Andrey L. Ghindilis

Immunosensors: electrochemical sensing and other engineering approaches.

This article overviews the engineering approaches and the recent trends in the development of alternative immunoassay systems. A brief description of the main principles and limitations of conventional immunoassay is given. Immunosensing approaches overcoming these limitations are discussed. Alternatives to traditional immunoassay systems are discussed in terms of the enhancement of immunointeraction processes and in terms of the various detection principles. Applications of flow-injection techniques to the development of immunosensing systems are presented. Immunosensors are categorized based on the detection principle employed, as immunoelectrodes (electrochemical immunosensors), piezoelectric immunosensors, or as sensors based on optical detection of the immunointeraction. The discussion focuses on electrochemical immunosensors. In conclusion, the engineering issues involved in immunosensor development are outlined and trends towards practical applications are discussed.

Implantation of a refillable glucose monitoring-telemetry device☆

This study describes the components and short-term in vivo evaluation of an integrated implantable system consisting of an amperometric glucose biosensor, a miniature potentiostat, a FM signal transmitter, and a power supply. The device (dimensions: 5.0 x 7.0 x 1.5 cm) was implanted subcutaneously in healthy mongrel dogs. The biosensor performance was evaluated in vitro prior to implantation using standard solutions simulating the physiological environment. A linear response to glucose concentration was observed throughout the physiological and pathophysiological range (with an upper limit of 25 mM glucose, and a sensitivity of 0.5 microA/mM). The results of short-term subcutaneous implantation of the integrated system demonstrated good agreement between the glucose concentration measured by the biosensor and that obtained using standard glucose determination methods. The delay-time between the tissue glucose level (measured by the biosensor) and the blood glucose level (obtained by standard methodology) was 3-7 min. These results demonstrated the feasibility of data transmission by a telemetry system through the skin of a dog and allowed the commencement of chronic in vivo testing. During the chronic implantation the biosensor was refilled in vivo. A rejuvenation of the sensors response after refilling was observed suggesting the potential of such sensors for long-term implantation.

Potentiometric biosensors for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis.

A potentiometric method for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis has been developed. The method includes coimmobilization of three enzymes, butyrylcholinesterase, choline oxidase and peroxidase, on composite carbon electrodes. Catalytic hydrolysis of butyrylcholine and subsequent catalytic oxidation of choline result in the formation of hydrogen peroxide leads to a shift in the electrode potential. The detection limit for trichlorfon analysis is 2 x 10(-13) M. Electrodes remain stable for at least 4 weeks when stored at 277 K.

Direct electron transfer based tri-enzyme electrode for monitoring of organophosphorus pesticides

Abstract A potentiometric biosensor has been developed based on the ability of organophosphorus pesticides to inhibit the catalytic activity of the enzyme choline esterase. The detection of choline esterase activity is based on the principle of molecular transduction. Immobilized peroxidase acting as the molecular transducer, catalyzes the electroreduction of hydrogen peroxide by direct (mediatorless) electron transfer. The sensing element consists of a carbon based electrode covered by a layer of three co-immobilized enzymes: choline esterase, choline oxidase and peroxidase. A butyryl choline sensitive tri-enzyme has been developed employing highly dispersed teflonized carbon black as an electrode material. The immobilization procedure is based on physical adsorption of peroxidase and co-immobilization of choline oxidase and choline esterase using glutaraldehyde as a binding agent. The electrode retains 95% of its initial activity after 1 month of storage at 4°C. The parameters of the inhibitor assay procedure have been optimized. The procedure for measuring the electrode activity requires 3–5 min. Incubation of the electrode in a solution containing the organophosphorus pesticide, trichlorfon, for 10 min results in a notable decrease of electrode activity. This allows for the determination of trichlorfon in a nanomolar concentration range with a low detection limit of 5 nM (1.3 ppb).

Fast Amperometric Immunoassay Utilizing Highly Dispersed Electrode Material

Abstract An immunoassay technique based on a high surface area carbon immunoelectrode is described. Dispersed ULTI carbon serves as a carrier for immobilized antibodies and also as an electrode material. ‘Sandwich’ scheme of immunoassay of rabbit IgG (as a model analyte) has been used. Iodine formed as a result of the enzymatic oxidation of iodide by peroxidase-label has been detected amperometrically. Using dispersed carbon material as a solid support for immobilization of immunoagents and as an electrode material improves the efficiency of immuno-interaction and the sensitivity of electrochemical assay due to the high area-to-volume ratio of solid to liquid phases. The method based on ‘sandwich’ immunoassay is conducted at 21°C. The time of each stage of incubation (with analyte and with peroxidase-antibody conjugate) is 10 min. Electrochemical detection of peroxidase-labeled immuno-complex does not exceed several minutes. The technique allows fast determination of rabbit IgG with a low detection limit,...

Glucose potentiometric electrodes based on mediatorless bioelectrocatalysis. A new approach

A potentiometric method has been developed for analysis of glucose on the basis of glucose oxidase and peroxidase co-immobilized on the surface of an electrode made of composite carbonic material. Catalytic oxidation of glucose results in the formation of hydrogen peroxide. Mediatorless peroxidase catalysis of electroreduction of hydrogen peroxide leads to a shift in the electrode potential. The rate of the increase of the electrode potential is proportional to glucose concentration and calibration curve is linear at 0.025-2.0 mM. Electrodes permit at least 100 measurements without any loss in the activity. Electrodes remain stable for 90 days when stored at 277 K.

Potentiometric immunoelectrode for fast assay based on direct electron transfer catalyzed by peroxidase

Abstract A potentiometric immunoelectrode based on the direct electron transfer detection of immunointeraction is described. Enzyme peroxidase is used as a label. The ability of peroxidase to catalyze the electrode reaction of hydrogen peroxide electroreduction by a direct (mediatorless) mechanism is utilized. Peroxidase attached to the electrode surface in the presence of hydrogen peroxide results in a significant (hundreds of mV) increase of potential towards the equilibrium H 2 O 2 /H 2 O potential due to catalytic removal of overvoltage. Electrons are transferred directly from the electrode to the substrate molecules via the active site of the enzyme. Peroxidase labeled immunoconjugate interacting with the surface of a carbon electrode modified by antigen results in the increase of electrode potential. The rate of potential increase is proportional to the concentration of free conjugate in the solution. Free antigen in the reaction media inhibits the process of conjugate binding with immobilized antigen, resulting in a decrease in the rate of change of electrode potential. The percentage of inhibition is proportional to the concentration of free antigen (analyte). Rabbit IgG has been used as a model analyte. The method allows determination of IgG in the concentration range up to 400 μg/ml. Sensing of the immunointeraction is in a kinetic mode. The direct electron transfer detection of immunointeraction suggests a single stage analysis scheme. An analysis time does not exceed 25 min. Different kinds of carbon materials for electrode construction have been examined.

Flow-cell fibre-optic enzyme sensor for phenols

Abstract A solid-state fibre-optic luminescent oxygen sensor was used for flow-through measurements. It acts as a transducer in a new flow-cell enzyme sensor arrangement. This arrangement comprises a flow path, sample injector, microcolumn with the immobilized enzyme, oxygen membrane and fibre-optic connector joined together to form an integral unit. Laccase enzyme was used as a recognition system which provided specific oxidation of the substrates with the dissolved oxygen being monitored. The assay procedure was optimized and performance of the new system studied. The sensor was applied to the determination polyphenol content in tea, brandy, etc. (quality control test). The sensitivity to some important phenolic compounds was tested with the view of industrial wastewater control applications.

Development of an amperometric immunoassay based on an highly dispersed immunoelectrode

Abstract An amperometric immunoassay technique utilizing a highly dispersed immunoelectrode was developed. The assay method is based on the ‘sandwich’ scheme of immunointeraction. Rabbit IgG has been used as model analyte. Horseradish peroxidase labelled conjugates were employed coupled with amperometric detection of iodine reduction. Highly dispersed ULTI carbon serves as an immunosorbent and at the same time as an electrode material. It was found that the dependence of immunoelectrode response on the time of incubation during the second stage of ‘sandwich’ -based assay was complex. This is probably due to non-equilibrium immuno-agglomeration of sub-micron sized particles of immunosorbent. The improvement in the sensitivity of immunoelectrode by increasing the amount of immunosorbent was demonstrated. The increase in the magnitude of the immunoelectrode response is proportional to the increase of the amount of immunosorbent. A detection limit of 10 pM has been achieved with an overall assay time of less ...

Bifunctional hydrogen peroxide electrode as an amperometric transducer for biosensors

Abstract This work presents a novel approach to design an amperometric transducer capable to detect hydrogen peroxide in anodic (oxidation) and cathodic (reduction) mode of operation. The basic transducer employs non-platinum electrocatalyst—pyrolyzed cobalt–tetramethoxy–phenylporphyrin (CoTMPP)—for H 2 O 2 electrooxidation, and bio-electrocatalyst—horseradish peroxidase (HRP)—for H 2 O 2 electroreduction. The bifunctional hydrogen peroxide electrode was used as an amperometric transducer in designing a glucose biosensor utilizing glucose oxidase (GOD) as second enzyme. A technique for preparation of the transducer electrode with the catalyst and adsorbed enzymes (bienzyme electrode) from a pressed matrix (tablet) of Teflonized carbon black is described. This technique allows manufacturing of numerous sensors from the same pressed tablet, all of which demonstrate good reproducibility of their parameters (sensitivity, linear range, lifetime). Bifunctional glucose sensors were operated in H 2 O 2 oxidation mode at applied potentials from +0.2 to +0.6 V versus Ag/AgCl, and in H 2 O 2 reduction mode at 0.0 V versus the same reference electrode. Linearity of the sensor response to glucose concentrations up to 25 mM in H 2 O 2 reduction mode is reported. Electro-interference from some substances commonly present in physiological fluids was investigated in both reduction and oxidation modes. Sensors can be used for continuous glucose monitoring in vitro for at least 1 month with constant parameters of the signal output. Sensor demonstrated satisfactory performances in undiluted blood plasma in vitro.