CYP450 biosensors based on screen-printed carbon electrodes for the determination of cocaine

A new electrochemical method has been described and characterized for the determination of cocaine using screen-printed biosensors. The enzyme cytochrome P450 was covalently attached to screen-printed carbon electrodes. Experimental design methodology has been performed to optimize the pH and the applied potential, both variables that have an influence on the chronoamperometric determination of the drug. This method showed a reproducibility of 3.56% (n = 4) related to the slopes of the calibration curves performed in the range from 19 up to 166 nM. It has been probed the used of this kind of biosensors in the determination of cocaine in street samples, with an average capability of detection of 23.05 ± 3.53 nM (n = 3, α = β = 0.05).     

Electrochemical DNA biosensors based on platinum nanoparticles combined carbon nanotubes

Platinum nanoparticles were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced electrochemical DNA biosensor. Multi-walled carbon nanotubes and platinum nanoparticles were dispersed in Nafion, which were used to fabricate the modification of the glassy carbon electrode (GCE) surface. Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated daunomycin. Due to the ability of carbon nanotubes to promote electron-transfer reactions, the high catalytic activities of platinum nanoparticles for chemical reactions, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.0 × 10⁻¹¹ mol l⁻¹.     

Sulfite oxidase biosensors based on tetrathiafulvalene modified screen-printed carbon electrodes for sulfite determination in wine

Screen-printed carbon electrodes have been modified with tetrathiafulvalene and sulfite oxidase enzyme for the sensitive and selective detection of sulfite. Amperometric experimental conditions were optimized taking into account the importance of quantifying sulfite in wine samples and the inherent complexity of these samples, particularly red wine. The biosensor responds to sulfite giving a cathodic current (at +200 mV vs screen-printed Ag/AgCl electrode and pH 6) in a wide concentration range, with a capability of detection of 6 μM (α = β = 0.05) at 60 °C. The method has been applied to the determination of sulfite in white and red samples, with averages recoveries of 101.5% to 101.8%, respectively.     

Speciation of chromium using chronoamperometric biosensors based on screen-printed electrodes

Chronoamperometric assays based on tyrosinase and glucose oxidase (GOx) inactivation have been developed for the monitoring of Cr(III) and Cr(VI). Tyrosinase was immobilized by crosslinking on screen-printed carbon electrodes (SPCEs) containing tetrathiafulvalene (TTF) as electron transfer mediator. The tyrosinase/SPC_TTFE response to pyrocatechol is inhibited by Cr(III). This process, that is not affected by Cr(VI), allows the determination of Cr(III) with a capability of detection of 2.0 ± 0.2 μM and a reproducibility of 5.5%. GOx modified screen-printed carbon platinised electrodes (SPC_PtEs) were developed for the selective determination of Cr(VI) using ferricyanide as redox mediator. The biosensor was able to discriminate two different oxidation states of chromium being able to reject Cr(III) and to detect the toxic species Cr(VI). Chronoamperometric response of the biosensor towards glucose decreases with the presence of Cr(VI), with a capability of detection of 90.5 ± 7.6 nM and a reproducibility of 6.2%. A bipotentiostatic chronoamperometric biosensor was finally developed using a tyrosinase/SPC_TTFE and a GOx/SPC_PtE connected in array mode for the simultaneous determination of Cr(III) and Cr(VI) in spiked tap water and in waste water from a tannery factory samples.     

Trends in DNA biosensors

Biosensors have witnessed an escalating interest nowadays, both in the research and commercial fields. Deoxyribonucleic acid (DNA) biosensors (genosensors) have been exploited for their inherent physico-chemical stability and suitability to discriminate different organism strains. The main principle of detection among genosensors relies on specific DNA hybridization, directly on the surface of a physical transducer. This review covers the main DNA immobilization techniques reported so far, new micro- and nanotechnological platforms for biosensing and the transduction mechanisms in genosensors. Clinical applications, in particular, demand large-scale and decentralized DNA testing. New schemes for DNA diagnosis include DNA chips and microfluidics, which couples DNA detection with sample pretreatment under in vivo-like hybridization conditions. Higher sensitivity and specificity may arise from nanoengineered structures, like carbon nanotubes (CNTs) and DNA/protein conjugates. A new platform for universal DNA biosensing is also presented, and its implications for the future of molecular diagnosis are argued.     

Present and future of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves—surface plasmon-polaritons—to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.     

Cholinesterase immobilisation on the surface of screen-printed electrodes based on concanavalin A affinity

This paper reports a new method for the immobilisation of acetylcholine esterase (AChE) on the surface of screen-printed electrodes (SPEs) based on the affinity between the glycoprotein enzyme and concavalin A (Con A). The surface of the working electrode has been modified with a Nafion layer that contains graphite, the mediator 7,7,8,8-tetracyanoquinodimethane (TCNQ) and heptylamine. The enzyme-free SPEs were characterised by cyclic voltammetry in buffer solutions and amperometry using cysteamine as analyte. The AChE immobilisation process leads to the sandwich structure: electrode-carbohydrate-Con A-enzyme. The first step of the immobilisation is the covalent activation of an amino group bound in a Nafion layer. The following steps are based on the affinity. The non-specific adsorption has been totally eliminated using BSA solutions at two different pHs. Various amounts of enzyme, from 0.1 to more than 2 mIU AChE, have been loaded on the electrode surface. The method offers the advantage of a free diffusion, which allows obtaining a response time of less than 2 min. An operational stability of more than 10 measurements was registered, while the active surface of the electrode was successfully reloaded for three consecutive times without any important change of the analytical performances.     

Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors

Screen-printed electrodes coated with the nafion layer have been investigated for cholinesterase biosensor design. The butyrylcholinesterase (ChE) from horse serum was immobilised onto the nafion layer by cross-linking with glutaraldehyde vapours. The biosensors obtained showed better long-term stability and lower working potential in comparison to those obtained with no nafion coating. The sensitivity of a biosensor toward organophosphate pesticides is not affected by the nafion coating. The detection limits were found to be 3.5x10(-7) M for trichlorfon and 1.5x10(-7) M for coumaphos.     

Acetylcholinesterase sensor based on screen-printed carbon electrode modified with prussian blue

Acetylcholinesterase (ChE) sensor based on Prussian blue (PB) modified electrode was developed and tested for the detection of organophosphorus and carbamic pesticides. The signal of the sensor was generated in PB mediated oxidation of thiocholine recorded at+200 mv in DC mode. ChE from electric eel was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) on the surface of screen-printed carbon electrode covered with PB and Nafion. The content of the surface layer (specific enzyme activity, Nafion and BSA amounts) was optimized to establish high and reliable response toward the substrate and ChE inhibitors. The ChE/PB sensor makes it possible to detect Aldicarb, Paraoxon and Parathion-Methyl with limits of detection 30, 10 and 5 ppb, respectively (incubation 10 min). The feasibility of practical application of the ChE/PB sensor developed for the monitoring of degradation of the pesticides in wine fermentation was shown. To diminish matrix interferences, the electrolysis of the grape juice with Al anode and evaporation of ethanol were suggested, however the procedures decrease the sensitivity of pesticide detection and stability of the sample tested.     

Tuning the wetting angle of fluorinated polymer with modified nanodiamonds: towards new type of biosensors

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Development and evaluation of electrochemical glucose enzyme biosensors based on carbon film electrodes

Electrochemical glucose enzyme biosensors have been prepared on carbon film electrodes made from carbon film electrical resistors. Evaluation and characterisation of these electrodes in phosphate buffer saline solution has been carried out with and without pretreatment by cycling in perchloric acid or at fixed applied potential. Both pretreatments led to a reduction in the carbon surface oxidation peak and enabled better detection of hydrogen peroxide in the pH range of 5-7. Glucose oxidase enzyme was immobilised on the carbon surface by mixing with glutaraldehyde, bovine serum albumin and with and without Nafion. The performance of these two types of electrode was similar, that containing Nafion being more physically robust. Linear ranges were up to around 1.5 mM, with detection limits 60 μM, and pretreatment of the carbon film electrode at a fixed potential of +0.9 V versus SCE for 5 min was found to be the most beneficial. Michaelis-Menten constants between 5 mM and 10 mM were found under the different experimental conditions. Coating the immobilised enzyme layer with a thin layer of Nafion was found to give similar results in the determination of glucose to mixing it but with benefits against interferences for the analysis of complex matrices, such as wine. Potentialities, for a short-term-use or disposable sensors, are indicated.     

Amperometric biosensors for glucose based on carbon paste modified electrodes

The modification of carbon-paste electrodes by incorporation of the enzyme glucose oxidase (GOD) is described. The resulting probes can be operated as amperometric glucose sensors in the presence or absence of a mediator (1,1'-dimethylferrocene) mixed into the paste. Extended linear calibration ranges have been obtained up to 90 and 5OmM glucose respectively. The electrode responses were rapid, reaching steady-state values within 30-40 sec. Advantages of using a GOD-paste formulation are suggested. Plasma glucose assays were correlated with spectrophotometric determinations based on glucose oxidase (y = 1.07x - 0.16, r = 0.973, n = 17).     

Electrocatalytic reduction of hydrogen peroxide on modified graphite electrodes: application to the development of glucose biosensors

The electrocatalytic activities of a series of compact graphites modified with microquantities of platinum metals (Pd or Pt+Pd) towards the electrochemical reduction of hydrogen peroxide were characterised. Operational parameters such as the optimal working potential, the influence of temperature and the resulting electrode characteristics were examined. The benefits of using graphite modified with Pt+Pd (mixture ratio 30%:70%) as the basic transducer in a glucose biosensor with improved sensitivity were demonstrated. It was proven that, under the working conditions chosen, the selected electrode (whether bare or covered with an enzyme layer) did not respond to any glutathione, uric acid or ascorbic acid (which all normally occur in biological fluids) present.     

Electrochemical and piezoelectric DNA biosensors for hybridisation detection

DNA biosensors (or genosensors) are analytical devices that result from the integration of a sequence-specific probe and a signal transducer. Among other techniques, electrochemical and piezoelectric methods have recently emerged as the most attractive due to their simplicity, low instrumentation costs, possibility for real-time and label-free detection and generally high sensitivity.Focusing on the most recent activity of worldwide researchers, the aim of the present review is to give the readers a critical overview of some important aspects that contribute in creating successful genosensing devices. Advantages and disadvantages of different sensing materials, probe immobilisation chemistries, hybridisation conditions, transducing principles and amplification strategies will be discussed in detail. Dedicated sections will also address the issues of probe design and real samples pre-treatment. Special emphasis will be finally given to those protocols that, being implemented into an array format, are already penetrating the molecular diagnostics market.     

Amperometric sensors based on tyrosinase-modified screen-printed arrays

This paper describes the design, development and characteristics of a tyrosinase (polyphenol oxidase) modified amperometric screen-printed biosensor array, with the enzyme cross-linked in a redox-hydrogel namely the PVI₁₃-dmeOs polymer. Two types of Au-screen-printed four-channel electrode arrays, differing in design and insulating layer, were compared and investigated. Au-, graphite-coated-Au- and Carbopack C-coated-Au-surfaces, serving as the basis for tyrosinase immobilisation, were investigated and the performances of the different arrays were evaluated and compared in terms of their electrocatalytic characteristics, as well as operational- and storage stability using catechol as model substrate. It was found that the Carbopack C-coated array was the best choice for tyrosinase immobilisation procedure mainly due to a higher mechanical stability of the deposited enzyme layer, combined with good sensitivity and stability for up to 6 months of use. In the batch mode the biosensors responded linearly to catechol up to 30 μM with limits of detection from 0.14 μM. Parameters from cyclic voltammograms indicated that the reversibility of the direct electrochemical reaction for catechol on the three types of electrode surfaces (no tyrosinase modification) was not the limiting factor for the construction and performance of tyrosinase biosensors.     

Glucose and urea biosensors based on all solid-state PVC-NH2 membrane electrodes

Miniaturized urea and glucose sensors prepared by immobilization of glucose oxidase or urease directly onto all solid-state contact PVC-NH₂ membrane ammonium and hydrogen ion selective electrodes are described. The resulting biosensing membranes function equivalently to normal PVC membranes in terms of potentiometric response properties. The most important features of the glucose and urea sensors were high sensitivity, long life-time, easily built at a low cost, micro-construction and short response time. The characteristics of the glucose and urea sensors were examined in several buffer solutions at different concentrations and pH values. The influence of immobilization conditions on the dynamic response properties and life-time of the electrodes was studied. Under optimal conditions, the urea electrode showed a linear response between 5×10⁻² and 5×10⁻⁴ M urea, while the glucose electrode showed a linear response between 5×10⁻² and 1×10⁻⁴ M glucose.     

Bio-electrocatalysis of NADH and ethanol based on graphene sheets modified electrodes

Characterization and application of graphene sheets modified glassy carbon electrodes (graphene/GC) have been presented for the electrochemical bio-sensing. A probe molecule, potassium ferricyanide is employed to study the electrochemical response at the graphene/GC electrode, which shows better electron transfer than graphite modified (graphite/GC) and bare glassy carbon (GC) electrodes. Based on the highly enhanced electrochemical activity of NADH, alcohol dehydrogenase (ADH) is immobilized on the graphene modified electrode and displays a more desirable analytical performance in the detection of ethanol, compared with graphite/GC or GC based bio-electrodes. It also exhibits good performance of ethanol detection in the real samples. From the results of electrochemical investigation, graphene sheets with a favorable electrochemical activity could be an advanced carbon electrode materials for the design of electrochemical sensors and biosensors.     

Cholinesterase sensors based on screen-printed electrodes for detection of organophosphorus and carbamic pesticides

Cholinesterase sensors based on screen-printed electrodes modified with polyaniline, 7,7,8,8-tetracyanoquinodimethane (TCNQ), and Prussian blue have been developed and tested for detection of anticholinesterase pesticides in aqueous solution and in spiked grape juice. The influence of enzyme source and detection mode on biosensor performance was explored. It was shown that modification of the electrodes results in significant improvement of their analytical characteristics for pesticide determination. Thus, the slopes of the calibration curves obtained with modified electrodes were increased twofold and the detection limits of the pesticides were reduced by factors of 1.6 to 1.8 in comparison with the use of unmodified transducers. The biosensors developed make it possible to detect down to 2×10^–8 mol L^–1 chloropyrifos-methyl, 5×10^–8 mol L^–1 coumaphos, and 8×10^–9 mol L^–1 carbofuran in aqueous solution and grape juice. The optimal conditions for grape juice pretreatment were determined to diminish interference from the sample matrix.Abbreviations ChE Cholinesterase - TCNQ 7,7,8,8-Tetracyanoquinodimethane - ChO Choline oxidase - AChE Acetylcholinesterase - BChE Butyrylcholinesterase - BSA Bovine serum albumin - 2-PAM 2-Pyridine aldoxime methiodide     

基于碳纳米管修饰电极检测有机磷农药的生物传感器

报道了一种用于检测有机磷农药的安培型生物传感器,利用戊二醛交联法将乙酰胆碱酯酶和牛血清白蛋白固定在羧基化多壁碳纳米管修饰玻碳电极表面,制备了可应用于检测有机磷农药的新型生物传感器,并确定了最佳工作条件.该方法具有良好的重现性和回收率,当辛硫磷及氧化乐果的浓度分别在5.0×10-4～5.0×10-1 g/L和1.0×10-3～5.0×10-1 g/L范围内时,抑制率与其浓度的对数呈线性关系,检出限按抑制率为10%时的农药浓度计算,可分别达到3.6×10-4 g/L和5.9×10-4 g/L.