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.     

A mediator-free amperometric hydrogen peroxide biosensor based on HRP immobilized on a nano-Au/poly 2,6-pyridinediamine-coated electrode

A mediator-free amperometric hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) enzyme on colloidal Au modified platinum (Pt) wire electrode, which was modified by poly 2,6-pyridinediamine (pPA). The modified process was characterized by electrochemical impedance spectroscopy (EIS), and the electrochemical characteristics of the biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The biosensor displayed an excellent electrocatalytical response to reduction of H₂O₂ without the aid of an electron mediator, the linear range was 4.2 × 10⁻⁷–1.5 × 10⁻³ mol/L (r = 0.9977), with a detection limit of 1.4 × 10⁻⁷ mol/L. Moreover, the performance and factors influencing the resulted biosensor were studied in detail. The studied biosensor exhibited permselectivity, good stability and good fabrication reproducibility.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

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.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Amperometric third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

Amperometric hydrogen peroxide biosensor with sol-gel/chitosan network-like film as immobilization matrix

A new type of sol-gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H(2)O(2) biosensor. The composite film was used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By using fluorescent label, a protein density on sol-gel/chitosan has been calculated to be 3.14 x 10(12) moleculescm(-2). With the aid of catechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0 x 10(-9)-1.0 x 10(-7) mol l(-1) and a detection limit of 2 x 10(-9) mol l(-1). Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant K(M)(app) is found to be 1.30 micromol l(-1). The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol(-1). The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4 degrees C.     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Electrochemical synthesis of polyaniline nano-networks on p-aminobenzene sulfonic acid functionalized glassy carbon electrode Its use for the simultaneous determination of ascorbic acid and uric acid

A composite film of polyaniline (PAN) nano-networks/p-aminobenzene sulfonic acid (ABSA) modified glassy carbon electrode (GCE) has been fabricated via an electrochemical oxidation procedure and applied to the electro-catalytic oxidation of uric acid (UA) and ascorbic acid (AA). The ABSA monolayer at GCE surface has been characterized by X-ray photo-electron spectroscopy (XPS) and electrochemical techniques. Atomic force microscopy (AFM), field emission scanning electron microscope (SEM), electrochemical impedance spectroscopy (EIS), UV-visible absorption spectra (UV-vis) and cyclic voltammetry (CV) have been used to investigate the PAN-ABSA composite film, which demonstrates the formation of the composite film and the maintenance of the electroactivity of PAN in neutral and even in alkaline media. Due to its different catalytic effects towards the electro-oxidation of UA and AA, the modified GCE can resolve the overlapped voltammetric response of UA and AA into two well-defined voltammetric peaks with both CV and differential pulse voltammetry (DPV), which can be used for the selective and simultaneous determination of these species in a mixture. The catalytic peak currents are linearly dependent on the concentrations of UA and AA in the range of 50-250 and 35-175mumoll(-1) with correlation coefficients of 0.997 and 0.998, respectively. The detection limits for UA and AA are 12 and 7.5mumoll(-1), respectively. Besides the good stability and reproducibility of PAN-ABSA/GCE due to the covalent attachment of ABSA at GCE surface, the modified electrode also exhibits good sensitivity and selectivity.     

Electrochemical recognition for sugars on the chitosan-poly(diallyldimethylammonium chloride)-nano-Prussian blue/nano-Au/4-mercaptophenylboronic acid modified glassy carbon electrode

A new amperometric biosensor for the detection of sugars was prepared. A glassy carbon electrode was modified with Prussian blue (PB) nanoparticles protected by chitosan (CS) and poly(diallyldimethylammonium chloride) (PDDA), and then gold nanoparticles were assembled onto the electrode followed by the assembly of 4-mercaptophenylboronic acid (MPBA) onto the surface of gold nanoparticles through a sulfur–Au bond to fabricate a self-assembled biosensor. The PB nanoparticles protected by CS and PDDA were characterized using transmission electron microscopy and UV–vis absorption spectroscopy. The characterization of the self-assembled electrode was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The pK _a values of the MPBA monolayer before and after combining with sugars were determined. The fabricated electrode exhibited excellent performances for determining d(+)-glucose, d(+)-mannose, and d(−)-fructose on the basis of the change in i _p of the Fe(CN)₆^3−/4− ion in the presence of sugars.     

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.     

Preparation and properties of chitosan/carbon nanotube nanocomposites using poly(styrene sulfonic acid)-modified CNTs

Poly(styrene sulfonic acid)-functionalized carbon nanotubes (CNT-PSSA), which was obtained with atom transfer radical polymerization (ATRP), was utilized in preparation of chitosan/CNT nanocomposites (CH/CNT-PSSA). Chemical linkages between chitosan and CNTs form in the nanocomposites through the reaction between the sulfuric acid groups of CNT-PSSA and the amino groups of chitosan, to warrant the homogenous dispersion of CNTs. The CH/CNT-PSSA nanocomposites were superior to the neat chitosan polymer in thermal and mechanical properties, water and solvent uptakes, bond water ratios, and electrical conductivity. The attractive property of the CH/CNT-PSSA nanocomposites also implied their application potentials for separation membranes and sensor electrodes.     

Amperometric hydrogen peroxide biosensor based on covalently immobilizing thionine as a mediator

A novel amperometric hydrogen peroxide biosensor based on the immobilization of hemoglobin on the 2,6-pyridinedicarboxylic acid (PDC) polymer, thionine and nano-Au was successfully fabricated. In this strategy, PDC polymer acted as the matrices to covalently immobilize the thionine, and then hemoglobin was successfully adsorbed on the nano-Au which was electro-deposited on to thionine modified electrode surface. The preparation process of modified electrode was characterized with electrochemical impedance spectroscopy and atomic force microscope. The analytical performance of proposed biosensor toward H₂O₂ was investigated by cyclic voltammetry and chronoamperometry. The resulted biosensor exhibited fast amperometric response (within 6 s) to H₂O₂, and linear range was from 9.1 μM to 5.0 mM with the detection limit of 2.6 μM (S/N = 3). The apparent Michaelis–Menten constant (K _M^app) was evaluated to be 3.2 mM. Furthermore, the resulted biosensor showed good stability and reproducibility.     

Amperometric ethanol biosensor based on poly(vinyl alcohol)-multiwalled carbon nanotube-alcohol dehydrogenase biocomposite

A novel amperometric ethanol biosensor was constructed using alcohol dehydrogenase (ADH) physically immobilized within poly(vinyl alcohol)–multiwalled carbon nanotube (PVA–MWCNT) composite obtained by a freezing–thawing process. It comprises a MWCNT conduit, a PVA binder, and an ADH function. The measurement of ethanol is based on the signal produced by β-nicotinamide adenine dinucleotide (NADH), the product of the enzymatic reaction. The homogeneity of the resulting biocomposite film was characterized by atomic force microscopy (AFM). The performance of the PVA–MWCNT–ADH biocomposite modified glassy carbon electrode was evaluated using cyclic voltammetry and amperometry in the presence of NADH and in the presence of ethanol. The ethanol content in standard solutions was determined and a sensitivity of 196 nA mM⁻¹, a linear range up to 1.5 mM, and a response time of about 8 s were obtained. These characteristics allowed its application for direct detection of ethanol in alcoholic beverages: beer, red wine, and spirit.     

Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.     

Amperometric biosensor for hydrogen peroxide based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite

A novel amperometric biosensor for hydrogen peroxide (H(2)O(2)) was developed by entrapping horseradish peroxidase (HRP) in a new ormosil composite doped with ferrocene monocarboxylic acid-bovine serum albumin conjugate and multiwall carbon nanotubes (MWNTs). The ormosil was prepared using 3-(aminopropyl)triethoxysilane and 2-(3,4 epoxycyclohexyl)-ethyltrimethoxy silane as monomers. The encapsulated conjugate showed excellent electrochemistry and acted as an electron transfer mediator. The presence of MWNTs improved the conductivity of the composite film. This matrix showed a biocompatible microenvironment for retaining the native activity of the entrapped HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H(2)O(2). The proposed H(2)O(2) biosensor exhibited a linear range of 0.02-4.0 mM with a detection limit of 5.0 microM (S/N = 3) and a K(M)(app) value of 2.0 mM. It could be used for flow injection analysis of hydrogen peroxide with a liner range from 0.02 to 4.5 mM, sensitivity of 0.042 microA/mM and analytical time of 20 s per sample. This biosensor possessed good analytical performance and storage stability.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

An amperometric hydrogen peroxide sensor based on immobilization of hemoglobin in poly(o-aminophenol) film at iron-cobalt hexacyanoferrate-modified gold electrode

A series of hybrid iron-cobalt hexacyanoferrate (FeCoHCF) films were electrodeposited on gold electrodes from solutions containing 6mM Fe(CN)(6)(3-) with different concentrations of Co(2+) and Fe(3+). FeCoHCF films deposited from solutions with different molar ratios of iron were studied by cyclic voltammetry, and their solid states were characterized by Fourier transform infrared spectroscopy. The kind of FeCoHCF film that deposited from a solution with a molar ratio of iron of 0.4 showed the largest response current to H(2)O(2) and was characterized by energy-dispersive X-ray spectroscopy. Therefore, the optimized FeCoHCF film was combined with nonconducting poly(o-aminophenol) (POAP) film that entrapped the hemoglobin (Hb) to construct hydrogen peroxide biosensor. The response current of the Hb/POAP/FeCoHCF/Au electrode (29.8 nA) was nearly 40 and was 1.5 times that of the Hb/POAP/Au (0.7 nA) and POAP/FeCoHCF/Au (20 nA) electrodes, respectively. The Michaelis-Menten constant of Hb in the Hb/POAP/FeCoHCF/Au film was 9.31 mM. These results show that the immobilized Hb in the Hb/POAP/FeCoHCF/Au film exhibits higher catalytic activity and larger response current to H(2)O(2) by the mediation of FeCoHCF. In addition, effects of applied potential, solution pH, and electroactive interferent on the response current of the Hb/POAP/FeCoHCF/Au electrode were investigated in detail.     

Development of DNA electrochemical biosensor based on immobilization of ssDNA on the surface of nickel oxide nanoparticles modified glassy carbon electrode

A sensitive electrochemical method for DNA hybridization based on immobilization of DNA probe and [Ru(NH₃)₅Cl]PF₆ complex onto nickel oxide nanomaterials (NiOx_np) modified glassy carbon electrode was developed. Due to strong affinity of NiOx_np for phosphate groups, oligonucleotides probe with a terminal 5′-phosphate group was attached to the surface of the modified electrode. DNA immobilization and hybridization were characterized by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry using K₃Fe(CN)₆/K₄Fe(CN)₆ and [Ru(NH₃)₅Cl]PF₆ as probe and indicator, respectively. The Ru-complex current response indicates only the complementary sequence showing an obvious current signal in comparison to non-complementary and three or single point mismatched sequences. The fabricated biosensor possessed good selectivity and sensitivity for complementary probe, taxon: 32630 tumor necrosis factor (TNF). The linear dynamic range, sensitivity and detection limit of the proposed biosensor were 4 × 10⁻¹⁰ M to 1 × 10⁻⁸ M, 34.32 nA nM⁻¹ and 6.8 × 10⁻¹¹ M, respectively. Excellent reproducibility and stability, quite simple and inexpensive preparation are the other advantages of proposed biosensor.