A hydroquinone biosensor using modified core-shell magnetic nanoparticles supported on carbon paste electrode

A hydroquinone biosensor was developed and used to determine hydroquinone concentration in compost extracts based on the immobilization of laccase on the surface of modified magnetic core-shell (Fe(3)O(4)-SiO2) nanoparticles. Laccase was covalently immobilized on the magnetic nanoparticles by glutaraldehyde, which was modified with amino groups on its surface. The obtained magnetic bio-nanoparticles were attached to the surface of carbon paste electrode with the aid of a permanent magnet to determine hydroquinone. A good microenvironment for retaining the bioactivity of laccase was provided by the immobilization matrix. The linear range for hydroquinone determination was 1 x 10(-7) to 1.375 x 10(-4)M, with a detection limit of 1.5 x 10(-8)M. The current reached 95% of the steady-state current within about 60s. Hydroquinone concentration in compost extracts was determined by laccase biosensor and HPLC, the results of the two methods were approximately the same.     

The carbon paste electrode encrusted with a microreactor as glucose biosensor.

The amperometric biosensors based on carbon paste electrodes (CPEs) encrusted with single microreactor (MR) have been constructed for the determination of glucose. The MRs were prepared from CPC-silica carrier (CPC) and were loaded with glucose oxidase (GO), mediator (M) and acceptor (A). As the mediator cation radical of 5,10-dimethyl-5, 10-dihydrophenazine (DMDHP), N-methylphenazonium methyl sulfate (PMS) and o-benzoquinone (BQ) and as the acceptor Fe[EDTA]- or Fe(CN)6(3-) was used. The biosensors acted at electrode potential 0.15-0.27 V versus Ag-AgCl electrode. The calibration graphs of the biosensors were linear in the range from 1.5 to 50 mM of glucose. The sensitivity of the biosensors did not change at pH 6-8. The dissolved oxygen little (7%) influenced the biosensors response and 1 mM of ascorbic acid produced the response that was of equal value to 0.5 mM of glucose. The biosensors showed high stability; no change of the response of the biosensors prepared by using the novel microreactor was observed at least for 6 months by keeping the loaded CPC at room temperature in silica container. An optimization of the biosensors response against the GO, the mediator and the polymer amount was performed. The digital modeling of the biosensors action is following.     

Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine

An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds.     

Amperometric mediated carbon paste biosensor based on D-fructose dehydrogenase for the determination of fructose in food analysis

A new mediated amperometric biosensor for fructose is described. The sensor is based on a commercially available D-fructose dehydrogenase. The enzyme is incorporated in a carbon paste matrix containing Os(bpy)₂Cl₂ as redox mediator that achieves electron transfer at 0·1 V (versus Ag/AgCl) with maximum apparent current densities of 1·2 mA/cm². The dependence of the steady-state current on the loading of the mediator and the enzyme, other electrode construction parameters, the operating potential, the pH and the temperature was studied. In the steady-state mode the response current was directly proportional to D-fructose concentration from 0·2 to 20mM with a detection limit of 35 μM (signal-to-noise ratio, S/N, 3). In the flow injection analysis mode the response current was directly proportional to D-fructose concentration from 0·5 to 15 M with a detection limit of 115 μM (S/N 3). The sensor was used for the determination of fructose in food samples in a flow injection system and validated with a commercial enzyme kit.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H₂O₂. The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H₂O₂ in the linear range from 2.6 × 10^− 6 mol/L to 8.8 × 10^− 3 mol/L with a detection limit of 6.4 × 10^− 7 mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Amperometric ascorbic acid sensor based on doped ferrites nanoparticles modified glassy carbon paste electrode

In this study, a novel electrochemical sensor for quantification of ascorbic acid with amperometric detection in physiological conditions was constructed. For this purpose, cobalt and nickel ferrites were synthesized using microwave and ultrasound assistance, characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and X-ray powder diffraction (XRPD), and used for modification of glassy carbon paste electrode (GCPE). It was shown that introducing these nanoparticles to the structure of GCPE led to increasing analytical performance. Co ferrite modified GCPE (CoFeGCPE) showed better characteristics toward ascorbic acid sensing. The limit of detection (LOD) obtained by sensor was calculated to be 0.0270 mg/L, with linear range from 0.1758 to 2.6010 mg/L. This sensor was successfully applied for practical analysis, and the obtained results demonstrated that the proposed procedure could be a promising replacement for the conventional electrode materials and time-consuming and expensive separation methods.     

Amperometric choline biosensor based on multiwalled carbon nanotubes/zirconium oxide nanoparticles electrodeposited on glassy carbon electrode

Perturbation of the tubulin/microtubule dynamic in cells is perhaps the single most important mode of action of anticancer drugs. Standard methods for identifying and evaluating compounds for their ability to alter tubulin polymerization are low throughput, labor intensive, expensive, or make their assessment in vitro. Here we report a method to rapidly quantify the extent of tubulin polymerization in whole cells using flow cytometry, and we use this technique to evaluate compounds that stabilize and destabilize microtubule formation. This facile method is useful for conveniently, quantitatively, and cost-effectively comparing small molecules that perturb tubulin polymerization.     

Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp.

The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 μM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 μM p-nitrophenol and mineralized about 60% of 720 μM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode

A new amperometric biosensor, based on adsorption of glucose oxidase (GOD) at the platinum nanoparticle-modified carbon nanotube (CNT) electrode, is presented in this article. CNTs were grown directly on the graphite substrate. The resulting GOD/Pt/CNT electrode was covered by a thin layer of Nafion to avoid the loss of GOD in determination and to improve the anti-interferent ability. The morphologies and electrochemical performance of the CNT, Pt/CNT, and Nafion/GOD/Pt/CNT electrodes have been investigated by scanning electron microscopy, cyclic voltammetry, and amperometric methods. The excellent electrocatalytic activity and special three-dimensional structure of the enzyme electrode result in good characteristics such as a large determination range (0.1-13.5mM), a short response time (within 5s), a large current density (1.176 mA cm(-2)), and high sensitivity (91mA M(-1)cm(-2)) and stability (73.5% remains after 22 days). In addition, effects of pH value, applied potential, electrode construction, and electroactive interferents on the amperometric response of the sensor were investigated and discussed. The reproducibility and applicability to whole blood analysis of the enzyme electrode were also evaluated.     

Detection of pesticides using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic system

A new highly sensitive amperometric method for the detection of organophosphorus compounds has been developed. The method is based on a ferophthalocyanine chemically modified carbon paste electrode coupled with acetylcholinesterase and choline oxidase co-immobilized onto the surface of a dialysis membrane. The activity of cholinesterase is non-competitively inhibited in the presence of pesticides. The highest sensitivity to inhibitors was found for a membrane containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetylcholine as substrate; choline produced by hydrolysis in the enzymatic layer was oxidized by choline-oxidase and subsequently H(2)O(2) produced was electrochemically detected at +0.35 V vs. Ag/AgCl. The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, up to 10(-10) M of paraoxon and carbofuran can be detected.     

Amperometric glucose biosensor based on single-walled carbon nanohorns

The biosensing application of single-walled carbon nanohorns (SWCNHs) was demonstrated through fabrication of an amperometric glucose biosensor. The biosensor was constructed by encapsulating glucose oxidase in the Nafion-SWCNHs composite film. The cyclic voltammograms for glucose oxidase immobilized on the composite film displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of -0.453 V. The biosensor had good electrocatalytic activity toward oxidation of glucose. To decrease detection potential, ferrocene monocarboxylic acid was used as a redox mediator. The mediated glucose biosensor shows a linear range from 0 to 6.0 mM. The biosensor shows high sensitivity (1.06 microA/mM) and stability, and can avoid the commonly coexisted interference. Because of impressive properties of SWCNHs, such as high purity and high surface area, SWCNHs and their composites are expected to be promising material for biomolecular immobilization and biosensing applications.     

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.     

Development of a carbon nanotube paste electrode osmium polymer-mediated biosensor for determination of glucose in alcoholic beverages

This paper describes a new amperometric biosensor for glucose monitoring. The biosensor is based on the activity of glucose dehydrogenase (GDH) and diaphorase (DI) co-immobilized with NAD(+) into a carbon nanotube paste (CNTP) electrode modified with an osmium functionalized polymer. This mediator was demonstrated to shuttle the electron transfer between the immobilized diaphorase and the CNTP electrode, thus, showing a good electrocatalytic activity towards NADH oxidation at potentials around +0.2V versus Ag|AgCl, where interfering reactions are less prone to occur. The biosensor exhibits a detection limit of 10 micromol L(-1), linearity up to 8 x 10(-4) mol L(-1), a sensitivity of 13.4 microA cm(-2)mmol(-1)L(-1), a good reproducibility (R.S.D. 2.1%, n=6) and a stability of about 1 week when stored dry at 4 degrees C. Finally, the proposed biosensor was applied for the determination of glucose in different samples of sweet wine and validated with a commercial spectrophotometric enzymatic kit.     

Amperometric biosensor based on a functionalized gold electrode for the detection of antioxidants

A method for the electrochemical detection of antioxidants has been developed, which is based on a radical measurement with a cytochrome c modified electrode. A controlled enzymatic production system for superoxide radicals based on xanthine oxidase was used. The addition of antioxidants facilitated the decomposition of the radical in addition to the spontaneous dismutation. The steady-state of superoxide generation and decomposition was thus shifted to a new situation due to the higher decomposition rate after antioxidant addition. This resulted in a decreased current level at the electrode. Antioxidant activity could be quantified from the response of the sensor electrode by the percentage of the signal decrease. The 50% inhibition value (IC(50)) for different antioxidants was calculated and the antioxidant activity of numerous substances was compared. Thus, a hierarchy of superoxide radical scavenging abilities of flavonoids was established: flavanols>flavonols>flavones>flavonones>isoflavonones.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Amperometric glucose biosensor utilizing FAD-dependent glucose dehydrogenase immobilized on nanocomposite electrode

Amperometric glucose biosensors utilizing commercially available FAD-dependent glucose dehydrogenases from two strains of Aspergillus species are described. Enzymes were immobilized on nanocomposite electrode consisting of multi-walled carbon nanotubes by entrapment between chitosan layers. Unlike the common glucose oxidase based biosensor, the presented biosensors appeared to be O(2)-independent. The optimal amount of enzymes, working potential and pH value of working media of the glucose biosensors were determined. The biosensor utilizing enzyme isolated from Aspergillus sp. showed linearity over the range from 50 to 960 μM and from 70 to 620 μM for enzyme from Aspergillus oryzae. The detection limits were 4.45 μM and 4.15 μM, respectively. The time of response was found to be 60 s. The biosensors showed excellent operational stability - no loss of sensitivity after 100 consecutive measurements and after the storage for 4 weeks at 4 °C in phosphate buffer solution. When biosensors were held in a dessicator at room temperature without use, they kept the same response ability at least after 6 months. Finally, the results obtained from measurements of beverages and wine samples were compared with those obtained with the enzymatic-spectrophotometric and standard HPLC methods, respectively. Good correlation between results in case of analysis of real samples and good analytical performance of presented glucose biosensor allows to use presented concept for mass production and commercial use.     

Amperometric biosensor based on tyrosinase immobilized on a boron-doped diamond electrode

A novel method has been developed to immobilize tyrosinase onto the surface of boron-doped diamond (BDD) electrode. The hydrogen-terminated BDD (HBDD) surface was first functionalized by photochemically linking vinyl groups of allylamine, producing covalently linked amine-terminated active BDD (ABDD) surface. Then the tyrosinase was immobilized onto the ABDD surface by carbodiimide coupling reaction. The amperometric response was measured as a function of concentration of phenolic compounds in 0.1M phosphate buffer solution (pH 6.5). The tyrosinase-modified ABDD electrode gave a linear response range of 1-175, 1-200 and 1-200 microM and sensitivity of 80.0, 181.4 and 110.0 mA M(-1)cm(-2) for phenol, p-cresol, 4-chlorophenol, respectively. Moreover, selective detection of dopamine (DA) in the presence of ascorbic acid (AA) has been demonstrated with the tyrosinase-modified ABDD electrode. Linearity was observed within the range of 5-120 microM. The above enzyme electrode could maintain 90% of its original activity after intermittent use for 1 month when storing in a dry state at 4 degrees C.     

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.