Selective detection method derived from a controlled diffusion process at metal-modified diamond electrodes

A novel, selective methodology is derived based on the difference between the diffusion processes at microelectrodes (i.e., hemispherical diffusion) and the macroelectrode (i.e., linear diffusion) in a metal-implanted boron-doped diamond electrode (metal-BDDs). As an example, the selective detection of glucose in a solution containing interference species such as ascorbic acid and uric acid is demonstrated. The electrochemical properties of BDD, which are low background current, extremely high stability, and (especially) inactivity toward glucose, play an important role in realizing these differences in the diffusion characteristics. The present methodology can be applied not only to selective glucose detection by the metal-BDD system but also to other selective detection systems.     

Integrated electrophoresis chips/amperometric detection with sputtered gold working electrodes

An on-chip electrochemical detector for micromachined capillary electrophoresis (CE) systems, based on sputtering a gold working electrode directly onto the capillary outlet, is described. The new on-chip detector preparation requires no microfabrication or alignment procedures nor a decoupling mechanism. The attractive performance of the integrated electrophoresis chips/amperometric detection was demonstrated for the anodic detection of neurotransmitters. The response for dopamine was linear from 20 to 200 μM, with a LOD of 1.0 μM and a sensitivity of 52 pA/μM. Such intimate coupling of capillary electrophoresis chips and electrochemical detection facilitates the realization of complete integrated microanalytical devices.     

Development of amperometric immunosensor using boron-doped diamond with poly(o-aminobenzoic acid)

An alternative method of a protein immunosensor has been developed at boron-doped diamond (BDD) electrode material. In order to construct the base of the immunosensor, o-aminobenzoic acid (o-ABA) was electropolymerized at an electrode by cyclic voltammetry. The poly-o-ABA-modified BDD was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The XPS result found that carboxyl groups were formed at the electrode surface. The carboxyl groups were then used to covalently attach protein probes. The amperometric sensing of mouse IgG (MIgG) was selected as the model at the poly-o-ABA-modified BDD to compare to the poly-o-ABA-modified glassy carbon (GC) at the same condition. An antimouse IgG from goat (GaMIgG) was covalently immobilized at a poly-o-ABA-modified BDD electrode which used a sandwich-type alkaline phosphatase (ALP) catalyzing amperometric immunoassay with 2-phospho-L-ascorbic acid (AAP) as substrate. The ALP enzyme conjugated at the immunosensor can generate AAP to the electroactive species of ascorbic acid (AA), which can be determined by amperometric detection. The signal was found to be proportional with the quantity of MIgG. The limits of detection (LODs) of 0.30 (3 SD) and 3.50 ng mL(-1) (3 SD) for MIgG at BDD and GC electrodes were obtained. It also was found that the dynamic range of 3 orders of magnitude (1-1000 ng mL(-1)) was obtained at BDD, whereas at GC, the dynamic range was more narrow (10-500 ng mL(-1)). The method was applied to a real mouse serum sample that contains MIgG.     

Palladium film decoupler for amperometric detection in electrophoresis chips

Demonstrated in this article is that a palladium metal film can be applied to decouple the electric circuitry of electrochemical detection from that of the electrophoretic separation in an electrophoresis chip. The Pd solid-state field decoupler, as well as the working electrodes, is thermally evaporated onto the plastic chip and oriented vertically across the separation channel. After the sample zones flow over the Pd decoupler, their electrochemical response is measured at working electrodes in the downstream pathway. Because the electrodes are on the separation channel, the electrode channel alignment is no longer a problem. For a separation channel of roughly 200 microm in width and 75 microm in depth in 10 mM phosphate (pH 5.1), the noise level at the working electrode is < 15 pA at an electric field of 570 V/cm.     

Electrochemical detection of carbamate pesticides at conductive diamond electrodes

Conductive boron-doped diamond thin-film electrodes were used for the electrochemical detection of selected N-methylcarbamate pesticides (carbaryl, carbofuran, methyl 2-benzimidazolecarbamate, bendiocarb) after liquid chromatographic separation. Two kinds of detection methods were adopted in this study. In the first method, a direct detection of underivatized pesticides was carried out at an operating potential of 1.45 V versus Ag/AgCl, which resulted in the detection limits of 5-20 ng/mL (or 5-20 ppb) with S/N = 2 due to the low background current and wide potential window of the diamond electrode. In the second method, the detection limits were improved by subjecting the pesticide samples to alkaline hydrolysis in a separate step prior to injection. The phenolic derivatives obtained by alkaline hydrolysis oxidize at a relatively lower potential (0.9 V vs Ag/AgCl), which increases the sensitivity drastically. The advantage of the diamond electrode for the detection of phenolic derivatives is that it offers excellent stability in comparison to other electrodes. This method gives the detection limits of 0.6-1 ng/mL (or 0.6-1 ppb), which are well below the maximum residue levels allowed for carbaryl, carbofuran, and bendiocarb. While the lowest detection limits (LOD) obtained by the direct detection of pesticides are comparable to the those reported by the well-established HPLC-fluorescence, the LODs of the alkaline hydrolysis method are found to be even lower than the reported limits. On-line reactivation of the diamond electrode surface was shown to be possible by an anodic treatment of the electrode at approximately 3 V for 30 min in case of electrode fouling, which may occur after a prolonged use. Such a treatment damages the glassy carbon (GC) and metal electrodes, while the diamond electrode remains stable. These results suggest that the diamond electrode is superior to the other previously used electrodes such as GC and Kelgraf type for highly sensitive and stable detection of carbamate pesticides.     

Uricase-catalyzed oxidation of uric acid using an artificial electron acceptor and fabrication of amperometric uric acid sensors with use of a redox ladder polymer.

Electrochemical oxidation of uric acid catalyzed by uricase (uric acid oxidase, UOx; EC 1.7.3.3) was studied using several redox compounds including 5-methylphenazinium (MP) and 1-methoxy-5-methylphenazinium (MMP) as electron acceptors for UOx, which does not contain any redox cofactor. It was found that MP and MMP were useful to mediate electrons from UOx to an electrode in the enzymatic oxidation of uric acid. A novel redox polymer, poly(N-methyl-o-phenylenediamine)(poly-MPD), containing the MP units was also found to possess the mediation ability for UOx, and poly-MPD was immobilized together with UOx onto an electrode substrate covered with a self-assembled monolayer of 2-aminoethanethiolate with use of glutaraldehyde as a binding agent. The resulting electrode (poly-MPD/UOx/Au) exhibited amperometric responses to uric acid with very fast response of approximately 30 s, allowing reagentless amperometric determination in a concentration range covering that in the blood of a healthy human being. Kinetic parameters of the apparent Michaelis constant and the maximum current response obtained at the poly-MPD/UOx/Au suggested that electrochemical oxidation of uric acid was controlled by diffusion of uric acid into the enzyme film and that the redox polymer worked well in mediating between active sites of UOx molecules and the electrode substrate.     

Zeptomole voltammetric detection and electron-transfer rate measurements using platinum electrodes of nanometer dimensions

The characterization of quasi-hemispherical Pt electrodes of nanometer dimensions (radius 2-150 nm), prepared by electrophoretic coating of etched Pt wires with poly-(acrylic acid), is described. The goals of these experiments are to estimate the accuracy of using steady-state voltammetric limiting currents (i(lim)) in determining the true electrode area and to develop new electrochemical methods for rapidly screening individual electrodes for non-ideal geometries. Electrochemical active areas were determined by measuring the electrical charge (Q) associated with oxidation of adsorbed bis(2,2'-bipyridine)chloro(4,4'-trimethylenedipyridine)osmium(II) in fast-scan voltammetric measurements (scan rate 1000 V/s). Voltammetric peaks corresponding to oxidation of as few as approximately 7000 molecules (approximately 11 zmol) at individual electrodes are reported, allowing precise measurement of electrode areas as small as approximately 10(-10) cm2. A plot of i(lim) (for a soluble redox species) versus Q1/2 (for an adsorbed redox species), constructed from i(lim)-Q1/2 data pairs obtained as a function of the electrode radius, is shown to be linear if the electrode geometry is independent of electrode radius; departure of experimental values from the straight-line plot is a diagnostic indicator of a nonideal electrode geometry. The results indicate that approximately 50% of the electrodes prepared by the electrophoretic polymer-coating procedure are quasi-hemispherical, the remaining being recessed slightly below the polymer coating. The heterogeneous electron-transfer rate constant for the oxidation of the ferrocenylmethyltrimethylammonium cation in H2O/ 0.2 M KCl was also determined from steady-state voltammetry using the method of Mirkin and Bard and found to be 4.(8) +/- 3.(2) cm/s with alpha = 0.6(4) +/- 0.1(5).     

Selective detection of o-diphenols on copper-plated screen-printed electrodes

Selective detection of o-diphenols (e.g., catechol, dopamine, and pyrogallol) in the presence of simple phenols, m- and p- derivative diphenols, and ascorbic acid has been demonstrated on copper-plated screen-printed electrodes (CuSPEs) in pH 7.4 phosphate buffer solution. The CuSPE showed an unusual catalytic response at -0.05 V versus Ag/AgCl selectively to o-diphenolic compounds. The o-diphenols can thus be determined amperometrically through direct electrochemical oxidation in low potentials (approximately 0 V), where the CuSPE is much less subject to interfering reactions. Such a catalytic phenomenon cannot be observed on conventional Pt and glassy carbon electrodes. The selective mechanism is explained in terms of the formation of cyclic five-member complex intermediate (Cu(II)-o-quinolate). Most important of all, the common drawbacks of electrode fouling through polymerization were completely overcome in this system.     

Stripping voltammetric behavior of nitrogen-doped tetrahedral amorphous carbon thin film electrodes in NaCl solutions

Conductive nitrogen-doped tetrahedral amorphous carbon (ta-C:N) thin films fabricated with a filtered cathodic vacuum arc technique were investigated for their ability to detect multiple trace heavy metals such as mercury, copper and lead by linear sweep anodic stripping voltammetry in sodium chloride aqueous solutions. The ta-C:N film electrodes exhibited a significant stripping response for determination of individual elements (Pb²⁺ and Hg²⁺) and multiple elements (Pb²⁺ + Hg²⁺ and Cu²⁺ + Hg²⁺), indicating that these electrodes have a great potential for simultaneously tracing multiple heavy metals in aqueous solutions.     

Electrochemical oxidation of oxalic acid at highly boron-doped diamond electrodes

Electrochemical oxidation of oxalic acid has been investigated at bare, highly boron-doped diamond electrodes. Cyclic voltammetry and flow injection analysis with amperometric detection were used to study the electrochemical reaction. Hydrogen-terminated diamonds exhibited well-defined peaks of oxalic acid oxidation in a wide pH range. A good linear response was observed for a concentration range from 50 nM to 10 microM, with an estimated detection limit of approximately 0.5 nM (S/N = 3). In contrast, oxygen-terminated diamonds showed no response for oxalic acid oxidation inside the potential window, indicating that surface termination contributed highly to the control of the oxidation reaction. An investigation with glassy carbon electrodes was conducted to confirm the surface termination effect on oxalic acid oxidation. Although a hydrogen-terminated glassy carbon electrode showed an enhancement of signal-to-background ratio in comparison with untreated glassy carbon, less stability of the current responses was observed than that at hydrogen-terminated diamond.     

Experimental and theoretical investigations on the adsorption of 2'-deoxyguanosine oxidation products at oxidized boron-doped diamond electrodes

Electrochemical oxidation of 2'-deoxyguanosine has been performed on boron-doped diamond (BDD) electrodes, resulting in a strong adsorption of the formed oxidized products onto the BDD surface. The adsorption behavior has been investigated by studying the electrochemical behavior of a redox probe ([IrCl6]3-) using cyclic voltammetry. The most probable situations are the formation of (A) an insulating adsorbed film resulting in a partially blocked electrode behavior, (B) a porous film, or (C) an overall conductive film. Different parameters such as the standard rate constant, the charge-transfer coefficient, the electrode/adsorbed products/solution interface resistance, and the formal potential of the redox couple were determined. Through comparison of theoretical current-potential curves obtained by analytical calculations with experimental cyclic voltammograms, we found that the oxidized products of 2'-deoxyguanosine form a continuous conductive film on BDD.     

Nanostructured carbon fiber disk electrodes for sensitive determinations of adenosine and uric acid

Nanostructured carbon fiber microdisk electrodes were prepared by a combination of mechanical polishing and electrolytic treatment, where the latter involved moderate oxidation of the surface, followed by a reduction. A high density of surface defects contributed to a high capacitance of the nanostructured electrodes. Facilitated proton transfer was observed at the nanostructured surface and was associated with cation-exchanged oxide defects. The nanostructured surfaces intercalated uric acid and adenosine and engaged in fast electron/proton transfer in the oxidation of both analytes. As a result, electrolytic treatment followed by fast-scan voltammetry determinations led to a sensitive response to both analytes in physiological buffers. The nanostructured electrodes showed remarkable stability and could be easily regenerated and reused. With long use, electrode activity decreased. Kinetic discrimination of the surface-mediated reaction of ascorbate was achieved at high scan rates.     

Three-electrode electrochemical detector and platinum film decoupler integrated with a capillary electrophoresis microchip for amperometric detection

This article demonstrates that a three-electrode electrochemical (EC) detector and an electric decoupler could be fabricated in the same glass chip and integrated with an O2-plasma-treated PDMS layer using microfabrication techniques to form the capillary electrophoresis (CE) microchip. The platinized decoupler could mostly decouple the electrochemical detection circuit from the interference of an separation electric field in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5) solution. The baseline offset of background current recorded from the working electrode with and without application of a separation electric field was maintained at less than 0.05 pA in 10 mM MES. In addition, the platinized pseudoreference electrode was demonstrated to offer a stable potential in electrochemical detection. As a consequence, the limit of detection of dopamine was 0.125 microM at a S/N = 4. The responses for dopamine to different concentrations were found to be linear between 0.25 and 50 microM with a correlation coefficient of 0.9974 and a sensitivity of 11.76 pA/microM. The totally integrated CE-EC microchip should be able to fulfill the ideal of miniaturization and commercialization.     

Chlorinated phenol analysis using off-line solid-phase extraction and capillary electrophoresis coupled with amperometric detection and a boron-doped diamond microelectrode

The analysis of chlorinated phenols (2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol) in river water was accomplished using off-line solid-phase extraction (SPE) and capillary electrophoresis coupled with electrochemical detection. A key to the sensitive, reproducible, and stable detection of these pollutants was the use of a boron-doped diamond microelectrode in the amperometric detection mode. An off-line SPE procedure was utilized to extract and preconcentrate the pollutants prior to separation and detection, with ENVI-Chrom P, a highly cross-linked styrene-divinylbenzene copolymer, being employed as the sorbent. Pollutant recoveries in the 95-100% range with relative standard deviations of 1-4% were achieved. The diamond microelectrode provided a low and stable background current with low peak-to-peak noise. The oxidative detection of the pollutants was accomplished at +1.05 V vs Ag/AgCl without the need for electrode pretreatment. The method was evaluated in terms of the linear dynamic range, sensitivity, limit of quantitation, response precision, and response stability. A reproducible electrode response was observed during multiple injections of the chlorinated phenol solutions with a relative standard deviation of < or =5.4%. Good electrode response stability was observed over many days of continuous use with no significant electrode deactivation or fouling. The separation efficiencies for all six pollutants were greater than 170,000 plates/m. The minimum concentration detectable for all six ranged from 0.02 to 0.2 ppb (S/N > or = 3) using a 250:1 preconcentration factor.     

Electrochemical oxidation of chlorophenols at a boron-doped diamond electrode and their determination by high-performance liquid chromatography with amperometric detection

Anodically pretreated diamond electrodes have been used for the detection of chlorophenols (CPs) in environmental water samples after high-performance liquid chromatographic (HPLC) separation. The anodization of as-deposited boron-doped polycrystalline diamond thin-film electrodes has enabled the stable determination of phenols over a wide concentration range. Prior to the HPLC analysis, a comparative study with ordinary glassy carbon, as-deposited diamond, and anodized diamond was made to examine the oxidative behavior of phenols by cyclic voltammety and flow injection analysis with amperometric detection. At anodized diamond electrodes, reproducible, well-defined cyclic voltammograms were obtained even at high CP concentration (5 mM), due to a low proclivity for adsorption of the oxidation products on the surface. In addition, after prolonged use, the partially deactivated diamond could be reactivated on line by applying a highly anodic potential (2.64 Vvs SCE) for 4 min, which enabled the destruction of the electrodeposited polymer deposits. Hydroxyl radicals produced by the high applied potential, in which oxygen evolution occurs, are believed to be responsible for the oxidation of the passivating layer on the surface. When coupled with flow injection analysis (FIA), anodized diamond exhibited excellent stability, with a response variability of 2.3% (n = 100), for the oxidation of a high concentration (5 mM) of chlorophenol. In contrast, glassy carbon exhibited a response variability of 39.1%. After 100 injections, the relative peak intensity, for diamond decreased by 10%, while a drastic decrease of 70% was observed for glassy carbon. The detection limit obtained in the FIA mode for 2,4-dichlorophenol was found to be 20 nM (S/N = 3), with a linear dynamic range up to 100 microM. By coupling with the column-switching technique, which enabled on-line preconcentration (50 times), the detection limit was lowered to 0.4 nM (S/N = 3). By use of this technique, anodized diamond electrodes were demonstrated for the analysis of CPs in drainwater that was condensed from the flue gas of waste incinerators.     

Electrochemical detection of arsenic(III) using iridium-implanted boron-doped diamond electrodes

Iridium-modified, boron-doped diamond electrodes fabricated by an ion implantation method have been developed for electrochemical detection of arsenite (As(III)). Ir+ ions were implanted with an energy of 800 keV and a dose of 10(15) ion cm(-2). An annealing treatment at 850 degrees C for 45 min in H2 plasma (80 Torr) was required to rearrange metastable diamond produced by an implantation process. Characterization was investigated by SEM, AFM, Raman, and X-ray photoelectron spectroscopy. Cyclic voltammetry and flow injection analysis with amperometric detection were used to study the electrochemical reaction. The electrodes exhibited high catalytic activity toward As(III) oxidation with the detection limit (S/N = 3), sensitivity, and linearity of 20 nM (1.5 ppb), 93 nA microM(-1) cm(-2), and 0.999, respectively. The precision for 10 replicate determinations of 50 microM As(III) was 4.56% relative standard deviation. The advantageous properties of the electrodes were its inherent stability with a very low background current. The electrode was applicable for analysis of spiked arsenic in tap water containing a significant amount of various ion elements. The results indicate that the metal-implanted method could be promising for controlling the electrochemical properties of diamond electrodes.     

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.     

Algae columns with anodic stripping voltammetric detection

The use of silica-immobilized algal cells for on-line column separation in conjunction with continuous monitoring of trace metals is described. Algae-silica preparations are highly suitable for flow analysis as they couple the unique reactivity patterns and high binding capacity of algal biomass with the hydrodynamic and mechanical features of porous silica. Such advantages are illustrated by using on-line anodic stripping voltammetry and the alga Chlorella pyrenidosa. Selective and exhaustive removal of interfering constituents circumvents common problems such as overlapping peaks and intermetallic effects. Effects of flow rate, pH, operation time, and other variables are reported. The system is characterized by high durability, simplicity, and economy and offers an attractive alternative to prevalent columns used for flow analysis.     

Conformational change detection in nonmetal proteins by direct electrochemical oxidation using diamond electrodes

In this report, we established a new electrochemical method for the detection of conformational changes in large, non-metalloproteins such as bovine serum albumin, using flow injection analysis coupled with hydrogen-terminated, boron-doped diamond electrodes. The oxidation current was used as a signal reporter in the monitoring of urea-induced BSA denaturation. In the denatured state at high urea concentrations, the electrochemical signal increased, and the amperometric responses for the oxidation potential at 1300 mV were consistent with the results of conventional methods of denaturation monitoring using fluorescence spectroscopy. The oxidation involved at least five redox-active species (cysteine, tryptophan, tyrosine, methionine, and disulfide bonds). Furthermore, the method also showed high sensitivity for quantitative analysis of protein. A linear dynamic in the concentration range 50-400 microg/mL (r(2) = 0.977) with a lower detection limit of 190 ng/mL was achieved for BSA. Direct electrochemical detection of conformation changes of proteins using BDD electrodes can be performed with advantages in terms of simplicity and sensitivity.     

Development and characterization of microfabricated disposable gold working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection

We have developed a new type of microfabricated thin-film electrode on polymeric substrates. The microfabrication process allows for inexpensive and reproducible mass production of disposable working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection (IPAD). These microfabricated electrodes are disposable and have been optimized for use in flow-through low-dead-volume electrochemical cells. The analytical performance of microfabricated gold electrodes was characterized with the help of the IPAD method for amino acid detection under alkaline conditions required for anion-exchange separations. When used with a new optimized six-potential IPAD waveform, the electrodes functioned properly for weeks. Compared to nondisposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration, and reproducibility. Disposable electrodes make it possible to avoid polishing and reconditioning, which are required with nondisposable electrodes.