New CNT/poly(brilliant green) and CNT/poly(3,4-ethylenedioxythiophene) based electrochemical enzyme biosensors

A combination of the electroactive polymer poly(brilliant green) (PBG) or conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) with carbon nanotubes to obtain CNT/PBG and CNT/PEDOT modified carbon film electrodes (CFE) has been investigated as a new biosensor platform, incorporating the enzymes glucose oxidase (GOx) as test enzyme, alcohol oxidase (AlcOx) or alcohol dehydrogenase (AlcDH). The sensing parameters were optimized for all biosensors based on CNT/PBG/CFE, CNT/PEDOT/CFE platforms. Under optimized conditions, both GOx biosensors exhibited very similar sensitivities, while in the case of AlcOx and AlcDH biosensors, AlcOx/CNT/PBG/CFE was found to give a higher sensitivity and lower detection limit. The influence of dissolved O₂ on oxidase-biosensor performance was investigated and was shown to be different for each enzyme. Comparisons were made with similar reported biosensors, showing the advantages of the new biosensors, and excellent selectivity against potential interferents was successfully demonstrated. Finally, alcohol biosensors were successfully used for the determination of ethanol in alcoholic beverages.     

Recent Progress in the Development of Conducting Polymer‐Based Nanocomposites for Electrochemical Biosensors Applications: A Mini‐Review

Among various immobilizing materials, conductive polymer‐based nanocomposites have been widely applied to fabricate the biosensors, because of their outstanding properties such as excellent electrocatalytic activity, high conductivity, and strong adsorptive ability compared to conventional conductive polymers. Electrochemical biosensors have played a significant role in delivering the diagnostic information and therapy monitoring in a rapid, simple, and low cost portable device. This paper reviews the recent developments in conductive polymer‐based nanocomposites and their applications in electrochemical biosensors. The article starts with a general and concise comparison between the properties of conducting polymers and conducting polymer nanocomposites. Next, the current applications of conductive polymer‐based nanocomposites of some important conducting polymers such as PANI, PPy, and PEDOT in enzymatic and nonenzymatic electrochemical biosensors are overviewed. This review article covers an 8‐year period beginning in 2010.     

Immobilization of Invertase in a Novel Proton Conducting Poly(vinylphosphonic acid) – poly(1-vinylimidazole) Network

A novel proton conducting polymer blend was prepared by mixing poly(vinylphosphonic acid) (PVPA) with poly(1-vinylimidazole) (PVI) at various stoichiometric ratios via changing molar ratio of monomer repeating unit to achieve the highest protonation. The polymer network having the most suitable stoichiometric ratio for substantial proton conductivity was prepared and characterized by FT-IR spectroscopy and proton conductivity measurements. The network was used for immobilization of invertase and some important kinetic parameters such as the maximum reaction rate (V_max) and Michaelis-Menten constant (K_m) were investigated for the immobilized invertase. Additionally, optimum temperature and pH were determined to acquire the best conditions for the highest enzyme activity. Operational stability of the entrapped enzyme was also examined. The results reveal that the most stable and highly proton conducting polymer network may play a pioneer role in the biosensors applications as given by FT-IR, elemental analysis, impedance spectroscopy and storage stability experiments.     

Biosensors Based on Aligned Carbon Nanotubes Coated with Inherently Conducting Polymers

The use of multiwalled aligned carbon nanotubes provides a novel electrode platform for inherently conducting polymer based biosensors. The example used here to highlight the usefulness of such a platform is the polypyrrole based glucose oxidase system for detection of glucose. The use of these three dimensional electrodes offers advantages in that large accessible enzyme loadings can be obtained within an ultrathin layer. It has also been found that the detection of H₂O₂ at these new electrode structures containing iron loaded nanotube tips can be achieved at low anodic potentials. The result is a sensitive and selective glucose sensor.     

生物电子学—现代分析化学新发展的重要领域

生物电子学技术是基于电子学与生物技术的发展而诞生的新领域。本文阐述并展望在这领域的最新发展。包括:生物计算机与分子计算机,生物传感器,酶电极的生物催化作用,基于离子电极和生物离子学的生物传感器,导电聚合物传感器等。     

Electrochemical Glucose Biosensors: Whole Cell Microbial and Enzymatic Determination Based on 10-(4H-Dithieno[3,2-b:2′,3′-d]Pyrrol-4-yl)Decan-1-Amine Interfaced Glassy Carbon Electrodes

The fabrication of amperometric biosensors based on whole cell Gluconobacter oxydans DSMZ 2343 (G. oxydans) and glucose oxidase (GOx) was performed for the detection of glucose. Glassy carbon electrodes (GCE) were coated with a 10-(4H-dithiyeno [3,2-b:2’,3’-d]pyroll-4-il)decan-1-amine (DTP-alkyl-NH₂) polymer using an electropolymerization method and the formed interface was used to connect the bacteria and the enzyme to the electrode. The transfer of electrons from enzyme to electrode was successfully demonstrated by the biocatalytic activity and unique morphology of the conducting polymer. Characterization of the biosensors was assessed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM) analyses. The detection limits of the enzyme and microbial based biosensors for glucose were 0.022 and 0.081?mM, respectively. The broad linear dynamic ranges of the GOx and G. oxydans biosensors were observed to be 0.045–50.0 and 0.19–50.0?mM, respectively. The analytical performances of biosensors were compared according to the following figures of merit: detection limits, limits of quantification, pH and current response time. In addition, to demonstrate the applicability of the biosensors, real-time measurements and recovery studies were evaluated.     

An amperometric biosensor based on covalent immobilization of ascorbate oxidase on biocompatiable and low-toxic poly(thiophene-3-acetic acid) matrix

The biocompatiable and low-toxic poly（thiophene-3-acetic acid）（PTAA） matrix was successfully electrosynthesized in ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate（BmimPF₆） in comparison with the electrosynthesis of PTAA matrix in acetonitrile（ACN）.Ascorbate oxidase（AO） was used as a model for the development and application of biosensor.Vitamin C（VC） biosensors were facilely fabricated by the covalent immobilization of AO molecules on PTAA matrices electrosynthesized in ACN containing tetrabutylammonium tetrafluoroborate and BmimPF₆, respectively.Electrochemical impedance spectroscopy,scanning electron microscopy and FTIR spectroscopy indicated that AO molecules were covalently immobilized on PTAA matrices.Parameters of the as-obtained biosensors such as working potential,pH and temperature have been optimized.The amperometric biosensor based on PTAA matrix electrosynthesized in BmimPF₆ exhibited wider linear range,lower detection limit,higher sensitivity and bioaffinity,and better operational and storage stability than that electrosynthesized in ACN under optimal conditions.The as-obtained biosensor based on PTAA matrix electrosynthesized in BmimPF₆ was employed for the detection of VC content in commercial juices,and the result was close to the data given by manufacturers.Excellent results indicate that the PTAA matrix electrosynthesized in ionic liquid is a promising platform for the covalent immobilization of biologically-active species and the development of biosensors.     

Conducting Redoxpolymer-Based Reagentless Biosensors Using Modified PQQ-Dependent Glucose Dehydrogenase

Reagentless, oxygen-independent glucose biosensors based on an Os-complex-modified polypyrrole matrix and on soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus are described.As the soluble form of glucose dehydrogenase from Acinetobacter calcoaceticus is a hydrophilic enzyme with a positive net charge, its entrapment into the positively charged hydrophobic polypyrrole film is much more complicated than that of the corresponding membrane enzyme or the negatively charged and very stable glucose oxidase. Possible ways for using soluble PQQ-dependent glucose dehydrogenase in combination with conducting polymer films are seen in the modulation of the enzyme properties by covalent binding of suitable compounds to the protein shell together with the adjustment of the properties of the conducting polymer film. This can be done by neutralising the net charge of the protein and/or optimising the electron-transfer pathway between enzyme and electrode surface by covalent binding of suitable redox relays to the protein surface.In addition, methods for increasing the hydrophilicity of the polymer film, such as the co-entrapment of high-molecular weight hydrophilic additives and copolymerisation of hydrophilic pyrrole derivatives are presented. It is demonstrated that the replacement of the parent monomer pyrrole by a suitable hydrophilic pyrrole derivative facilitates the entrapment of the modified soluble PQQ-dependent glucose dehydrogenase into the Os-complex-modified polymer and hence allows for the development of reagentless biosensors.     

电化学式生物传感器的近期发展动向

电化学式生物传感器是生物传感器的很重要组成部分。新材料用于生物传感器是其新发展的重要研究方向,它包括PVC膜的替代材料,离子交换聚合物膜,有机导电聚合物和氧化还原聚合物等。各学科的相互渗透,使生物传感器出现新颖的设想和概念,包括氧化还原酶的电子导通,超薄组成膜和化学敏感微电化学装置及离子通道传感器与膦酯膜电极。电子鼻和组电极以及生物传感器的微型化都是生物传感器的新发展动向。     

Electroconductive Green Metal-polyaniline Nanocomposites: Synthesis and Application in Sensors

Polyaniline (PANI) is one of the most extensively used conducting polymer due to its fascinating properties including conducting, thermal, optical, magnetic and electrochemical properties, simple synthesis procedure and low cost of monomer. It has attracted major attention in a variety of applications including electrochemical sensors, catalysts, supercapacitors and biosensors. However, its limitations such as insolubility in common solvents, low process-ability and poor mechanical properties have led to the development of new approaches to improve it properties. Metal nanoparticles (MNPs) such as silver, gold, copper and palladium have been combined with PANI to improve on its properties which has led to a new class of materials known as metal/PANI nanocomposites. These hybrid nanocomposites incorporate advantages of both MNPs and polymers which effectively improves the properties of the individual materials. Various synthesis techniques including in situ polymerization, ɤ-radiolysis, electrodeposition, complexation, vacuum deposition and interfacial polymerization have been used in the formation of metal/PANI nanocomposites. These nanocomposites have been used in various sensor and biosensor applications due to their excellent conductivity, ease of synthesis, excellent redox potentials, chemical and thermal stability. This review highlights the various metal/PANI nanocomposites, their various synthesis techniques and their application in sensors and biosensors. The importance of these nanocomposites in sensing and signaling various toxic heavy metals such as mercury, lead and silver and toxic gases such as hydrogen sulphide, ammonia and chloroform has been discussed. In addition the review covers the applications of metal/PANI nanocomposites in biosensor systems for the detection of glucose, DNA, protein, cholesterol, drugs and hydrogen peroxide.     

Kinetic study of the performance of third-generation biosensors

A kinetic study of the performance of third-generation biosensors for glucose based on glucose oxidase immobilized on a microporous matrix of the conducting polymer poly(pyrrole) is presented. The mechanism of the enzymatically catalyzed oxidation of glucose will be different in this type of biosensor as the natural electron acceptor oxygen is replaced by the conducting polymer. Different kinetic parameters are found for the immobilized glucose oxidase than for the enzyme in solution. Mediation by the conducting polymer is found to be very effective and no significant electron transfer to oxygen is observed. In addition to substrate transport limitation in the microporous matrix, the enzymatic reaction in the biosensors is limited by the applied potential.     

A New Bienzymatic Biosensor Based on Butyrylcholine Esterase‐Sulfhydryl Oxidase Enzymes

Abstract

Over the last decades, cholinesterase (ChE) biosensors have emerged as a sensitive and rapid technique for toxicity analysis in environmental monitoring, food, and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. In this study, a new bienzymatic biosensor based on butyrylcholinesterase (BuChE) and sulfhydryl oxidase (SOX) enzymes was developed. This system makes use of the biocatalyzed hydrolysis of butyrylthiocholine to butyric acid and thiocholine that acts as a SOX substrate. Measurements were performed by following of the consumed oxygen level related to butyrylthiocholine concentration in the enzymatic reactions. Bienzymatic system was characterized and applied for detection of donepezil which is a reversible inhibitor of cholinesterase and belongs to a new class of cholinesterase inhibitors having an N‐benzylpiperidine and an indanone moiety, by following of the decrease in biosensor response as a result of competitive inhibition.     

Glucose biosensors prepared by electropolymerization of p-chlorophenylamine with and without Nafion

Two different glucose biosensors for the amperometric determination of glucose, based on poly(p-chlorophenylamide) (PCPA) and bilayer film of PCPA and Nafion (PCPA/Nafion), are successfully developed. These two biosensors show linear amperometric responses to glucose ranging from 2.0×10⁻⁴ to 3.5×10⁻² mol l⁻¹ and 5.0×10⁻⁴ to 7.5×10⁻² mol l⁻¹, respectively, with the same correlation coefficient of 0.9988. Effects of polymerization potential and polymerization time on the performance of enzyme sensors are studied. It is found that PCPA, as a non-conducting polymer, can largely reduce the influence of electroactive interferents. Introduction of inner Nafion membrane not only further eliminates the influence of ascorbic acid on the sensor response but also increases electrode stability.     

An improved biosensor for acetaldehyde determination using a bienzymatic strategy at poly(neutral red) modified carbon film electrodes

Improved biosensors for acetaldehyde determination have been developed using a bienzymatic strategy, based on a mediator-modified carbon film electrode and co-immobilisation of NADH oxidase and aldehyde dehydrogenase. Modification of the carbon film electrode with poly(neutral red) mediator resulted in a sensitive, low-cost and reliable NADH detector. Immobilisation of the enzymes was performed using encapsulation in a sol-gel matrix or cross-linking with glutaraldehyde. The bienzymatic biosensors were characterized by studying the influence of pH, applied potential and co-factors. The sol-gel and glutaraldehyde biosensors showed a linear response up to 60 μM and 100 μM, respectively, with detection limits of 2.6 μM and 3.3 μM and sensitivities were 1.7 μA mM⁻¹ and 5.6 μA mM⁻¹. The optimised biosensors showed good stability and good selectivity and have been tested for application for the determination of acetaldehyde in natural samples such as wine.     

Conducting polymers in chemical sensors and arrays

The review covers main applications of conducting polymers in chemical sensors and biosensors. The first part is focused on intrinsic and induced receptor properties of conducting polymers, such as pH sensitivity, sensitivity to inorganic ions and organic molecules as well as sensitivity to gases. Induced receptor properties can be also formed by molecularly imprinted polymerization or by immobilization of biological receptors. Immobilization strategies are reviewed in the second part. The third part is focused on applications of conducting polymers as transducers and includes usual optical (fluorescence, SPR, etc.) and electrical (conductometric, amperometric, potentiometric, etc.) transducing techniques as well as organic chemosensitive semiconductor devices. An assembly of stable sensing structures requires strong binding of conducting polymers to solid supports. These aspects are discussed in the next part. Finally, an application of combinatorial synthesis and high-throughput analysis to the development and optimization of sensing materials is described.     

无标记DNA在氨基改性导电聚吡咯表面的固定/杂交

通过吡咯(Py)与其衍生物——6-吡咯己胺(PyHA)的共聚物聚(吡咯-co-6-吡咯己胺)[poly(Py-co-PyHA)]的合成研究,并采用电化学循环伏安法来考察体系的电化学活性.在缓冲溶液中,由于探针DNA链上的负电荷与共聚物分子链上的正电荷之间存在强烈的静电吸引力,使得DNA能够固定在导电聚合物膜上.实验结果证明,目标DNA和聚吡咯薄膜之间不存在非特异性吸附,而能和探针DNA进行顺利杂交.此结果为以后研究更为敏感的DNA固定及导电聚合物敏感膜提供了实验基础.     

Direct biosensor coating with a photopolymerised microlayer through a controllable step-procedure

Biosensors are based on the intimate association of a transducer and of a sensing layer. The latter can be a preformed membrane further connected to the transducer, or a thin film directly deposited on its surface. As the stability is a key parameter to be considered, a polymer with high potentialities for this purpose was chosen to form a direct surface coating: a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). Choline oxidase was entrapped in this photo-cross-linkable gel for making an enzyme electrode through controllable steps. The influence of the ratio PVA-SbQ/enzyme was studied and the stability of the resulting modified electrodes was determined. After deposition of minute volumes (10–45 l) including no more than one unit of choline oxidase and 0.3 mg of polymer, an efficient choline sensor was obtained. It exhibited a fast response time lower than 30 s, a low detection limit of 2.5 nM choline, a wide linear range extending up toca. 10^–4 M and good stability, both operational and on storage. This method appears promising for making miniaturized biosensors.     

Nanowire‐Based Electrochemical Biosensors

We review recent advances in biosensors based on one‐dimensional (1‐D) nanostructure field‐effect transistors (FET). Specifically, we address the fabrication, functionalization, assembly/alignment and sensing applications of FET based on carbon nanotubes, silicon nanowires and conducting polymer nanowires. The advantages and disadvantages of various fabrication, functionalization, and assembling procedures of these nanosensors are reviewed and discussed. We evaluate how they have been used for detection of various biological molecules and how such devices have enabled the achievement of high sensitivity and selectivity with low detection limits. Finally, we conclude by highlighting some of the challenges researchers face in the 1‐D nanostructures research arena and also predict the direction toward which future research in this area might be directed.     

Improvement of poly(amphiphilic pyrrole) enzyme electrodes via the incorporation of synthetic laponite-clay-nanoparticles

The electropolymerization of an enzyme-amphiphilic pyrrole ammonium-laponite nanoparticles mixture preadsorbed on the electrode surface provides the simultaneous immobilization of the enzyme and the hydrophilic laponite-clay-nanoparticles in a functionalized polypyrrole film. The presence of incorporated laponite particles within the electrogenerated polymer induces a strong improvement of the analytical performances (I_max and sensitivity) of amperometric biosensors based on polyphenol oxidase. These beneficial effects have been attributed to a marked enhancement of the apparent specific activity of the immobilized enzyme (from 0.21 to 0.85% of the specific activity of the free enzyme), the permeability of the host polymer being unchanged. This strategy of biosensor performance improvement was tested with cholesterol oxidase as an enzyme model. The presence of laponite additive in the poly(amphiphilic pyrrole) host matrix induces a similar enhancement of sensitivity and I_max for cholesterol biosensing as well as a large improvement of the storage stability of the polypyrrole-cholesterol oxidase electrode.     

A comparative study of immobilization methods of a tyrosinase enzyme on electrodes and their application to the detection of dichlorvos organophosphorus insecticide

The use of several designs of amperometric enzymatic biosensors based on the immobilized tyrosinase enzyme (Tyr) for determining dichlorvos organophosphate pesticide are described. The biosensors are based on the reversible inhibition of the enzyme and the chronocoulometric measurement of the charge due to the charge-transfer mediator 1,2-naphthoquinone-4-sulfonate (NQS). Tyr becomes active when reducing the quinone form of the mediator molecule (NQS) to the reactive o-diol form substrate of Tyr (H₂NQS) at the working electrode, thus permitting modulation of the catalytic activity of the enzyme and measurement of the inhibition produced by the pesticide. The full activity of the enzyme reversibly recovers after removal of the pesticide and re-oxidation of H₂NQS.Tyr was immobilized onto electrodes using different procedures: (i) entrapment within electropolymerized conducting and non-conducting polymers, (ii) covalent attachment to self-assembled monolayers (SAM), (iii) cross-linking with glutaraldehyde (and nafion covering) and (iv) dispersion within carbon-paste electrodes. The mediator was co-immobilized onto the working electrode next to the enzyme and reagentless biosensors were subsequently constructed. In the SAM design (ii) NQS was added to the solution. The analytical properties of the different biosensors based on the competitive inhibition produced by dichlorvos were then compared. A detection limit of about 0.06 μM was obtained for dichlorvos with entrapment of NQS and Tyr within electropolymerized poly(o-phenylenediamine) polymer (oPPD), which was the design that proved to have the best analytical performance.