Amperometric cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on conducting polypyrrole films

Fabrication of an amperometric cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto conducting polypyrrole (PPY) films using electrochemical entrapment technique is described. Electrochemical polymerization was carried out using a two-electrode cell configuration at 0.8 V. Characterization of resulting amperometric biosensor for the estimation of cholesterol has been experimentally determined in terms of linear response range, optimum pH, applied potential, temperature, and shelf-life. These PPY/ChEt/ChOx electrodes can be used for cholesterol ester estimation from 1 to 8 mM and have shelf-life of about 4 weeks at 4 °C during which about 15 estimations of cholesterol ester could be made. The sensitivity of PPY/ChEt/ChOx electrode has been found to be 0.15 μA/mM and the apparent K_m value for this electrode is 9.8 mM. Conductivity of the polymer films found to be about 3×10⁻³ S/cm.     

An inhibition type amperometric biosensor based on tyrosinase enzyme for fluoride determination

In this study, a new biosensor based on the inhibition of tyrosinase for the determination of fluoride is described. To construct the biosensor tyrosinase was immobilized by using gelatine and cross-linking agent glutaraldehyde on a Clark type dissolved oxygen (DO) probe covered with a teflon membrane which is sensitive for oxygen. The phosphate buffer (50 mM, pH 7.0) at 30 °C were established as providing the optimum working conditions. The method is based on the measurement of the decreasing of dissolved oxygen level of the interval surface that related to fluoride concentration added into reaction medium in the presence of catechol. Inhibitor effect of fluoride results in decrease in dissolved oxygen concentration. The biosensor response depends linearly on fluoride concentration between 1.0 and 20 μM with a response time of 3 min.In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity and storage stability were carried out. From the experiments, the average value (x), Standard deviation (S.D) and coefficient of variation (C.V %) were found as 10.5 μM, ± 0.57 μM, 5.43%, respectively for 10 μM fluoride standard.     

Amperometric glucose biosensor based on a gold nanorods/cellulose acetate composite film as immobilization matrix

We report on the utilization of gold nanorods to create a highly responsive glucose biosensor. The feasibility of an amperometric glucose biosensor based on immobilization of glucose oxidase (GOx) in gold nanorod is investigated. GOx is simply mixed with gold nanorods and cross-linked with a cellulose acetate (CA) medium by glutaraldehyde. The adsorption of GOx on the gold nanorods is confirmed by X-ray photoelectron spectroscopy (XPS) measurements. Circular dichroism (CD) and UV-spectrum results show that the activity of GOx was preserved after conjugating with gold nanorods. The current response of modified electrode is 10 times higher than that of without gold nanorods. Under optimal conditions, the biosensor shows high sensitivity (8.4 μA cm⁻² mM⁻¹), low detection limit (2 × 10⁻⁵ M), good storage stability and high affinity to glucose (). A linear calibration plot is obtained in the wide concentration range from 3 × 10⁻⁵ to 2.2 × 10⁻³ M.     

Development of a biosensor for caffeine

We have utilized a microbe, which can degrade caffeine to develop an Amperometric biosensor for determination of caffeine in solutions. Whole cells of Pseudomonas alcaligenes MTCC 5264 having the capability to degrade caffeine were immobilized on a cellophane membrane with a molecular weight cut off (MWCO) of 3000-6000 by covalent crosslinking method using glutaraledhyde as the bifunctional crosslinking agent and gelatin as the protein based stabilizing agent (PBSA). The biosensor system was able to detect caffeine in solution over a concentration range of 0.1 to 1 mg mL⁻¹. With read-times as short as 3 min, this caffeine biosensor acts as a rapid analysis system for caffeine in solutions. Interestingly, successful isolation and immobilization of caffeine degrading bacteria for the analysis of caffeine described here was enabled by a novel selection strategy that incorporated isolation of caffeine degrading bacteria capable of utilizing caffeine as the sole source of carbon and nitrogen from soils and induction of caffeine degrading capacity in bacteria for the development of the biosensor. This biosensor is highly specific for caffeine and response to interfering compounds such as theophylline, theobromine, paraxanthine, other methyl xanthines and sugars was found to be negligible.Although a few biosensing methods for caffeine are reported, they have limitations in application for commercial samples. The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. The optimum pH and temperature of measurement were 6.8 and 30 ± 2 °C, respectively. Interference in analysis of caffeine due to different substrates was observed but was not considerable. Caffeine content of commercial samples of instant tea and coffee was analyzed by the biosensor and the results compared well with HPLC analysis.     

Polyurethane Enzyme Membranes for Chip Biosensors

Abstract

The coverage of electrochemical chip sensors based on silicon technology with a polyurethane enzyme membrane is described. After crosslinking of the surface by polyfunctional isocyanates the enzyme membrane shows good adhesion, complete retention of the enzyme molecules, and low diffusional resistance to both analytes and products. Using thin film noble metal electrodes and ion sensitive field effect transistors, glucose and urea sensors with good long term stability and short response time have been prepared.     

Immobilization and stabilization of biomaterials for biosensor applications

Biosensors are finding applications in a variety of analytical fields. A biosensor basically consists of a transducer in conjunction with a biologically active molecule that converts a biochemical signal into a quantifiable electric response. The specificity of the biosensor depends on the selection of the biomaterial. Enzymes, antibodies, DNA, receptors, organelles, microorganisms as well as animal and plant cells or tissues have been used as biologic sensing materials. Advances in biochemistry, molecular biology, and immunochemistry are expected to lead to a rapid expansion in the range of biologic recognition elements to be used in the field of biosensors. Biomaterials that are stable and function even in highly acidic, alkaline, hydrophobic, or oxidizing environments as well as stable to high temperature and immune to toxic substrates in the processing stream will play an important role. Techniques for immobilization of the biomaterials have played a significant role in the biosensor field. Immobilization not only brings about the intimate contact of the biologic catalysts with the transducer, but also helps in the stabilization of the biologic system, thus enhancing its operational and storage stability. A number of techniques have been developed in our laboratory for the immobilization of enzymes, multienzyme systems, cells, and enzyme-cell conjugates. Some of these aspects that are of significance in biosensor applications have been highlighted.     

Light-emitting devices based on solid electrolytes and polyelectrolytes

The properties of light-emitting diodes (LEDs) based on organic layers containing mobile ions, so-called light-emitting electrochemical cells (LECs), are reviewed. These devices have some unique properties: their current–voltage characteristics are antisymmetric with respect to the origin and they emit light under both forward and reverse bias. The physical processes involved in the emission from LECs are discussed in terms of a thermodynamic model. Recent work on blends of luminescent and ion-conducting polymers is summarized. In addition, the properties of novel single-component LECs and polyelectrolyte-based devices are presented. The results show that LECs with performances superior to that of conventional LED devices can be fabricated, but questions concerning the transient behavior and degradation mechanisms persist. © 1998 John Wiley & Sons, Ltd.     

Advanced carbon nanomaterials for electrochemiluminescent biosensor applications

Electrochemiluminescent biosensors are nowadays an established technology in the field of immunosensors and diagnostics. Along with the advent of nanotechnology, the marriage between electrochemiluminescence and nanomaterials results in promising enhancing strategies in many biosensor applications. Among nanomaterials, carbon-based ones are the most used, as (i) scaffolds, (ii) luminophores and (iii) electrode materials of the sensor. In this review, we describe the importance of a rational modification and functionalization of carbon nanomaterials to optimize electrochemiluminescence signal, and we also resume the latest and most relevant applications of electrochemiluminescent biosensors based on carbon nanomaterials.     

Enzyme-functionalized mesoporous silica for bioanalytical applications

The unique properties of mesoporous silica materials (MPs) have attracted substantial interest for use as enzyme-immobilization matrices. These features include high surface area, chemical, thermal, and mechanical stability, highly uniform pore distribution and tunable pore size, high adsorption capacity, and an ordered porous network for free diffusion of substrates and reaction products. Research demonstrated that enzymes encapsulated or entrapped in MPs retain their biocatalytic activity and are more stable than enzymes in solution. This review discusses recent advances in the study and use of mesoporous silica for enzyme immobilization and application in biosensor technology. Different types of MPs, their morphological and structural characteristics, and strategies used for their functionalization with enzymes are discussed. Finally, prospective and potential benefits of these materials for bioanalytical applications and biosensor technology are also presented. Figure Enzyme-functionalized mesoporous silica fibers and their integration in a biosensor design. The immobilization process takes place essentially in the silica micropores.     

Amperometric biosensor for rapid measurement of 3-hydroxybutyrate in undiluted whole blood and plasma

A simple, rapid (< 30 s) electrochemical method for the determination of 3-hydroxybutyrate in whole blood or plasma is described, which uses NAD⁺-dependent d-3-hydroxybutyrate dehydrogenase immobilized at novel platinized carbon electrodes. The steady-state oxidation current produced by enzymatically generated NADH is measured at + 150 mV vs. Ag/AgCl. Enzyme electrodes produced by direct adsorption were stable for at least 3 months. Undiluted whole blood measurement with the sensor was compared with routine spectrophotometric analysis of plasma and perchloric acid extracts of whole blood.     

Development of a hypoxanthine biosensor based on immobilized xanthine oxidase chemically modified electrode

An amperometric biosensor for hypoxanthine determination has been developed. The sensor uses a Nafion-paraquat chemically modified glassy-carbon electrode. It detects the oxygen consumed by the enzymatic reaction catalyzed by xanthine oxidase which is immobilized on the electrode surface. The sensor responds linearly to hypoxanthine over the concentration range of 1 × 10⁻⁶ M −2 × 10⁻⁴ M. The biosensor can be reused for more than 100 times without significant deterioration in performance. After 32 days storage at 3–5°C, the sensor response remains at 68% of the initial level. The high sensitivity, selectivity and stability of this biosensor demonstrates its practical applicability for a simple, rapid and economical determination of hypoxanthine in fish samples.     

Dual amperometric-potentiometric biosensor detection system for monitoring organophosphorus neurotoxins

A dual-transducer flow-injection biosensor detection system for monitoring organophosphorus (OP) neurotoxins is described. Such simultaneous use of different physical transducers in connection to the same (organophosphorous hydrolase (OPH)) enzyme enhances the information content and provides discrimination between various subclasses of OP compounds. While the potentiometric biosensor responds favorably to all OP compounds, reflecting the pH changes associated with the OPH activity, the amperometric device displays well-defined signals only towards OP substrates (pesticides) liberating the oxidizable p-nitrophenol product. The potentiometric detection has been accomplished with a silicon-based pH-sensitive electrolyte-insulator-semiconductor (EIS) transducer, operated in the constant-capacitance (ConCap) mode. Both transducers are prepared by a thin-film fabrication technology, and respond rapidly and independently to sudden changes in the level of the corresponding OP compound, with no apparent cross reactivity. Relevant experimental variables were evaluated and optimized. Such development holds great promise for field screening of OP neurotoxins in connection to various defense and environmental scenarios. The multiple-transduction concept could be extended for increasing the information content of other ‘class-enzyme’ biosensor systems.     

介孔材料及其改性材料中酶的固定化研究进展

介孔材料具有高的比表面积、高的孔体积、均一可调的孔径、有序的孔道结构以及易于表面功能化等优点,可广泛用于酶的固定化.介孔材料中酶的固定化方法主要包括物理吸附、物理包埋和化学吸附.综述了介孔材料中不同固定化酶方法的优缺点、酶的固定化影响因素及固定化酶的应用,并对固定化酶的发展前景进行了展望.     

An Electrochemical Acetylcholinesterase Biosensor Based on Fluorene(bisthiophene) Comprising Polymer for Paraoxon Detection

In this study, a novel conductive polymer comprising biosensor based on poly-2,2′-(9,9-dioctyl-9 h-fluorene-2,7-diyl)bistiophene (Poly(BT)) and acetylcholinesterase (AChE) was reported for the determination of paraoxon. This practical biosensor allowed to catalyze electrochemical oxidation of acetylthiocholine (+0.6 V vs. Ag reference). The detection range for acetylcholine chloride (AThCl) with Poly(BT)/AChE was found to be 0.025–4 mM. In pesticide analysis, wide linear ranges from 0.5 to 1 μg/L and 1 to 14 μg/L, and a low detection limit of 0.033 μg/L were estimated. Under optimum operating conditions, the developed biosensor was used for pesticide detection in milk and tap water samples, effectively.     

A new photoimaging system based on a trisdiazonium salt as a photocrosslinker for sulfonated polyelectrolytes

A photocrosslinker based on a trisdiazonium salt has been synthesized from 4,4′,4″-triaminotriphenylamine. Polyelectrolyte complexes can be obtained by mixing the trisdiazonium crosslinker with poly(styrenesulfonate) (PSS). The polyelectrolyte complex precipitates when PSS is added in an ionic ratio of around 1:1 to a trisdiazonium salt solution. However, the addition of an excess of PSS to the mixture leads to redissolution of the precipitate. A mixture of PSS and the photocrosslinker (27 wt.%) can be processed from solution to give thin films by spin coating. When the films are irradiated with UV light, a change in the solubility of the material is observed, which is due to the decomposition of diazonium groups and the transformation of the ionic bond into a covalent one, which in turn transforms the polyelectrolyte complex into a covalent network. The process has been characterized by UV and IR spectroscopy. Photoimaging experiments have been performed on this material and polymeric relief structures have been obtained through a photolithographic process.     

核酸工具酶辅助的信号放大技术在生物分子检测中的应用

核酸工具酶具有催化能力高、序列识别能力强、反应条件温和以及生物相容性好等优势,因而被广泛地应用于核酸、蛋白质和小分子等生物活性分子的分析检测。本文主要对核酸工具酶进行介绍,并对应用于生物分子检测的一些核酸工具酶辅助的信号放大技术进行较为全面的综述,并展望了该技术的应用前景。     

溶胶凝胶复合材料的研究及对生物酶的固定

研究了一种新型的氧化铝/聚乙二醇（Al2O3/PEG）溶胶凝胶复合材料,利用红外光谱、探针电镜对材料的性能进行了表征。试验结果表明：复合膜中含有大量的羟基和氢键,且两种组分形成互穿网络,该材料刚柔相济的特点使其适合于做生物传感器的固定化材料。PEG是极性高聚物,在溶胶凝胶的体系中,可以起到加速反应及防止膜开裂的作用。Al2O3/PEG复合材料对酶具有较好的生物相容性和较高的稳定性,将生物酶固定其中制备成的生物传感器具有良好的反应活性,同时,还证明了这种生物传感器具有稳定性和灵敏度高的特点。     

酶注射式葡萄糖生物传感器建模方法研究

建立了酶注射式生物传感器的机理模型,并通过实验验证模型精确性.用传感器检测1和2 mg/mL葡萄糖溶液得到电压数据,通过数据拟合确定模型参数.将浓度值3 mg/mL带入模型得到预测曲线,再将其与传感器检测数据拟合后曲线进行比较,验证模型精确性.结果表明,参加反应的酶液米氏常数Km为1.97,数学模型与实际传感器工作模型相关系数(R2)为0.998.     

TTF-TCNQ complex based printed biosensor for long-term operation

A printed amperometric glucose sensor based on glucose oxidase adsorbed on crystals of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) is described. The sensitivity and the stability of the sensor are affected by the binder and solvent used for the preparation of the GOD.TTF-TCNQ paste. The sensors are continuously used in a flow injection analysis (FIA) system under continuous polarization at 0.15 V (vs Ag/AgCl) at 37°C. The developed sensors exhibit a large response current, an extended linear range and oxygen independence. The sensors can be used for more than 3 months. The GOD.TTF-TCNQ paste is suitable for the preparation of planar sensor by screen printing method.     

Biosensor for determination of hydrogen peroxide based on catalase activity of human erythrocytes

A hydrogen peroxide biosensor based on human erythrocytes is described. Erythrocytes are retained on the surface of an oxygen electrode by a semipermeable membrane. The response is based on the catalase activity of the erythrocytes. The sensitivity of 10^?4 mol 1^?1 and linearity from 1.5×10^?4 to 5×10^?3 mol^?1 are comparable to those of analogous enzyme biosensors for hydrogen peroxide determination. The greatest advantages of this biosensor are its easy preparation and a lifetime of 2 months together with good reproducibility (relative standard deviation <5%) and selectivity; only ascorbic acid appeared to interfere with the measurements.