Immunoassays based on electrochemical detection using microelectrode arrays.

We show that CombiMatrix's VLSI arrays of individually addressable electrodes, using conventional CMOS integrated circuitry, can be used in detecting various analytes via immunoassay protocols. These microarrays provide over 1000 electrodes per square centimeter. The chips are coated with a porous material on which specific affinity tags are synthesized proximate to selected electrode sites. CombiMatrix microarrays are used to develop spatially multiplexed assay formats for biological entities over a wide range of sizes, from small molecules to cells. Antibodies are tagged with coded affinity labels and then allowed to self-assemble on the appropriate electrode assay sites. Each analyte-specific antibody is chaperoned to individual, predetermined locations by the self-assembly process. The resulting chip can perform numerous different analyte-specific immunoassays, simultaneously. We present new detection technologies based upon the use of the active individually addressable microelectrodes on the chip: redox enzyme amplified electrochemical detection. The results for human alpha1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spores, and fluorescein indicate that this method is one of the most sensitive available, with limits of detection in the attomole range. The detection range is 4-5 logs of analyte concentration, with an assay volume of 50 microl or less. The system provides for a host of multiplexed immunoassays because of the large number of electrodes available. We show how the assays can be optimized for maximum performance on the CombiMatrix microarray platform.

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.

Flow-through amperometric immunosensor: fast 'sandwich' scheme immunoassay.

A flow-through immunosensor based on a high-surface-area carbon immunoelectrode has been developed. Dispersed carbon material serves as a carrier for immobilized antibodies and at the same time as an electrode material. The 'sandwich' scheme of immunoassay has been used. Iodine formed as a result of the enzymatic oxidation of iodide by a peroxidase label has been detected amperometrically. The immunosensor consists of a disposable sensing element (immunocolumn) containing dispersed carbon material with immobilized antibodies which also acts as a working electrode. A current collector connects the working electrode to the measuring device. The electrochemical detection time of the peroxidase-labeled immuno-complex does not exceed several minutes. The overall time of analysis including flowing of analyte, flowing of antigen, washing and detection stages is as low as 22 min. This technique allows fast determination of rabbit IgG (used as a model analyte) with a low detection limit in the picomolar range and also the determination of human IgM in blood plasma with a low detection limit in the nanomolar range.

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 sensor's response after refilling was observed suggesting the potential of such sensors for long-term implantation.

Short-term canine implantation of a glucose monitoring-telemetry device.

In this study we report the development and short-term in vivo evaluation of an integrated implantable device consisting of an amperometric glucose biosensor, a miniature potentiostat, a FM signal transmitter, and power supply. The device (dimensions: 5.0 x 7.0 x 1.5 cm) was implanted under the skin of medium-size anaesthetized dog. The experimental set-up included several methods for data collection: analog recording via wired X-T chart recorders; data collection by wearable microprocessor--data logger, and remote data collection via antenna and receiver linked to a computer-based data acquisition system. The device (sensor) performance was evaluated in vitro prior to implantation, using different model solutions simulating the physiological environment. A linear response to glucose concentration was obtained up to 25 mM glucose, with a sensitivity of 0.5 microA/mM. The results of short-term subcutaneous implantation of the integrated device reveal adequate monitoring of an artificially-induced glycaemia. The delay-time was 3-7 minutes. These tests demonstrate the feasibility of data transmission by the telemetry system through the skin of a medium-sized dog and allow the commencement of chronic in vivo experimentation.

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.

A new sensitive and simple method for detection of catecholamines from adrenal chromaffin cells.

A biosensor was used for the analysis of catecholamines in media and lysates of cultured bovine adrenal chromaffin cells. The sensor is composed of coimmobilised laccase and glucose dehydrogenase coupled with an oxygen electrode, using the catalytic effect of cate cholamines for glucose oxidation in this system. The analysis time is almost 5 min. The correlation between the biosensor and HPLC determination is 0.99.

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.

Laccase-based biosensor for determination of polyphenols: determination of catechols in tea.

A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized laccase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practical validity of the method was demonstrated by tannin analysis in tea of different brands.

A new approach to the construction of potentiometric immunosensors.

An electrochemical immunosensor based on a new detection principle was developed. Laccase, which is able to catalyse the electroreduction of oxygen via the direct (mediatorless) mechanism was used as an enzyme label. The new detection method does not require the presence of an electrochemically active mediator, and the reaction substrates are atmospheric oxygen and electrons, the latter being taken by the active site of the enzyme label directly from the electrode. The formation of the complex between laccase-labelled antibody and antigen on the electrode surface resulted in a considerable (more than 300 mV) shift of the electrode potential. The rate of the increase of the electrode potential was inversely proportional to the concentration of the free antigen in the sample. The non-specific adsorption of conjugate and other proteins on the electrode could be eliminated by using a polyethylenimine-based polymer on the electrode surface. Insulin was used as a model analyte. The sensitivity limit for this antigen was approximately 3 micrograms ml-1.

Immunopotentiometric electrodes based on bioelectrocatalysis in the absence of mediators.

A new method of immunoelectrochemical analysis employing laccase as the enzyme label is described. The ability of the enzyme to catalyze electroreduction of oxygen via a direct mechanism allows the detection of the biospecific interaction of a laccase-labeled receptor, or antibody, with a ligand-modified electrode. Formation of a complex between the laccase-labeled antibody and the antigen on the electrode surface resulted in a considerable (greater than 300 mV) change in the electrode potential. Analysis was performed in a competitive scheme, and a single measurement could be made within 20 min in the absence of an electrochemically active mediator. The reaction substrates were atmospheric oxygen and electrons that were transferred directly from the electrode to the active site of the enzyme label. The use of a composite carbon material containing a polyethyleneimine-based polymer eliminated nonspecific interactions between the reaction components and the electrode surface. Insulin and mouse immunoglobulin were used as a model analytes.