Prussian blue based nanoelectrode arrays for H(2)O(2) detection

We propose to form nanoelectrode arrays by deposition of the electrocatalyst through lyotropic liquid crystalline templates onto inert electrode support. Whereas Prussian Blue is known to be a superior electrocatalyst in hydrogen peroxide reduction, carbon materials used as electrode support demonstrate only a minor activity. We report on the possibility for nanostructuring of Prussian Blue by its electrochemical deposition through lyotropic liquid crystalline templates, which is noticed from atomic force microscopy images of the resulting surfaces. The resulting Prussian Blue based nanoelectrode arrays in flow injection analysis mode demonstrate a sub-part-per-billion detection limit (1 x 10(-)(8) M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of H(2)O(2) concentrations (1 x 10(-)(8) to 1 x 10(-)(2) M), which are the most advantageous analytical performances in hydrogen peroxide electroanalysis.     

Peroxidase model electrodes: heme peptide modified electrodes as reagentless sensors for hydrogen peroxide

A peroxidase model electrode was devised for reagentless sensing of hydrogen peroxide (H2O2). A small model molecule, which mimics the vicinity of the reaction center of a redox enzyme, can communicate electrochemically with an electrode. Heme nonapeptide (MW congruent to 1600) having peroxidase activity was adopted as a peroxidase model compound and was covalently immobilized on a tin oxide (SnO2) electrode as a roughly monomolecular layer. The modified electrode thus obtained responded to H2O2 at concentrations down to 10(-6) M without electron mediator or promoter, at a mild potential of +150 or +300 mV vs Ag/AgCl. In a batch system, the response reached a steady state in a few seconds. Measurements were possible also in a flow system with an assay time of 0.5-1.0 min/sample. The steady-state response of the electrode was kinetically analyzed.     

Nonconducting polymers on Prussian Blue modified electrodes: improvement of selectivity and stability of the advanced H/sub 2/O/sub 2/ transducer

An approach to improve the analytical performance of a Prussian Blue (PB)-based hydrogen peroxide transducer is described. In support of this objective, both the stabilizing and anti-interferent properties of nonconducting films were used. Electropolymerization on the top surface of PB modified electrodes is possible due to the high oxidizing ability of Berlin Green, and the growth of nonconductive polymers may be independently monitored by investigating the redox activity of the inorganic polycrystal. The best performance characteristics, which are advantageous over existing H/sub 2/O/sub 2/ sensors, were obtained for PB electrodes covered with electropolymerized o-phenylenediamine (1,2-diaminobenzene). The reported transducer remained at the 100% response state for more than 20 h under continuous flow of 0.1-mM hydrogen peroxide (flow rate 1 mlmin/sup -1/), which improves the stability level among the selective H/sub 2/O/sub 2/ sensors by one order of magnitude. The selectivity factor of the PB-poly (1,2-diaminobenzene) based transducer relative to ascorbate is nominally 600. PB-poly(1,2-diaminobenzene) modified electrode allows the detection hydrogen peroxide in the flow-injection mode down to 10/sup -7/ M with sensitivity of 0.3 AM/sup -1/cm/sup -2/, which is two times lower compared to the uncovered PB-based transducer.     

Continuous on-line measurement of cerebral hydrogen peroxide using enzyme-modified ring-disk plastic carbon film electrode

An amperometric method suitable for the continuous on-line measurement of cerebral hydrogen peroxide from a microdialysate has been successfully performed for the first time by using an enzyme-modified ring-disk plastic carbon film electrode (PCFE) in a thin-layer radial flow cell. PCFE consists of a ring electrode modified with horseradish peroxidase to detect H2O2 at 0.0 V (vs Ag/ AgCl) and a disk electrode coated with ascorbate oxidase (AOx) to preoxidize ascorbic acid (AA) and thus suppress interference via direct oxidation. Analytes in solution (brain dialysates or standards) are mixed on-line with a phosphate-buffered solution containing dissolved oxygen and chelating agent, EDTA. The buffered solution is used to provide the O2 necessary for the AOx catalytic reaction, stabilize the changes in dialysate pH that are associated with the in vivo formation of H2O2, and remove heavy metal ion impurities and thus suppress reactions between AA and H2O2. This procedure enables trace levels of H2O2 to be readily monitored, virtually interference-free from physiological levels of AA, uric acid, electroactive neurotransmitters and their principle metabolites, in a continuous-flow system.     

Operation of a miniature redox hydrogel-based pyruvate sensor in undiluted deoxygenated calf serum

An amperometric sensor for the detection of pyruvate in biological fluids was formed by modifying the tip of a 0.25 mm gold wire with a layer of electrically "wired" recombinant pyruvate oxidase (POP). The sensor did not require O2 for its operation. The electroactive area of the tip of the microwire was increased by electrodeposition of platinum black. The POP was adsorbed on the platinum black and then "wired" with the cross-linked, subsequently deposited poly(4-vinylpyridine), part of the pyridine functions of which were complexed with [Os(bpy)2Cl](+/2+) and part quaternized with 2-bromoethylamine. In the resulting thin layer the POP was well "wired". When the electrode was poised at 0.4 V vs Ag/ AgCl, the sensitivity at pH 6 was 0.26 A cm(-2) M(-1) and the current increased linearly with the pyruvate concentration through the 2 x 10(-6) - 6 x 10(-4) M range. Thiamine diphosphate, flavin adenine dinucleotide, and MgCl2 were not required for the assay, but stabilized the stored enzyme electrode. Placement of a dialysis membrane (MWCO 3500) on the electrode alleviated the severe interference of ascorbate. In calf serum, the detection limit was 30 microM, suggesting that the electrode might be used in the continuous monitoring of pyruvate in hypoxic organs.     

Electrocatalytic detection of NADH and glycerol by NAD(+)-modified carbon electrodes

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.     

Enzyme-amplified amperometric sandwich test for RNA and DNA.

A one-step enzyme-amplified amperometric sandwich hybridization test for RNA and DNA is described. The test utilizes a carbon electrode, modified with a film of co-electrodeposited avidin and redox polymer; the redox polymer electrically "wiring" horseradish peroxidase (HRP) reaction centers upon contact. The film is made specific for the particular RNA or DNA sequence tested by conjugating its avidin with a biotinylated oligonucleotide, complementary to the assayed sequence. This oligonucleotide-modified redox polymer film, prepared prior to the test, forms the base of the sandwich. The center layer of the sandwich, added in the test, is the analyte RNA or DNA; its top is a second complemetary oligonucleotide, which is HRP-labeled, and is cohybridized in the test. The test consists of mixing the analyte DNA or RNA solution, the HRP-labeled oligonucleotide solution, and a hydrogen peroxide solution, immersing the base-layer carrying electrode applying a potential of 0 V versus Ag/AgCl, and measuring the H2O2 electroreduction current. Completion of the sandwich brings the HRP label into electrical contact with the redox polymer, converting the nonelectrocatalytic base layer into an electrocatalyst for the electroreduction of H2O2 to water. Flow of H2O2 electroreduction current when the electrode is poised near Ag/AgCl potential indicates the presence of the analyte RNA or DNA. The current density for the maximally sandwich-covered electrode was 250 microA cm(-2), exceeding more than a 100-fold the current density flowing upon nonspecific binding of the HRP-labeled oligonucleotide. High concentrations of irrelevant DNA and diluted serum did not interfere with the assay. When the electrodes were rotated in order to make the solution-phase mass transport rapid, the test was completed in approximately 30 min. The test was applied in probing for the presence of a 60-base E. coli mRNA sequence.     

Flow injection analysis of an ultratrace amount of arsenite using a Prussian blue-modified screen-printed electrode

We report here a new electrochemical method for the selective detection of ultratrace amount of arsenite (AsO(2)(-), As(3+)) using a Prussian blue-modified screen-printed electrode (designated as PBSPE) by flow injection analysis (FIA) in 0.1 M, pH 4 KCl/HCl carrier solution. The Prussian yellow/Prussian blue redox couple of the PBSPE was found to mediate the As(3+) oxidation. Various factors influencing the determination of As(3+) were thoroughly investigated in this study. Under the optimized FIA conditions, a linear calibration plot in the range of 50 nM-300 microM with a detection limit (S/N = 3) of 25 nM (i.e., 64.9 pg in 20-microL loop) was observed at an operation potential of +0.6 V vs Ag/AgCl. The sensitivity was good enough to detect arsenite at levels lower than the current EPA standard. This modified electrode showed good resistance to interference from common ions, especially Cl(-), which is generally considered as a major interference in the determination of As(3+) by ICPMS. The practical utility of the PBSPE to detect As(3+) was demonstrated in "blackfoot" disease endemic village groundwater from the southwestern coast area of Taiwan (Pei-Men).     

Voltammetric reduction and determination of hydrogen peroxide at an electrode modified with a film containing palladium and iridium

Cyclic voltammetry of a mixture containing 0.2 mM Na2IrCl6, 0.1 mM PdCl2, 0.2 M K2SO4, and 0.1 M HCl between 1.2 and -0.3 V vs Ag/AgCl for five cycles at 50 mV s-1 yields a stable film on a glassy carbon electrode. The reduction of hydrogen peroxide in 0.1 M KCl is diffusion controlled at that modified electrode. Calibration curves obtained at a 100 mV s-1 scan rate are linear in the range 0.2-1.8 mM H2O2. The slope, 28 microA L mmol-1, is independent of film thickness. Since dissolved oxygen is reduced at about the same potential as H2O2, -0.3 V, at the modified electrode, it will act as an interferent in solutions that are not deaerated; however, the currents are additive. A second limitation of the described procedure is that with the KCl electrolyte the immobilized film must be reoxidized prior to each measurement. Preliminary data are described which suggest that this problem is alleviated by switching to a basic supporting electrolyte.     

Direct electrochemistry and electrocatalytic activity of cytochrome c covalently immobilized on a boron-doped nanocrystalline diamond electrode

Cytochrome c (Cyt c) was covalently immobilized on a boron-doped nanocrystalline diamond (BDND) electrode via surface functionalization with undecylenic acid methyl ester and subsequent removal of the protecting ester groups to produce a carboxyl-terminated surface. Cyt c-modified BDND electrode exhibited a pair of quasi-reversible and well-defined redox peaks with a formal potential (E(0)) of 0.061 V (vs Ag/AgCl) in 0.1 M phosphate buffer solution (pH 7.0) and a surface-controlled process with a high electron transfer constant (ks) of 5.2 +/- 0.6 s(-1). The electrochemical properties of as-deposited and Cyt c-modified boron-doped microcrystalline diamond (BDMD) electrodes were also studied for comparison. Investigation of the electrocatalytic activity of the Cyt c-modified BDND electrode toward hydrogen peroxide (H2O2) revealed a rapid amperometric response (5 s). The linear range of response to H2O2 concentration was from 1 to 450 microM, and the detection limit was 0.7 microM at a signal-to-noise ratio of 3. The stability of the Cyt c-modified BDND electrode, in comparison with that of the BDMD and glassy carbon counterpart electrodes, was also evaluated.     

A nanostructured electrode of IrOx foil on the carbon nanotubes for supercapacitors

IrO(x) nanofoils (IrO(x)NF) of high surface area are sputtered on multi-wall carbon nanotubes (CNT) in the preparation of a structured electrode on a stainless steel (SUS) substrate for supercapacitor applications. This IrO(x)/CNT/SUS electrode is featured with intriguing IrO(x) curved foils of 2-3 nm in thickness and 400-500 nm in height, grown on top of the vertically aligned CNT film with a tube diameter of ～ 40 nm. These nanofoils are moderately oxidized during reactive sputtering and appeared translucent under the electron microscope. Detailed structural analysis shows that they are comprised of contiguous grains of iridium metal, iridium dioxide, and glassy iridium oxide. Considerable Raman line broadening is also evidenced for the attributed nanosized iridium oxides. Two capacitive properties of the electrode are significantly enhanced with addition of the curved IrO(x) foils. First, IrO(x)NF reduces the electrode Ohmic resistance, which was measured at 3.5 Ω cm(2) for the CNT/SUS and 2.5 Ω cm(2) for IrO(x)NF/CNT/SUS using impedance spectroscopy. Second, IrO(x)NF raises the electrode capacitance from 17.7 F g(-1) (CNT/SUS) to 317 F g(-1) (IrO(x)/CNT/SUS), measured with cyclic voltammetry. This notable increase is further confirmed by the galvanostatic charge/discharge experiment, measuring 370 F g(-1) after 2000 uninterrupted cycles between - 1.0 and 0.0 V (versus Ag/AgCl).     

Electro-catalyzed oxidation of reduced glutathione and 2-mercaptoethanol by cobalt phthalocyanine-containing screen printed graphite electrodes

Electro-catalytic behavior of screen printed graphite electrodes modified with cobalt phthalocyanine (CoPc) towards the oxidation of reduced glutathione (GSH) and 2-mercaptoethanol (2-ME) is reported. We find, by using cyclic voltammetry, that the oxidation of 2-ME occurs at 0.2 V vs Ag/AgCl and − 0.3 vs Ag/AgCl V at pH = 7 and pH = 13, respectively and that of GSH occurs at 0.4 V vs Ag/AgCl and 0.0 V vs Ag/AgCl at pH = 7 and 13, respectively. The electro-catalytic activity depends on the method of electrode modification and the amount of catalyst incorporated in the ink used to fabricate the SPCEs. The highest activity was obtained with electrodes prepared with 2.5% (w:w) of CoPc.     

Amperometric determination of hydrogen peroxide by functionalized carbon nanotubes through EDC/NHS coupling chemistry

The electrochemistry of the redox mediator Toluidine blue (TB) which was covalently linked to the carboxyl group of the multiwalled carbon nanotubes (MWNTs) by coupling reactions, in which N-hydroxysuccinimide was used to assist 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride catalyzed amidation reaction is described. The results from cyclic voltammetry (CV) and amperometry suggested that the redox mediator is linked to the surface of the MWNTs and the nanotubes showed an obvious promotion for the direct electron-transfer between the redox mediator and the electrode. A couple of well-defined redox peak of TB was observed in a phosphate buffer solution (pH 7.0). The redox mediator immobilized to MWNTs exhibits remarkable electrocatalytic activity for the reduction of hydrogen peroxide (H2O2). The analytical applicability of the modified electrode for the determination of hydrogen peroxide was examined. A linear response in the concentration range of 6.8 x 10(-7)-3.4 x 10(-2) M (r = 0.9958) was obtained with detection limit of 3.4 x 10(-7) M for the determination of hydrogen peroxide. The modified electrode has advantages of being highly stable, sensitive, ease of construction and use.     

An electrochemical method for making enzyme microsensors. Application to the detection of dopamine and glutamate

A novel method of microbiosensor fabrication is described. It is based on the electrochemical polymerization of an enzyme-amphiphilic pyrroleammonium solution on the surface of a microelectrode in the absence of supporting electrolyte. By trapping glutamate oxidase (GMO) or polyphenol oxidase (PPO) in such polypyrrole films, we made microbiosensors for the amperometric determination of glutamate or dopamine, respectively. The response of the GMO microelectrode to glutamate was based on the amperometric detection of the enzymically generated hydrogen peroxide at 0.6 V vs SCE. The detection limit and sensitivity of this microbiosensor were 1 μM and 32 mA M(-1) cm(-2), respectively. The response of the PPO microelectrode to dopamine was based on the amperometric detection of the enzymically generated quinoid product at -0.2 V. The calibration range for dopamine measurement was 5 × 10(-8)-8 × 10(-5) M and the detection limit and sensitivity were 5 × 10(-8) M and 59 mA M(-1) cm(-2), respectively.     

Flexible carbon cloth electrode modified by hollow core-mesoporous shell carbon as a novel efficient bio-anode for biofuel cell

A new approach is described to produce an efficient electrode material for biofuel cells using flexible carbon cloth (FCC) and hollow core-mesoporous shell carbon (HCMSC) nanospheres as bio-anode materials. The bio-electrochemical activity of glucose oxidase (GOx) enzyme adsorbed on this bio-anode was evaluated, with the maximum anodic current density varying from 80 microA cm(-2) to 180 microA cm-2 for glucose concentrations up to 5.0 mmol L(-1) for the FCC modified electrode with HCMSCs. The open circuit cell voltage was E(0) = 380 mV, and the catalytic electro-oxidation current of glucose reached 0.1 mA cm(-2) at 0.0 V versus Ag/AgCl. This new system employing HCMSC-based FCC is promising toward novel bio-anodes for biofuel cells using glucose as a fuel.     

In situ measurement of Cl- concentrations and pH at the reinforcing steel/concrete interface by combination sensors

This paper presents an in situ, nondestructive method of monitoring Cl- concentrations and pH values at the steel/concrete interface. The Ag/AgCl electrodes prepared by the electrochemical anodization and the Ir/IrO2 electrodes prepared by thermal oxidation in carbonate served as Cl- concentration and pH sensors, respectively. The potentiometric response of the Ag/AgCl electrode to the logarithm of Cl- concentrations ranging from 1 x 10(-4) to 2 M in saturated Ca(OH)2 solution simulating the inner electrolytic medium of concrete shows good linearity. The Ir/IrO2 electrode also exhibits an ideal Nernstian response in the range of pH 1-14. The Ag/AgCl and Ir/IrO2 electrodes were combined into a multiplex Cl-/pH sensor, and the sensor was embedded in concrete close to the steel/concrete interface to realize an in situ and long-term measurement of Cl- concentrations and pH values. The results indicate that the combined sensor is robust and sensitive enough to in situ measure Cl- concentrations and pH quantitatively at the steel/concrete interface, which is of indispensable importance to the study of corrosion and protection of the steel in concrete.     

Electron-transfer reactivity and enzymatic activity of hemoglobin in a SP Sephadex membrane

Hemoglobin can exhibit a direct electron-transfer reaction after being entrapped in a SP Sephadex membrane. A pair of stable and well-defined redox waves are obtained at a hemoglobin-SP sephadex modified pyrolytic graphite electrode. The anodic and cathodic peak potentials are located at -0.244 and -0.336 V (vs SCE), respectively. On the other hand, the peroxidase activity of the protein in the membrane is also greatly enhanced. The apparent Michaelis-Menten constant is calculated to be 1.9 mM, which shows a large catalytic activity of hemoglobin in the SP Sephadex membrane toward hydrogen peroxide (H2O2). According to the direct electron-transfer property and enhanced peroxidase activity of Hb in the membrane, a Hb/SP Sephadex membrane-based H2O2 biosensor is prepared, with a linear range approximately 5.0 x 10(-6) to 1.6 x 10(-4) mol/L.     

Development of a flow amperometric enzymatic method for the determination of total glucosinolates in real samples

The first amperometric flow analyzer, based on the biosensor concept, capable of determining total glucosinolates in real samples, is described. Myrosinase was immobilized on aminopropyl-modified controlled pore glass, which was then used for the construction of a packed-bed reactor. Myrosinase catalyzes the hydrolysis of glucosinolates (sinigrin) to glucose (among the other products), which is then oxidized by the action of glucose oxidase to produce hydrogen peroxide. The glucose enzyme electrode is based on a multimembrane architecture and was mounted on an amperometric flow cell (hydrogen peroxide detection at a platinum anode poised at +0.65 V vs Ag/AgCl/3M KCl). Different membrane types and different activation procedures were tested. The system was optimized to various working parameters, either as a glucose electrode or as a glucosinolate analyzer. The interference effect of various compounds was also investigated. Application of the method to real samples was carried out using glucose/glucose, hydrolyzed sinigrin and glucose/sinigrin solution as calibrators of the glucose electrode and the glucosinolate analyzer. Deviations due to the enantioselectivity of glucose oxidase to the beta-glucose anomer were observed, and a data elaboration protocol is proposed. The possibility of the simultaneous determination of glucose and glucosinolates is also demonstrated.     

Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: improvement of first-generation biosensors

An optimal environment for glucose oxidase (GOx) in Nafion membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nafion is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 microM level, along with high sensitivity (0.05 A M(-1) cm(-2)), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.     

Detection of glucose at 2 fM concentration

We report the amperometric detection of glucose at 2 fM concentration in a physiological buffer solution at 1 atm O2 pressure. The sensitive assay is based on the close to absolute electroreductive stripping of O2 from the solution near the glucose electrooxidizing anode. The glucose was detected by its electrooxidation on a stationary glassy carbon disk surrounded by an also stationary platinum ring. The disk was coated with a film of glucose oxidase (GOx), electrically "wired" with PVP-[Os(N,N'-dimethyl-2,2'-biimidazole)3]2+/3+ (polymer I), having a redox potential of -0.19 V versus Ag/AgCl. The ring was coated with bilirubin oxidase (BOD) "wired" with PAA-PVI-[Os(4,4'-dichloro-2,2'-bipyridine)2Cl]+/2+ (polymer II), having a redox potential of + 0.36 V versus Ag/AgCl. The ring-disk electrode was held facing up, and a 30-microL drop was placed on it for the assay, with the ring poised at -0.3 V/ AgAgCl and the disk poised at -0.1 V/ Ag/AgCl. Even though the atmosphere over the drop was O2 at 1 atm pressure, the wired BOD disk scavenged the O2 so effectively that the glucose-reduced FADH2 of GOx was not oxidized by O2, the natural cosubstrate of the enzyme.