Qijin Chi

Amperometric biosensors based on the immobilization of oxidases in a Prussian blue film by electrochemical codeposition

Abstract Prussian blue has been formed by cyclic voltammetry onto the basal pyrolytic graphite surface to prepare a chemically modified electrode which provides excellent electrocatalysis for both oxidation and reduction of hydrogen peroxide. It is found for the first time that glucose oxidase or D -amino oxidase can be incorporated into a Prussian blue film during its electrochemical growth process. Two amperometric biosensors were fabricated by electrochemical codeposition, and the resulting sensors were protected by coverage with a thin film of Nafion. The influence of various experimental conditions was examined for optimum analytical performance. The glucose sensor responds rapidly to substrates with a detection limit of 2 × 10−6 M and a linear concentration range of 0.01–3 mM. There was no interference from 2 mM ascorbic acid or uric acid. Another ( D -amino acid) sensor gave a detection limit of 3 × 10−5 M D -alanine, injected with a linear concentration range of 7.0 × 10−5-1.4 × 10−2 M. Glucose and D -amino acid sensors remain relatively stable for 20 and 15 days, respectively. There is no obvious interference from anion electroactive species due to a low operating potential and excellent permselectivity of Nafion.

Direct electrochemistry and surface characterization of glucose oxidase adsorbed on anodized carbon electrodes

The glassy carbon electrode (gce) and highly oriented pyrolytic graphite (hopg) were electrochemically anodized at a potential of +2.0V (vs. Ag/AgCl) to create active sites and to improve the adsorption of glucose oxidase (GOD) and flavin adenine dinucleotide (FAD) on the electrode surface. The GOD and FAD irreversibly adsorbed onto the anodized electrodes to form very stable modified surfaces. Direct electron-transfer reaction takes place between the adsorbed GOD and the anodized electrodes, but the adsorbed enzyme cannot catalyze its substrate to oxidize due to its denaturation. Two possible denatured processes have been proposed. Based on the analyses of scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS) and scanning tunneling microscope (STM), it is confirmed that the adsorbed GOD molecules extend to the unfolded structure owing to strong interaction between the enzyme and the electrode surface, but the FAD redox centers of the enzyme do not divorce from the apoenzyme.

ELECTROCATALYTIC OXIDATION OF REDUCED NICOTINAMIDE COENZYMES AT METHYLENE GREEN-MODIFIED ELECTRODES AND FABRICATION OF AMPEROMETRIC ALCOHOL BIOSENSORS

Abstract Chemically modified electrodes with Methylene Green adsorbed on the graphite surface and incorporated into carbon paste exhibit excellent electrocatalytic ability for oxidation of NADH. Alcohol dehydrogenase, nicotinamide adenine dinucleotide (NAD + ) and mediator were incorporated into a carbon paste matrix to yield an alcohol sensor. The enzyme and cofactor retain their bioactivity within the matrix. The surface of the sensor is protected by coverage with a poly(ester sulphonic acid) cation exchanger to form a membrane, which effectively prevents the aqueous soluble species from dissolving out from the enzyme electrode and reduces or eliminates the interference from electroactive anions. The oxidation current of the NADH formed by enzymatic reaction serves as the response for target analytes. The reagentless sensor shows steady-state signals within 50 s, owing to the intimate contact between the biocatalytic and sensing sites. The influence of various experimental conditions was explored for optimum analytical performance. The sensor remained relatively stable for about 15 days.

Flow-injection analysis of glucose at an amperometric glucose sensor based on electrochemical deposition of palladium and glucose oxidase on a glassy carbon electrode

A glassy carbon electrode (GCE) modified with palladium provides excellent electrocatalytic oxidation of hydrogen peroxide. When the electrolyte contains palladium chloride and glucose oxidase, the GCE can be modified by electrochemical codeposition at a given potential. The resulting modified surface was coated with a thin film of Nation to form a glucose sensor. Such a glucose sensor was successfully used in the flow-injection analysis of glucose with high stability and anti-poisoning ability. It gave a detection limit of 1 X 10(-7) M injected glucose, with a linear concentration range of 0.001-8 mM. There is no obvious interference from substances such as ascorbate and saccharides.

Electrochemistry of Self-Assembled Monolayers of the Blue Copper Protein Pseudomonas Aeruginosa Azurin on Au(111)

Abstract We report the self-assembly and electrochemical behaviour of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) electrodes in aqueous acetate buffer (pH=4.6). The formation of monolayers of this protein is substantiated by electrochemical measurements. Capacitance results indicate qualitatively that the protein is strongly adsorbed at sub-μM concentrations in a broad potential range (about 700 mV). This is further supported by the attenuation of a characteristic cyclic voltammetric peak of Au(111) in acetate solution with increasing azurin concentration. Reductive desorption is clearly disclosed in NaOH solution (pH=13), strongly suggesting that azurin is adsorbed via its disulphide group. An anodic peak and a cathodic peak associated with the copper centre of azurin are finally observed in the differential pulse voltammograms. These peaks are, interestingly, indicative of long-range electrochemical electron transfer such as paralleled by intramolecular electron transfer between the disulphide anion radical and the copper atom in homogeneous solution, and anticipated by theoretical frames. Together with reported in situ scanning tunnelling microscopy (STM) results they constitute the first case for electrochemistry of self-assembled monolayers of azurin, even redox proteins. This integrated investigation provides a new approach to both structure and function of adsorbed redox metalloproteins at the molecular level.

In situ electrochemical scanning tunnelling microscopy investigation of structure for horseradish peroxidase and its electricatalytic property

Immobilization of protein molecules is a fundamental problem for scanning tunnelling microscopy (STM) measurements with high resolution. In this paper, an electrochemical method has been proved to be an effective way to fix native horseradish peroxidase (HRP) as well as inactivated HRP from electrolyte onto a highly oriented pyrolytic graphite (HOPG) surface. This preparation is suitable for both ex situ and in situ electrochemical STM (ECSTM) measurements. In situ STM has been successfully employed to observe totally different structures of HRP in three typical cases: (1) in situ ECSTM reveals an oval-shaped pattern for a single molecule in neutral buffer solution, which is in good agreement with the dimension determined as 6.2 x 4.3 x 1.2. nm(3) by ex situ STM for native HRP; (2) in situ ECSTM shows that the adsorbed HRP molecules on HOPG in a denatured environment exhibit swelling globes at the beginning and then change into a V-shaped pattern after 30 min; (3) in situ ECSTM reveals a black hole in every ellipsoidal sphere for inactivated HRP in strong alkali solution. The cyclic voltammetry results indicate that the adsorbed native HRP can directly catalyse the reduction of hydrogen peroxide, demonstrating that a direct electron transfer reduction occurred between the enzyme and HOPG electrode, whereas the corresponding cyclic voltammograms for denatured HRP and inactivated HRP adsorbed on HOPG electrodes indicate a lack of ability to catalyse H2O2 reduction, which confirms that the HRP molecules lost their biological activity. Obviously, electrochemical results powerfully support in situ STM observations.

Single-Molecule Mapping of Long-range Electron Transport for a Cytochrome b562 Variant

Cytochrome b(562) was engineered to introduce a cysteine residue at a surface-exposed position to facilitate direct self-assembly on a Au(111) surface. The confined protein exhibited reversible and fast electron exchange with a gold substrate over a distance of 20 Å between the heme redox center and the gold surface, a clear indication that a long-range electron-transfer pathway is established. Electrochemical scanning tunneling microscopy was used to map electron transport features of the protein at the single-molecule level. Tunneling resonance was directly imaged and apparent molecular conductance was measured, which both show strong redox-gated effects. This study has addressed the first case of heme proteins and offered new perspectives in single-molecule bioelectronics.

Long-range interfacial electron transfer of metalloproteins based on molecular wiring assemblies

We address some physical features associated with long-range interfacial electron transfer (ET) of metalloproteins in both electrochemical and electrochemical scanning tunneling microscopy (ECSTM) configurations, which offer a brief foundation for understanding of the ET mechanisms. These features are illustrated experimentally by new developments of two systems with the blue copper protein azurin and enzyme nitrite reductase as model metalloproteins. Azurin and nitrite reductase were assembled on Au(111) surfaces by molecular wiring to establish effective electronic coupling between the redox centers in the proteins and the electrode surface for ET and biological electrocatalysis. With such assemblies, interfacial ET proceeds through chemically defined and well oriented sites and parallels biological ET. In the case of azurin, the ET properties can be characterized comprehensively and even down to the single-molecule level with direct observation of redox-gated electron tunnelling resonance. Molecular wiring using a pi-conjugated thiol is suitable for assembling monolayers of the enzyme with catalytic activity well-retained. The catalytic mechanism involves multiple-ET steps including both intramolecular and interfacial processes. Interestingly, ET appears to exhibit a substrate-gated pattern observed preliminarily in both voltammetry and ECSTM.

Spontaneous and Fast Growth of Large-Area Graphene Nanofilms Facilitated by Oil/Water Interfaces

An efficient wet-chemical method based on soft interfacial self-assembly is developed for spontaneous, fast growth of large-area graphene nanofilms on various substrates. The graphene nanofilms produced show tunable optical properties and a highly reversible optoelectronic response. Complementary to chemical vapor deposition, this method could offer a fast, simple, and low-cost chemical strategy to produce graphene nanofilms.

Direct observation of native and unfolded glucose oxidase structures by scanning tunnelling microscopy

Native and unfolded glucose oxidase (GOD) structures have been directly observed with scanning tunnelling microscopy (STM) for the first time. STM images show an opening butterfly-shaped pattern for the native GOD. When GOD molecules are extended on anodized, highly ordered pyrolytic graphite (HOPG), a helical structure composed of double-stranded chains was obtained under STM. These results are in good agreement with previous description of the GOD molecular structure. A simple model of the unfolding process for GOD molecules was proposed to explain these observations. Electrochemical evidence was provided to support the results obtained with STM and the proposed model.