D-fructose detection based on the direct heterogeneous electron transfer reaction of fructose dehydrogenase adsorbed onto multi-walled carbon nanotubes synthesized on platinum electrode

Multi-walled carbon nanotubes (MWCNTs) were synthesized on platinum plate electrodes by the chemical vapor deposition (CVD) method. From the results of X-ray photoelectron spectroscopy and voltammetric investigation, the iron nanoparticles used as a catalyst for the MWCNT synthesis were enclosed with MWCNTs. The MWCNTs synthesized on the Pt plate (MWCNTs/Pt) electrode were immediately immersed into solutions of d-fructose dehydrogenase (FDH) to immobilize the enzyme onto the MWCNTs/Pt electrode surfaces. After the FDH was immobilized onto the MWCNTs/Pt electrode, a well-defined catalytic oxidation current based on FDH was observed from ca. -0.15V (versus Ag/AgCl/sat'd KCl), which was close to the redox potential of heme c as a prosthetic group of FDH. From an analysis of a plot of the catalytic current versus substrate, the calibration range for the fructose concentration was up to ca. 40mmoldm(-3), and the apparent Michaelis-Menten constant was evaluated to be 11+/-1mmoldm(-3).     

Interaction of K7Fe3+P2W17O62H2 with supported bilayer lipid membranes on platinum electrode

The influence of K(7)Fe(3+)P(2)W(17)O(62)H(2) on l-alpha-phosphatidylcholine/cholesterol bilayer lipid membrane on Pt electrode was studied by voltammetry and AC impedance spectroscopy. The interaction of the polyoxometalates with the BLM can promote the access of Ru(NH(3))(6)(3+) and [Fe(CN)(6)](3-/4-) to the electrode surface. It was found that some kind of pores had been formed on the BLM by AFM. The phenomenon is attributed to the interaction of K(7)Fe(3+)P(2)W(17)O(62)H(2) with phosphatidylcholine phosphate groups located in its outer leaflet. Experimental results are helpful to understand the biological activity of the polyoxometalates in vivo.     

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.     

A mimic peroxidase biosensor based on calcined layered double hydroxide for detection of H2O2

An enzymeless biosensor was explored from Cu-Mg-Al calcined layered double hydroxide (CLDH) modified electrode in this study. The Cu-Mg-Al CLDH greatly promotes the electron transfer between H(2)O(2) and GCE, and it is exemplified toward the non-enzymatic sensing of H(2)O(2). The results indicate that the Cu-Mg-Al CLDH exhibits excellent electrocatalytic property, high sensitivity, good reproducibility, long-term stability, and fast amperometric response toward reduction of H(2)O(2), thus is promising for the future development of man-made mimics of enzyme in H(2)O(2) sensors. This work opens a way to utilize simply Cu-Mg-Al CLDH as an electron mediator to fabricate an efficient H(2)O(2) biosensor, which exhibits great potential applications in varieties of simple, robust, and easy-to-make analytical approaches in the future.     

Highly sensitive and selective hydrogen peroxide biosensor based on hemoglobin immobilized at multiwalled carbon nanotubes-zinc oxide composite electrode

A highly sensitive and selective amperometric hydrogen peroxide (H(2)O(2)) biosensor based on immobilization of hemoglobin (Hb) at multiwalled carbon nanotubes-zinc oxide (MWCNT/ZnO) composite modified glassy carbon electrode (GCE) is reported. ZnO microsponges were electrochemically grown on MWCNT surface by the simple, cost-effective, green, electrochemical method at room temperature. The MWCNT/ZnO/Hb composite film showed a pair of well-defined, quasi-reversible redox peaks with a formal potential (E°') of -0.336V, characteristic features of heme redox couple of Hb. The electron transfer rate constant (k(s)) of immobilized Hb was 1.26s(-1). The developed biosensor showed a very fast response (>2s) toward H(2)O(2) with good sensitivity, wide linear range, and low detection limit of 0.02μM. The fabricated biosensor showed interesting features, including high selectivity, acceptable stability, good reproducibility, and repeatability along with excellent conductivity, facile electron mobility of MWCNT, and good biocompatibility of ZnO. The fabrication method of this biosensor is simple and effective for determination of H(2)O(2) in real samples with quick response, good sensitivity, high selectivity, and acceptable recovery.     

Evaluation of different micro/nanobeads used as amplifiers in QCM immunosensor for more sensitive detection of E. coli O157:H7

Micro/nanobeads with different materials (magnetic, silica and polymer) and different sizes (diameters from 30nm to 970nm) were investigated for their use as amplifiers in a quartz crystal microbalance (QCM) immunosensor for more sensitive detection of Escherichia coli O157:H7. The micro/nanobeads were conjugated with anti-E. coli antibodies. E. coli O157:H7 cells were first captured by the first antibody immobilized on the electrode surface, and then micro/nanobeads labeled secondary antibodies attached to the cells, and finally the complexes of antibody-E. coli-antibody modified beads were formed. The results showed that antibody-labeled beads lead to signal amplification in both the change in frequency (ΔF) and the change in resistance (ΔR). Since the penetration depth of the oscillation-induced shear-waves for a ～8MHz crystal is limited to 200nm, the interpretation of how the signal is amplified by the adsorbed particles was represented in terms of the coupled-oscillator theory. The amplification is not sensed in terms of increase in mass on the sensor surface. Amplification is sensed as a change in bacterial resonance frequency when the spheres adsorb to the bacteria. The change in the values of ΔF caused by different micro/nanobeads (amplifiers) attaching on target bacterial cells is indicative of the ratio between the resonance frequency of the absorbed bacterial-particle complex (ω(s)), and the resonance frequency of the crystal (ω).     

Improvement of the performance of H2O2 oxidation at low working potential by incorporating TTF-TCNQ into a platinum wire electrode for glucose determination

A micro-biosensor was constructed by incorporating the organic conducting salt tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) into a platinized platinum (Pt) wire and further covering with the electrochemical polymerical heteropolypyrrole film, in which glucose oxidase (GOx) was entrapped. The enzyme electrode can sensitively determine glucose at a low working potential, mainly based on the oxidation of H2O2. The incorporated TTF-TCNQ can significantly improve the oxidation of H2O2 on the electrode, although a part of the TTF-TCNQ functions as a mediator. Compared with the same electrode prepared without TTF-TCNQ incorporated, the TTF-TCNQ modified electrode had better performance characteristics at a working potential of 200 mV (versus SCE). The response time to 90% of the steady value was shortened from about 40 s to less than 10 s, the lower limit of the linear response was greatly extended from about 1.6 mM to 10 microM, the linear range was shifted from 1.6-10.0 to 0.01-5 mM and the sensitivity was increased from about 1 to 1.5 microA/mM. The electrode was quite stable. For continuous operation, the electrode could work for about 5 weeks and only lost 60% of its original sensitivity. Stored at 4 degrees C for intermittent determinations, the electrode kept 80% sensitivity for over 6 months. Due to covering the electrode with a non-conductive heteropolypyrrole film, ascorbate, urate and 4-acetamidophenol caused only negligible current response at an applied potential of 200 mV.     

Erythroid precursors cultured from adult mice are sensitive to H2O2 toxicity

Summary The growth of late erythroid precursors (CFU-Es) from adult bone marrow is inhibited when Iscove's modified Dulbecco's medium supplied in liquid form is used. Catalase and other H₂O₂ destroying compounds restore the capacity of culture medium to support colony development. However early precursors from adult bone marrow and fetal liver CFU-Es were resistant to H₂O₂. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and the Fédération Nationale des Centres de Lutte contre le Cancer.     

A highly sensitive fluorescence sensor based on lucigenin/chitosan/SiO2 composite nanoparticles for microRNA detection using magnetic separation

In this paper, a convenient reverse‐phase microemulsion method for the synthesis of SiO₂ nanoparticles (NPs) by simply introducing the chitosan and fluorescent dye of lucigenin during the formation reaction of SiO₂ NPs was proposed. Addition of chitosan can make the SiO₂ NPs porous, and increases lucigenin molecule incorporation into chitosan/SiO₂ NPs nanopores based on electrostatic interaction and supermolecular forces. Therefore, fluorescence quantum yield of the lucigenin/chitosan/SiO₂ composite nanoparticles was increased by introduction of chitosan and compared with lucigenin/SiO₂ NPs without chitosan. Because the number of negative charges carried when using single‐stranded DNA (ssDNA) was different from that of double‐stranded DNA (dsDNA), the numbers of lucigenin/chitosan/SiO₂ composite nanoparticles with positive charge adsorbed using ssDNA or dsDNA were different. Consequently, fluorescence intensity caused using ssDNA or dsDNA/miRNA was clearly discriminative. With increase in target DNA/miRNA concentration, the difference in fluorescence intensity also increased, resulting in a good linear relationship between fluorescence intensity sensitizing value and target miRNA concentrations. Therefore, a new fluorescence analysis method for direct detection of let‐7a in human gastric cancer cell samples without enzyme, label free and no immobilization was established using lucigenin/chitosan/SiO₂ composite nanoparticles as a DNA hybrid indicator. The proposed method had high sensitivity and selectivity, low cost and the detection limit was 10 fM (S/N = 3).     

Amperometric xanthine biosensors based on electrodeposition of platinum on polyvinylferrocenium coated Pt electrode

Novel xanthine biosensors were successfully fabricated by immobilizing xanthine oxidase on polyvinylferrocenium perchlorate matrix (PVF⁺ClO₄⁻) and platinum electrodeposited polyvinylferrocenium perchlorate matrix. PVF⁺ClO₄⁻ film was coated on Pt electrode at +0.7 V vs. Ag/AgCl by electrooxidation of polyvinylferrocene (PVF). Platinum nanoparticles were deposited on PVF⁺ClO₄⁻ electrode by electrochemical deposition in 2.0 mM H₂PtCl₆ solution at −0.2 V. Xanthine oxidase was incorporated into the polymer matrix via ion exchange process by immersing modified Pt electrodes in the enzyme solution. The amperometric responses of the biosensors were measured via monitoring oxidation current of hydrogen peroxide at +0.5 V. Under the optimal conditions, the linear ranges of xanthine detection were determined as 1.73 × 10⁻³–1.74 mM for PVF⁺XO⁻ and 0.43 × 10⁻³–2.84 mM for PVF⁺XO⁻/Pt. The detection limits of xanthine were 5.20 × 10⁻⁴ mM for PVF⁺XO⁻ and 1.30 × 10⁻⁴ mM for PVF⁺XO⁻/Pt. Moreover, the effects of applied potential, electrodeposition potential, H₂PtCl₆ concentration, amount of electrodeposited Pt nanoparticles, thickness of polymeric film, temperature, immobilization time, xanthine and xanthine oxidase concentrations on the response currents of the biosensors were investigated in detail. The effects of interferents, the operational and storage stabilities of biosensors and the applicabilities to drug samples of the biosensors analysis were also evaluated.     

Selective and sensitive biosensor for theophylline based on xanthine oxidase electrode

Milk and microbial xanthine oxidases (XOs) were used for the construction of amperometric enzyme electrodes. Substrate specificity differences of these enzymes were studied. Of the two enzymes, only the microbial XO was found to oxidize theophylline, but not theobromine and caffeine. The substrate specificity of microbial XO was affected by pH, where the optimum for xanthine was 5.5, while for theophylline it was in the range from 6.5 to 8.5. The theophylline biosensor showed a low detection limit of 2 x 10(-7) M and signal linearity up to 5 x 10(-5) M. The sensitivity of the microbial XO electrode to theophylline could be selectively eliminated by immersion in alkaline phosphate solution, thus allowing for the construction of a blank electrode for differential measurements. The feasibility of this approach has been demonstrated by the determination of free (unbound) and total theophylline in blood samples. The biosensor exhibited good operational (>6 h) and shelf (>3 months) stability when trehalose was used as a stabilizer of the biocatalytic layer.     

Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils

A fast and sensitive chemiluminescence assay for the determination of H₂O₂ in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide. Changes in H₂O₂ concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H₂O₂ without influence of H₂O₂ decomposition by cellular enzymes or newly produced H₂O₂ due to dismutation of superoxide anion radicals. Concentrations of H₂O₂ were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H₂O₂ concentration upon stimulation could be observed at 3000 cell/mL.     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

Electrochemical assay of protease activities based on polycation/polyanion complex as substrate and polyion sensitive membrane electrode detection

A novel electrochemical method to detect protease activities is demonstrated. The assay is based on the use of a macromolecular polycation/polyanion substrate; specifically, a complex of the arginine-rich peptide protamine and pentosan polysulfate (PPS), a highly sulfated polysaccharide. As the protease of interest cleaves the protamine within the complex into smaller fragments, free PPS is generated and detected potentiometrically via a polyanion sensitive membrane electrode. Thus, the rate of free PPS generation is proportional to the activity of the protease in the assay solution. The effect of the substrate concentration is examined, as is the influence of the protamine/PPS stoichiometry on the assay performance. Using the optimized composition and concentration of the complex, the determination of trypsin at levels down to 5 U/ml and plasmin at levels approaching 0.002 U/ml can be achieved in a 10 min period. The prospects of further adapting this scheme to determine clot-busting plasminogen activators (e.g. streptokinase, tissue plasminogen activator, etc.) in samples as complex in whole blood are discussed.     

Novel snowflake-like Pt-Pd bimetallic clusters on screen-printed gold nanofilm electrode for H2O2 and glucose sensing

Novel snowflake-like Pt-Pd bimetallic nanoclusters (Pt-PdBNC) were synthesized on a screen-printed gold nanofilm electrode (SPGFE) substrate by electrochemically reducing precursors with a new constant potential/multi-potential step deposition strategy. The electrocatalytic behavior of the modified electrode (SPGFE/Pt-PdBNC) towards H(2)O(2) was investigated. The results indicate that the as-prepared Pt-PdBNC significantly enhances the electrochemical reduction of H(2)O(2) in neutral media, exhibiting preferable electrocatalytic performance compared to Pt and Pd monometallic nanoclusters. Under optimum conditions, SPGFE/Pt-PdBNC offers linear responses for H(2)O(2) in the concentration range from 0.005 to 6 mM with an ultrahigh sensitivity of 804 mA M(-1) cm(-2) and excellent selectivity. Furthermore, glucose oxidase was immobilized on the Pt-PdBNC structure, and the fabricated biosensor presents favorable properties for glucose sensing.     

A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3O4@SiO2 magnetic nanoparticles

A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe₃O₄@SiO₂ magnetic nanoparticles (Fe–MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe–MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA‐CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe–MNPs (amine‐functionalized Fe₃O₄@SiO₂ and carboxylic acid‐functionalized Fe₃O₄@SiO₂). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC₅₀) values of the two methods were about 0.024 ng ml^?1 and 0.046 ng ml^?1 respectively and LODs were approximately 0.0002 ng ml^?1 and 0.001 ng ml^?1 respectively. These methods were much more sensitive than that of any traditional enzyme‐linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe–MNPs.     

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

Piezoelectric detection of bilirubin based on bilirubin-imprinted titania film electrode

A novel quartz crystal microbalance (QCM) sensor with a high selectivity and sensitivity has been developed for bilirubin determination, based on the modification of bilirubin-imprinted titania film onto a quartz crystal by molecular imprinting and surface sol-gel techniques. The performance of the developed bilirubin biosensor was evaluated and the results indicated that a sensitive bilirubin biosensor could be fabricated. The obtained bilirubin biosensor presents high-selectivity monitoring of bilirubin, better reproducibility, shorter response time (30 min), wider linear range (0.1-50 μM), and lower detection limit (0.05 μM). The analytical application of the bilirubin biosensor confirms the feasibility of bilirubin determination in serum sample.     

Magnetic Dynabeads detection by sensitive element based on giant magnetoimpedance

A GMI-biosensor prototype was designed and fabricated using an amorphous ribbon. As a first step, a GMI response was measured with a model liquid media: suspension containing magnetisable Dynabeads M-450. These beads can be used as biomolecular labels when coated with a specific antibody. The GMI change caused by their presence was measured with a biosensor prototype designed with a measuring cell containing an amorphous CoFeMoSiB-ribbon element. GMI was measured at a range of current frequencies from 0.3 to 10 MHz and at intensities of either Irms=3 or 4.5 mA. Commercial Dynabeads M-450 were supplied as a suspension in a phosphate buffered saline. A maximum difference of 25% in the GMI ratio measured with and without Dynabeads was obtained at a frequency of 3.5 MHz and Irms=3 mA. Some potential applications for GMI-biosensor and further directions are proposed.