Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10⁷ CFU mL^?1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

Development of a new biosensor for superoxide radicals

A superoxide dismutase (SOD) biosensor for determination of superoxide radicals has been developed by immobilization of superoxide dismutase within gelatin (G) on a Pt electrode surface. The properties of the biosensor have been investigated and optimum conditions–enzyme concentration, glutaraldehyde concentration, and pH–were determined. The response of the G-SOD biosensor was proportional to concentration and the detection limit was 0.01 mmol L⁻¹ at a signal-to-noise ratio of 3. The biosensor retained 89% and 60% of its sensitivity after use for three and four weeks, respectively. Immobilization of SOD on gelatin provides a biocompatible microenvironment around the enzyme and stabilizes the activity of the enzyme very efficiently. The superoxide dismutase biosensor was used to determine the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.     

Amperometric determination of alcohols, aldehydes and carboxylic acids with an immobilized alcohol oxidase enzyme electrode.

A new enzyme electrode has been developed for the rapid and simple measurements of blood ethanol. A platinum electrode was coupled with immobilized alcohol oxidase, sensing the dissolved oxygen consumption amperometrically. Blood ethanol can be measured within several minutes without any pretreatments. The only reagent required is phosphate buffer. The enzyme electrode was found to respond not only to alcohols but also to aldehydes and carboxylic acids.     

Determination of salicylate in beverages and cosmetics by use of an amperometric biosensor

A fast and selective enzymatic method for the determination of salicylate in beverages and cosmetics has been developed. The enzyme salicylate hydroxylase was immobilised covalently onto a glassy carbon working electrode of a wall-jet cell coupled with a flow-injection analysis system. The salicylate is enzymatically converted to catechol, which can be detected amperometrically on the glassy carbon electrode at +0.45 V. The response of the biosensor is linearly proportional to the concentration of salicylate between 725 nmol/l and 700 mol/l. A high sample throughput (60 h^-1) is possible, and the biosensor is stable for more than three months. Sample pretreatment for beverages and hair lotions is easy and fast. For creams, an extraction of salicylate is necessary. Relative standard deviations are less than 5.5% and the recoveries are between 95 and 105%.     

Glucose oxidase/hexokinase electrode for the determination of ATP

A hydrogen peroxide based enzyme electrode for the determination of ATP has been developed by the immobilization of glucose oxidase and hexokinase. Competition between the enzymes for the substrate glucose allowed the measurement of ATP. Different immobilization procedures and different types of hexokinase have been tested. Using a BSA-glutaraldehyde procedure and hexokinase from an overproducing strain of bakers' yeast, ATP was measured in the 0.05–0.5 mmol l⁻¹ range with a detection limit of 0.01 mmol l⁻¹. ATP concentrations comparable to those reported in the literature and a good recovery were obtained when the enzyme electrode was used with human erythrocyte hemolysate.     

A thermodynamic surface model for caesium sorption on bentonite

Caesium sorption on Wyoming bentonite MX-80 has been studied in solutions of NaCl, KCl, MgCl₂, CaCl₂, NaNO₃ and Ca (NO₃)₂ of concentrations varying between 0.025 and 1 mol/L, as well as in a weakly saline (I=0.004 ml/L) and a strongly saline (I=0.46 mol/L) natural groundwater. These experiments have been used to derive a thermodynamic model for the interaction of caesium with the bentonite surface in accordance with a surface chemical model, including acid/base reactions developed recently for montmorillonite. The sorption behaviour of caesium on bentonite can be described, within the experimental and model uncertainties, in terms of a one-site ion exchange model. The ion exchange constant obtained for the reaction NaX+Cs⁺CsX+Na⁺ (where X represents the ion exchange sites on montmorillonite) is log₁₀K⁰_ex=1.6. Impurities in the bentonite, influencing the concentrations of competing cations, such as Na⁺, K⁺, Mg²⁺ and Ca²⁺, have a crucial impact on the sorption of caesium. This impact can be adequately quantified with the present model. The model predictions compare well with sorption data published in the open literature on both Wyoming bentonite MX-80 and other types of bentonite. Distribution coefficients from the literature obtained from both batch and diffusion experiments and varying over four orders of magnitude are reproduced and explained successfully by the model.     

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

Self-assembly of acetylcholinesterase on gold nanoparticles electrodeposited on graphite

The immobilisation of AChE enzyme through chemisorption on Au-modified graphite was examined with view of its prospective application in the design of membraneless electrochemical biosensors for the assay of enzyme inhibitors. The developed immobilisation protocol has been based on a two-stage procedure, comprising i) electrodeposition of gold nanostructures on spectroscopic graphite; followed by ii) chemisorption of the enzyme onto gold nanoparticles. Both the coverage of the electrode surface with Au nanostructures and the conditions for enzyme immobilisation were optimised. The proposed electrode architecture together with the specific type of enzyme immobilisation allow for a long-term retaining of the enzyme catalytic activity. The extent of inhibition of the immobilised acetylcholinesterase enzyme by the organophosphorous compound monocrotophos has been found to depend linearly on its concentration over the range from 50 to 400 nmol mL^?1 with sensitivity 77.2% inhibition per 1 µmol mL^?1 of monocrotophos.      

Polyluminol/hydrogel composites as new electrochemiluminescent-active sensing layers

This paper reports on electrochemiluminescent sensors and biosensors based on polyluminol/hydrogel composite sensing layers using chemical or biological membranes as hydrogel matrices. In this work, luminol is electropolymerized under near-neutral conditions onto screen-printed electrode (SPE)-supported hydrogel films. The working electrode coated with a hydrogel film is soaked in a solution containing monomeric luminol units, allowing the monomeric luminol units to diffuse inside the porous matrix to the electrode surface where they are electropolymerized by cyclic voltammetry (CV). Sensors and enzymatic biosensors for H₂O₂ and choline detection, respectively, have been developed, using choline oxidase (ChOD) as a model enzyme. In this case, hydrogel is used both as the enzymatic immobilization matrix and as a template for the electrosynthesis of polyluminol. The enzyme was immobilized by entrapment in the gel matrix during its formation before electropolymerization of the monomer. Several parameters have been optimized in terms of polymerization conditions, enzyme loading, and average pore size. Using calcium alginate or tetramethoxysilane (TMOS)-based silica as porous matrix, H₂O₂ and choline detection are reported down to micromolar concentrations with three orders of magnitude wide dynamic ranges starting from 4?×?10^?7 M. Polyluminol/hydrogel composites appear as suitable electrochemiluminescence (ECL)-active sensing layers for the design of new reagentless and disposable easy-to-use optical sensors and biosensors, using conventional TMOS-based silica gel or the more original and easier to handle calcium alginate, reported here for the first time in such a configuration, as the biocompatible hydrogel matrix. Figure

Elaboration of electrochemiluminent polyluminol/hydrogel composite sensing layers     

Electrode interfaces switchable by physical and chemical signals for biosensing, biofuel, and biocomputing applications

This review outlines advances in designing modified electrodes with switchable properties controlled by various physical and chemical signals. Irradiation of the modified electrode surfaces with various light signals, changing the temperature of the electrolyte solution, application of a magnetic field or electrical potentials, changing the pH of the solutions, and addition of chemical/biochemical substrates were used to change reversibly the electrode activity. The increasing complexity in the signal processing was achieved by integration of the switchable electrode interfaces with biomolecular information processing systems mimicking Boolean logic operations, thus allowing activation and inhibition of electrochemical processes on demand by complex combinations of biochemical signals. The systems reviewed range from simple chemical compositions to complex mixtures modeling biological fluids, where the signal substrates were added at normal physiological and elevated pathological concentrations. The switchable electrode interfaces are considered for future biomedical applications where the electrode properties will be modulated by the biomarker concentrations reflecting physiological conditions.

Critical evaluation of a screening test for detection of herbicides by inhibiting photosynthesis of isolated chloroplasts

The possibility to determine herbicides in the aqueous environment by observation of inhibiting effects on the photosynthesis of isolated chloroplasts have been critically evaluated. The photosynthetical activities of freshly isolated chloroplasts from spinach (Spinacia oleracea L.) and peas (Pisum sativum L.) have been compared under well-defined conditions. The standard for the rate of photosynthesis was the oxygen evolution, which has been detected by a Clark-type electrode. The ratio between the oxygen production rate before and after a sample addition describes the chloroplast activity. In the presence of herbicides this ratio decreases. Increasing herbicide concentrations have been determined by a normalization procedure in semilogarithmic scales using a sigmoidal calibration plot.Photosynthesis inhibiting substances, both natural and anthropogenic, can be detected collectively. That is an advantage over enzyme immunoassays which can only detect single herbicides. Clearly disadvantageous are the insufficient detection limits (e.g. 8.9 g/l for atrazine, respectively 2.5 g/l for terbuthylazine) and therefore the necessary preconcentration.     

An enzyme electrode based on immobilized arylsulfatase for the selective assay of sulfate ion

An enzyme electrode has been constructed for the assay of sulfate ion based on inhibition of the reaction

The steady-state current arising from oxidation of the product, 4-nitrocatechol, is measured at +0.8 V vs. S. C. E. The competitive inhibition of this reaction by added sulfate ion causes a decrease in this steady-state current in a linear relationship to pSO₄ in the range 2–4. The enzyme arylsulfatase (arylsulfate sulfohydrolase, EC 3.1·6.1) is chemically immobilized in a layer on a platinum electrode. This enzyme electrode also gives linear calibration plots for phosphate ion (10^-2–10^-4 M) based on its competitive inhibition of the above reaction, and for fluoride ion (10^-2–10^-4 M) based on its activation of the reaction. The assay of 4-nitrocatechol sulfate (NCS) in the range 10^-6–10^-4 M is possible. By proper control of the NCS concentration the electrode can be made almost completely specific for sulfate: only molybdate interferes. To establish the best operating conditions for the electrode, the effect of pH on the V_m and K_m were determined.     

Radon and CN: Complementary tracers of polluted air masses at coastal and island sites

CN and radon concentrations have been measured at coastal and island sites in programs measuring baseline concentrations of atmospheric trace constituents. It is shown that addition of CN and radon concentrations to meteorological criteria for characterisation of an air mass, substantially improves the objectivity of baseline selection. Wind direction and CN concentration are both useful for indicating likelihood of pollution from sources within a few km. Beyond that the fetch over land is enough for radon to be useful. For sources more than a thousand km away, wind direction and CN become poor indicators, whereas radon concentrations still yield reliable indications of land contact.     

An analysis of avidin, biotin and their interaction at attomole levels by voltammetric and chromatographic techniques

The electroanalytical determination of avidin in solution, in a carbon paste, and in a transgenic maize extract was performed in acidic medium at a carbon paste electrode (CPE). The oxidative voltammetric signal resulting from the presence of tyrosine and tryptophan in avidin was observed using square-wave voltammetry. The process could be used to determine avidin concentrations up to 3 fM (100 amol in 3 l drop) in solution, 700 fM (174 fmol in 250 l solution) in an avidin-modified electrode, and 174 nM in a maize seed extract. In the case of the avidin-modified CPE, several parameters were studied in order to optimize the measurements, such as electrode accumulation time, composition of the avidin-modified CPE, and the elution time of avidin. In addition, the avidin-modified electrode was used to detect biotin in solution (the detection limit was 7.6 pmol in a 6 l drop) and to detect biotin in a pharmaceutical drug after various solvent extraction procedures. Comparable studies for the detection of biotin were developed using HPLC with diode array detection (HPLC-DAD) and flow injection analysis with electrochemical detection, which allowed biotin to be detected at levels as low as 614 pM and 6.6 nM, respectively. The effects of applied potential, acetonitrile content, and flow rate of the mobile phase on the FIA-ED signal were also studied.     

Calculating Electrochemical Activity of Regular-Structure Porous Electrodes with an Immobilized Enzyme

A porous electrode of regular structure with an immobilized enzyme is studied. The electrode carcass, which consists of substrate particles, is a system of two sets of mutually perpendicular planes crossing one another (cellular structure). A monomolecular layer of enzyme molecules is deployed on the inner surface of such a porous substrate. In the center of each cell of the substrate gas pores, which are cylinders of porous grains of a hydrophobizing agent one grain thick, are situated. The rest of the cell space is filled by a solid polymer electrolyte. The ultimate goal of calculations is to estimate the electrochemical activity of such an electrode. The estimation is done for an oxygen electrode with an enzyme whose characteristics are close to those of laccase. The calculation assumes that active centers of enzyme molecules undergo a direct, i.e. without participation of mediators, reduction. It is shown that at an overvoltage of 30 mV, it would be possible to obtain a current density of 0.44 A cm^–2 in an electrode 16 m thick.     

Miniaturized CE System Based on Amperometric Detection and Horizontal Sampling

A novel miniaturized capillary electrophoresis (CE)-amperometric detection system has been fabricated by integrating a self-positioning electrode system and a 10-cm piece of fused silica capillary on a Plexiglas base plate. The self-positioning electrode system was composed of a disc working electrode positioned opposite the outlet of the capillary, with an Ag/AgCl reference electrode, a platinum auxiliary electrode, and a platinum grounding electrode accommodated in a rectangular detection cell on the plate. The platinum wire, close to the inlet of capillary, served as a contact to the high-voltage power supply. Alternate placement of the capillary inlet in vials containing sample or buffer solutions permits volume-defined electrokinetic sample introduction in a horizontal arrangement. Performance of this unique system was demonstrated by separating sugars, amino acids, and two phenols used in the pharmaceutical industry. The factors influencing the analytical performance of the new microsystem for the separation of p-aminophenol and acetaminophen have been characterized and optimized. The two analytes could be separated on baseline within 160s in 50mM acetate buffer (pH 5.0). Responses for p-aminophenol and acetaminophen were linear over the range of 10 to 1000µM with detection limits (S/N=3) of 2.1 and 2.9µM.     

Calculating Electrochemical Activity of Porous Electrodes of a Filled-up Type with an Immobilized Enzyme and Regular Gas Pores

A model for a porous electrode of a filled-up type with an immobilized enzyme, in which a system of regular gas pores is formed, is studied. The system of regular gas pores is in fact a collection of equidistant cylinders whose thicknesses are equal to one grain of the hydrophobizing agent. Usually, the gas reactant passes into a porous electrode via a number of chains that comprise porous grains of the hydrophobizing agent. The latter is distributed in the electrode bulk in a random fashion. In this case, channels for supplying gas are formed at high concentrations of the hydrophobizing agent. As a result, the number of molecules of a catalyst in a porous electrode, and along with it the current, are not great. Should we pass to electrodes with regular gas pores, the required amount of a hydrophobizing agent would decrease. As a result, the number of molecules of the enzyme in the electrode and the current would increase. The calculation of the last quantity is the subject matter to which the paper pays a major attention. The calculation is performed for two versions of distribution of a hydrophobizing agent over the electrode bulk, specifically, a random distribution and a regular distribution. Concrete calculations are carried out for an oxygen porous electrode with an enzyme whose electrochemical characteristics are close to those obtained on laccase. It was assumed in the calculations that the enzyme operates without mediators. It is established that a porous electrode with a quasi-regular structure has a considerable advantage. With an electrode thickness of 13 m one can manage to obtain currents of nearly 0.74 A cm^–2 as early as at an overvoltage of about 30 mV.     

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Transients of the Current and Open-Circuit Potential Occurring during the Carbon Monoxide Adsorption on a Rhodium Electrode

Transients of the current and open-circuit potential are measured after bringing carbon monoxide in contact with an Rh/Pt electrode in 0.5 M H₂SO₄ and 1 M HCl solutions. To interpret these transients from positions of theoretical views on the transients of the current and open-circuit potential that were developed previously for the adsorption of neutral species on a hydrogen electrode, curves of dependences of the total electrode charge on the electrode potential are plotted in the presence and absence of chemisorbed CO on the surface. Good agreement of theory with experiment is established. In 1 M H₂SO₄, values of the potential of a zero total charge (PZTC) in the absence and presence of CO_ads and values of the reverse potential happen to be close, whereas the PZTC in 1 M HCl perceptibly shifts in the positive direction as a result of the CO adsorption, while the reverse potential, conversely, falls in the region of potentials of hydrogen evolution on Rh. As a result of the latter, in 1 M HCl, throughout the entire range of potentials where restrictions of the employed modeling notions are obeyed, in accordance with theory, integrated values of the current transients have only a negative sign, and those of the open-circuit potential transients have only a positive sign.     

Diamond like carbon coated films for enzyme electrodes; characterization of biocompatibility and substrate diffusion limiting properties

The biocompatibility and substrate diffusion limiting properties for a range of diamond like carbon (DLC) coated microporous polycarbonate and DLC coated dialysis (haemodialysis) membranes have been studied. This characterisation builds upon previous findings where DLC coated membranes imparted enhanced enzyme electrode performance. In this study electrode linear ranges have been extended from 10 mM glucose for a 0.01 μm pore size membrane to 160 mM. These findings correlated with the duration of DLC deposition and associated reductions in permeability for glucose. Permeability coefficient ratios for both microporous and dialysis membranes were also found to be important with low glucose/O₂ permeability ratios imparting extensions in glucose linear response range. DLC coated membranes employed within enzyme electrodes have also been shown to exhibit enhanced haemocompatibility as determined by both sensitivity change and surface deposition of blood components examined by scanning electron microscopy. Correlations are made between the reduced losses in sensor response to biofouling/ working electrode passivation processes, and extended linear ranges that DLC coated membranes may impart to enzyme electrode performance. Particular reference is made to the determination of glucose levels within whole blood.