin. The biosensor is shown to be suitable for the rapid detection of DNA damage and the assessment of the antioxidant capacity of sericin.     

Biosensor with Protective Membrane for the Detection of DNA Damage and Antioxidant Properties of Fruit Juices

With the purpose to prepare a DNA biosensor protected with an outer‐sphere membrane against high molecular weight interferences, a carbon film electrode was layer‐by‐layer modified with dsDNA and chitosan. Using cyclic and square‐wave voltammetry and impedance spectroscopy, the oxidative damage of DNA by the hydroxyl and superoxide anion radicals was detected which consists of opening of the helix structure followed by deep DNA chain degradation. The biosensor has been applied to the detection of the antioxidant effect of apple and orange juices. The investigation of the novel biosensor with a protective membrane represents a significant contribution to the field of DNA biosensors utilization.