Amperometric glucose microelectrodes prepared through immobilization of glucose oxidase in redox hydrogels.

Glucose microelectrodes have been formed with glucose oxidase immobilized in poly[(vinylpyridine)Os(bipyridine)2Cl] derivative-based redox hydrogels on beveled carbon-fiber microdisk (7 microns diameter) electrodes. In the resulting microelectrode, the steady-state glucose electrooxidation current density is 0.3 mA cm-2 and the sensitivity is 20 mA cm-2 M-1. The current density and sensitivity are 10 times higher than in macroelectrodes made with the same hydrogel. Furthermore, the current is less affected by a change in the partial pressure of oxygen. The higher current density and lower oxygen sensitivity point to the efficient collection of electrons through their diffusion in the redox hydrogel to the electrode surface. These results contrast with those observed for enzyme electrodes based on diffusing mediators, where loss of the enzyme-reduced mediator by radial diffusion to the solution decreases the current densities of microelectrodes relative to similar macroelectrodes.     

Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration.     

Glucose and lactate biosensors based on redox polymer/oxidoreductase nanocomposite thin films

Glucose and lactate enzyme electrodes have been fabricated through the deposition of an anionic self-assembled monolayer and subsequent redox polymer/enzyme electrostatic complexation on gold substrates. These surfaces were functionalized with a negative charge using 11-mercaptoundecanoic acid (MUA), followed by alternating immersions in cationic redox polymer solutions and anionic glucose oxidase (GOX) or lactate oxidase (LAX) solutions to build the nanocomposite structure. The presence of the multilayer structure was verified by ellipsometry and sensor function characterized electrochemically. Reproducible analyte response curves from 2 to 20 mM (GOX) and 2-10 mM (LAX) were generated with the standard deviation between multiple sensors between 12 and 17%, a direct result of the reproducibility of the fabrication technique. In the case of glucose enzyme electrodes, the multilayer structure was further stabilized through the introduction of covalent bonds within and between the layers. Chemical cross-linking was accomplished by exposing the thin film to glutaraldehyde vapors, inducing linkage formation between lysine and arginine residues present on the enzyme periphery with amine groups present on a novel redox polymer, poly[vinylpyridine Os(bisbipyridine)2Cl]-co-allylamine. Finally, an initial demonstration of thin-film patterning was performed as a precursor to the development of redundant sensor arrays. Microcontact printing was used to functionalize portions of a gold surface with a blocking agent, typically 1-hexadecanethiol. This was followed by immersion in MUA to functionalize the remaining portions of gold with negative charges. The multilayer deposition process was then followed, resulting in growth only on the regions containing MUA, resulting in a "positive"-type pattern. This technique may be used for fabrication of thin-film redundant sensor arrays, with thickness under 100 angstrom and lateral dimensions on a micrometer scale.     

L-alpha-glycerophosphate and L-lactate electrodes based on the electrochemical "wiring" of oxidases.

The title electrodes were constructed by coimmobilizing the respective FAD oxidases on solid electrode surfaces with a poly(vinyl pyridine) polymer which was N-derivatized with bromoethylamine and Os(bpy)2Cl2. The redox-polymer-enzyme hydrogels were cross-linked on the electrode surface using poly(ethylene glycol) diglycidyl ether. As in the case of glucose oxidase, the redox polymer acts as an electron relaying "wire" transferring electrons directly from the enzymes' FADH2 centers to the electrode. This transfer competes with the natural process of reoxidation of FADH2 by molecular oxygen. The variation of the response of these electrodes with the atmosphere (N2 or air), pH, and substrate concentration was determined. The pH profile of the electrocatalytic current differs from that of the activity of the free enzymes, exhibiting a broader maximum, shifted to higher pH values. The observed sensitivities and linear ranges are respectively 2 x 10(-2) A M-1 cm-2 and 2.7 mM for L-alpha-glycerophosphate, and 0.3 A M-1 cm-2 and 0.2 mM for L-lactate that may be compared to 2 x 10(-2) A M-1 cm-2 and 10 mM for glucose. The 0-90% response time for all electrodes is 1 s or less.     

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.     

Subnanoliter volume wall-jet cells combined with interdigitated microarray electrode and enzyme modified planar microelectrode

Miniaturized wall-jet type flow cells with an active volume of 0.042-15 nL were fabricated for use as highly sensitive electrochemical detectors for capillary electrophoresis/electrochemical detection and small on-line enzyme sensors. The cells consisted of three glass plates and a fused-silica capillary. Two of the plates had microfabricated flow channels and guide trenches for the capillary and working, reference, and counter electrodes. The other plate had a film electrode. When an interdigitated microarray electrode (total area, 66 microm x 64 microm; bandwidth and gap, 2 microm) was installed in the flow cell, the redox cycling enhanced the current at flow rates of less than 100 nL/min even though there were only eight pairs of microbands. A sharp dopamine peak enhanced by the redox cycling was observed when the cell was used for capillary electrophoresis. A square film electrode modified with glutamate oxidase and Os-poly(vinylpyridine) containing HRP was also installed in the flow cell and used to measure neurotransmitter release from cultured nerve cells. When the flow rate was relatively high, the response time of the modified electrode was comparable to that of a cylindrical carbon fiber electrode (33 microm o.d.) modified with the same enzyme and mediator. We observed a transient cathodic current response assigned to the glutamate release with the electrode in the flow cell in a suction mode measurement when we stimulated cultured nerve cells electrically with a dual microelectrode.     

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.     

In situ assembled mass-transport controlling micromembranes and their application in implanted amperometric glucose sensors

Micromembranes were assembled by sequentially chemisorbing polyanions and polycations on miniature (5 x 10(-4) cm2) enzyme electrodes. The sequential chemisorption process allowed the simultaneous tailoring of their sensitivity, dynamic range, drift, and selectivity. When assembled on tips of 250-microm-diameter gold wires coated with redox polymer-"wired" glucose oxidase, they allowed tailoring of the glucose electrodes for > 2 nA/mM sensitivity; 0-30 mM dynamic range; drift of < or =5% per 24 h at 37 degrees C at 15 mM glucose concentration; and < or =5% current increment by the combination of 0.1 mM ascorbate, 0.2 mM acetaminophen, and 0.5 mM urate. The membranes also retained transition metal ions that bound to and damaged the redox polymer "wiring" the enzyme. The electrodes were tested in the jugular veins and in the intrascapular subcutaneous region of anaesthetized and heparinized nondiabetic Sprague-Dawley rats, in which rapid changes of glycemia were forced by intravenous injections of glucose and insulin. After one-point in vivo calibration of the electrodes, all of the 152 data points were clinically accurate when it was assumed that after insulin injection the glycemia in the subcutaneous fluid lags by 9 min behind that of blood withdrawn from the insulin-injected vein.     

Spraying enzymes in microemulsions of AOT in nonpolar organic solvents for fabrication of enzyme electrodes

A new technique suitable for automated, large-scale fabrication of enzyme electrodes by air-spraying enzymes in organic inks is presented. Model oxidoreductases, tyrosinase (Tyr) and glucose oxidase (GOx), were adapted to octane-based ink by entrapment in a system of reverse micelles (RM) of surfactant AOT in octane to separate and stabilize the catalytically active forms of the enzymes in nonpolar organic media. Nonpolar caoutchouk polymer was also used to create a kind of "dry micelles" at the electrode/solution interface. Enzyme/RM/polymer-containing organic inks were air-brushed onto conductive supports and were subsequently covered by sprayed Nafion membranes. The air-brushed enzyme electrodes exhibited relevant bioelectrocatalytic activity toward catechol and glucose, with a linear detection range of 0.1-100 microM catechol and 0.5-7 mM glucose; the sensitivities were 2.41 A M(-1) cm(-2) and 2.98 mA M(-1) cm(-2) for Tyr and GOx electrodes, respectively. The proposed technique of air-brushing enzymes in organic inks enables automated construction of disposable enzyme electrodes of various designs on a mass-production scale.     

Electrochemical surface plasmon resonance measurement based on gold nanohole array fabricated by nanoimprinting technique

In this paper, we describe our development of an electrochemical surface plasmon resonance (EC-SPR) measurement device based on a bottom-filled gold nanohole array. The polymer based gold nanohole array was fabricated with a UV nanoimprint technique and electron beam gold deposition. Direct reflection mode measurement was used to monitor the SPR dip in the reflection spectra. A cyclic voltammogram was also operated by using the standard three electrodes containing working electrode having a gold nanohole array and counter and reference electrodes. The gold nanohole array was modified with an osmium-poly(vinylpyridine)-wired horseradish peroxidase (Os-gel-HRP) film, and its redox state induced by the change in potential was monitored simultaneously. The redox state of the local film was obtained simply by scanning the sample substrate stage. The substrate modified with Os-gel-HRP film was incorporated in a microfluidic chip, and then the hydrogen peroxide was determined in terms of the redox change in the Os complex mediator from the slope of the SPR dip shift. The linear relation of hydrogen peroxide from 10 to 250 μM was successfully monitored, and a high conversion efficiency was realized.     

Amperometric biosensors based on redox polymer-carbon nanotube-enzyme composites

Based on their size and unique electrical properties, carbon nanotubes offer the exciting possibility of developing ultrasensitive, electrochemical biosensors. In this study, we describe the construction of amperometric biosensors based on the incorporation of single-walled carbon nanotubes modified with enzyme into redox polymer hydrogels. The composite films were constructed by first incubating an enzyme in a single-walled carbon nanotube (SWNTs) solution and then cross-linking within a poly[(vinylpyridine)Os(bipyridyl)(2)Cl(2+/3+)] polymer film. Incorporation of SWNTs, modified with glucose oxidase, into the redox polymer films resulted in a 2-10-fold increase in the oxidation and reduction peak currents during cyclic voltammetry, while the glucose electrooxidation current was increased 3-fold to approximately 1 mA/cm(2) for glucose sensors. Similar effects were also observed when SWNTs were modified with horseradish peroxidase prior to incorporation into redox hydrogels.     

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.     

Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film

o-Phenylenediamine has been used for glucose oxidase (GOx) immobilization on Pt electrodes by electrochemical polymerization at +0.65 V vs SCE. By this approach the enzyme is entrapped in a strongly adherent, highly reproducible thin membrane, whose thickness is around 10 nm. This one-step procedure produces a glucose sensor with a response time less than 1 s, an active enzyme loading higher than 3 units/cm2 of electrode surface, a high sensitivity, and a sufficiently wide linear range. The glucose response shows an apparent Michaelis-Menten constant, K'm = 14.2 mM, and a limiting current density, jmax of 181 microA/cm2. The product kD of partition and diffusion coefficients of glucose in the polymer film is on the order of 10(-13) cm2/s. Due to permselectivity characteristics of the membrane, the access of ascorbate, a common interfering species, to the electrode surface is blocked. To our knowledge, this represents the first report of a membrane capable, at the same time, of immobilizing GOx and rejecting ascorbate. The interesting electrode behavior can be rationalized by using an existing model predicting the amperometric response of an immobilized GOx system.     

Transistor-like behavior of transition metal complexes

Electron transport through semiconductor and metallic nanoscale structures, molecular monolayers, and single molecules connected to external electrodes display rectification, switch, and staircase functionality of potential importance in future miniaturization of electronic devices. Common to most reported systems is, however, ultrahigh vacuum and/or cryogenic working conditions. Here we introduce a single-molecule device concept based on a class of robust redox active transition metal (Os(II)/(III)) complexes inserted between the working electrode and tip in an electrochemical scanning tunneling microscope (in situ STM). This configuration resembles a single-molecule transistor, where the reference electrode corresponds to the gate electrode. It operates at room temperature in a condensed matter (here aqueous) environment. Amplification on-off ratios up to 50 are found when the redox level is brought into the energy window between the Fermi levels of the electrodes by the overpotential ("gate voltage"). The current-voltage characteristics for two Os(II)/(III) complexes have been characterized systematically and supported by theoretical frames based on molecular charge transport theory.     

Electrical Communication between Electrodes and Enzymes Mediated by Redox Hydrogels

Different redox polymers based on poly(allylamine) with covalently attached ferrocene and pyridine groups that coordinate iron and ruthenium complexes were prepared, and hydrogels were obtained by cross-linking them with epichlorohydrin. Charge propagation from the underlying electrode, through the redox polymer and electrical communication with the enzyme FADH(2) of glucose oxidase, was studied by cyclic voltammetry and electrochemical impedance spectroscopy. The effects of electrolyte composition, concentration of enzyme and substrate, and electrode potential are reported. The role of different redox mediators covalently attached to the polymer backbone is discussed in terms of driving force and electrostatic barriers.     

A glucose oxidase electrode based on electropolymerized conducting polymer with polyanion-enzyme conjugated dopant

An enzyme immobilization method has been developed by electropolymerization chemistry of conducting polymer which results in a more effective and reproducible enzyme electrode. As a model system, in this study, glucose oxidase (GOD) was conjugated with a polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid), via a poly(ethylene oxide) spacer to improve the efficiency of enzyme immobilization into a conducting polymer. GOD was successfully conjugated with a high conjugation yield of more than 90%, and its bioactivity was preserved. The resulting polyanion-GOD conjugate was used as a dopant for the electrochemical polymerization of pyrrole. Polypyrrole was effectively deposited on a Pt wire working electrode with the polyanion-GOD conjugate. The enzyme electrode responded to glucose concentrations of up to 20 mM with a sensitivity of 40 nA/mM at an applied potential of 0.4 V within a response time of 30 s. Although the response signal decreased at the low applied potential of 0.3 V, the enzyme electrode showed sensitive response signals of about 16 nA/mM up to 20 mM in glucose concentration. Under the deoxygenated condition, reduced but clear response current signal was obtained. The results show that the current signal response of the enzyme electrode to glucose concentration may be produced by mixed mechanisms.     

Electroanalytical detection of glucose using a cyanometalate-modified electrode: requirements for the oxidation of buried redox sites in glucose oxidase

Modification of a nickel electrode with a mixture of iron and ruthenium cyanometalates allows one to efficiently turn over glucose oxidase in the presence of its substrate. A limit of detection for glucose of 25 μM can be obtained with an observed saturation concentration of 10 mM. Glucose detection is found to be very sensitive to the cationic environment of the electrolyte. The largest currents for glucose oxidation are observed in the presence of nonelectroactive multiply charged cations. The solid state nature of the surface-confined cyanometalate redox mediator argues against the widely held mechanism for enzyme oxidation in which the redox mediator is required to enter the enzyme active site.     

Covalent attachment of osmium complexes to glucose oxidase and the application of the resulting modified enzyme in an enzyme switch responsive to glucose

Pyridine-based osmium complexes bearing either a carboxylate or aldehyde group were covalently attached to glucose oxidase and were shown to work as mediators for the reoxidation of the enzyme. For the complex containing the carboxylate group, the binding was made through carbodiimide coupling to the amine residues in the protein. For the complex containing the aldehyde group, the reductive coupling was carried out by condensation with the amino groups on the protein in the presence of sodium cyanoborohydride. Electrochemical studies show evidence for both intramolecular and intermolecular redox mediation for the electrochemical reoxidation of the modified glucose oxidases in the presence of glucose. The modified enzymes adsorbed on glassy carbon and platinum show different electrochemical responses for the two electrode materials, suggesting that orientation of the adsorbed enzyme is induced due to the interaction of the osmium complex with the different surfaces. Construction of enzyme switches based on these modified enzymes was carried out, and their responses were compared with those obtained using native glucose oxidase and a soluble redox mediator.     

Elimination of electrooxidizable interferant-produced currents in amperometric biosensors.

Electrooxidizable ascorbate, urate, and acetaminophen that interfere with amperometric glucose assays are completely and rapidly oxidized by hydrogen peroxide in a multilayer electrode. The multilayer electrode is composed of an immobilized, but not electrically "wired", horseradish peroxidase (HRP) film coated onto a film of electrically "wired" glucose oxidase (GO). The "wired" enzyme is connected by a redox epoxy network to a vitreous carbon electrode. The current from the electrooxidizable interferants is decreased by their peroxidase-catalyzed preoxidation by a factor of 2500, and the glucose/interferant current ratio is increased 10(3)-fold. Undesired electroreduction of hydrogen peroxide can result when HRP is also "wired" to the electrode. Such unwanted "wiring" is prevented by incorporating an electrically insulating barrier layer between the wired GO film and the HRP film. The hydrogen peroxide necessary for elimination of interferants can be added externally, or when this is not possible, it can be generated in situ by means of a coupled enzyme reaction.     

LC-Biosensor System for the Determination of the Neurotoxin β-N-Oxalyl-l-α,β-diaminopropionic Acid

An analysis system is described for the determination of the neurotoxin β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP). The system is based on liquid chromatographic separation of β-ODAP from interfering amino acids on an anion exchange column coupled with an amperometric enzyme electrode for the registration of β-ODAP. The electrode is based on a graphite rod modified with an Os(2+/3+) redox polymer cross-linked with l-glutamate oxidase and horseradish peroxidase. This LC-bienzyme electrode analytical system enabled monitoring of as low as 4 μM β-ODAP (injection volume 100 μL). The β-ODAP content in real grass pea samples was measured to range between 3.3 and 5.2 g kg(-)(1) in dry grass pea.