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A combined, optospectroscopic and electrochemical assay system for the simultaneous monitoring of intra- and extracellular production of biologically important species has been developed and assessed. The present model system evaluates intra- and extracellular nitric oxide produced by stimulated glioblastoma multiform cell line (A172). The production of endogenous NO was induced by phorbol-12-myristate-13-acetate and inhibited by [N.sup.[omega]]-nitro-L-arginine methyl ester. Intracellular production of NO was monitored via fluorescence image analysis using a 4,5-diaminofluorescein probe, while extracellular NO release was monitored via a chemically modified electrode, which was incorporated into an optically transparent cell chip. The results indicated that there was no mutual interference between the optical and electrochemical measurement systems. The response time of the combined optical/electrochemical system was found to be in the range of a few tens of seconds.

This work reports on the successful integration of a one-step lateral flow immunoassay format and impedance detection of the specific affinity event using an electrochemical transducer coated with a pH-sensitive polymer layer. This approach was particularly applied to the development of a rapid single-use immunosensor for the sensitive detection of free and total prostate-specific antigen (f-PSA, t-PSA) tumor marker. Strips of nitrocellulose membrane were coated with appropriate antibodies to f-PSA and t-PSA and used as solid supports for the performance of noncompetitive immunoassays where PSA was allowed to react with both immobilized anti-PSA antibody and anti-PSA urease enzyme conjugate for less than 1 min. An additional piece within the device consisting of a storage blister filled with a urea solution allowed the rapid washing of unbound species from the membrane strips and simultaneous urea hydrolysis catalyzed by the bound urease conjugate in an automatic fashion. The hydrolysis of urea increased the pH of the reaction media, which in turn induced a breakdown of the polymer layer on the transducer and a consequent measurable change in capacitance of the system. This was easily recorded at a given frequency over a 30-min period. Overall, we describe a one-step immunosenser prototype that exhibits enough sensitivity to detect both forms of PSA at concentration levels down to 3 ng/mL. With the possibility of being portable and considering its ease of use, robustness, and simplicity, this device has great potential as a tool for the screening and early detection of prostate cancer.

Enzyme-catalyzed polymer transformation with electrochemical ac impedance detection has been employed for the measurement of urea and creatinine in serum samples. A polymer, based on poly(methylvinyl ether)/maleic anhydride modified by esterification with n-octanol, which is stable at pH 7.4 and which is transformed rapidly in response to alkaline pH changes, was linked to enzymatic reactions between urease and urea or creatinine deiminase and creatinine to produce a disposable sensor system. The polymer was screen-printed onto interdigitated screen-printed carbon electrodes and the electrodes overlaid with absorbent pads containing the relevant enzyme. Application of serum samples, 'spiked' with either urea or creatinine, resulted in rapid polymer transformation, and resultant changes in the capacitance of the polymer-coated electrodes were analyte-concentration dependent. Additional information on the mechanisms of polymer transformation was obtained from dynamic quartz crystal microbalance measurements.

A novel sensor approach based on ac impedance measurement of capacitance changes produced during enzyme-catalyzed dissolution of polymer coatings on electrodes, leading to a 4 orders of magnitude change in capacitance, is described. Electrodes were coated with an enteric polymer material, Eudragit S 100, which is based on methyl methacrylate, and dissolution was exemplified by utilizing the catalytic action of the enzyme urease. The resulting alkaline pH change caused dissolution of the polymer film with a consequent large increase in capacitance. A mechanism for polymer breakdown is proposed which has been validated experimentally using both ac impedance measurements and electron microscopy. The large changes in capacitance that are apparent using this technique allow much greater sensitivity of measurement than, for example, potentiometric electrodes. The potential broad clinical analytical application of this technique is demonstrated in this report by application to urea measurement and to enzyme immunoassay. Urea measurement between 2 and 100 mM has been achieved with a change in response over this concentration range by over 4 orders of magnitude. We have taken account of both the effect of protein adsorption on the surface of the polymer-coated and bare electrodes and the effect of buffer capacity when carrying out these measurements in buffered solutions containing 8% (w/v) protein and have demonstrated that the method should allow simple, interference-free measurement of urea in serum and whole blood. In addition, both competitive and noncompetitive enzyme immunoassays for human IgG based on the use of urease-antibody conjugates are reported. Human IgG, or goat anti-human IgG (Fab specific), were immobilized covalently onto cellulosic membranes via a diamine spacer group and the membranes placed over enteric polymer-coated electrodes. Specific measurement of IgG in both formats was achieved over the concentration range 0.0001-100 [[micro]gram] [[[micro]molar].sup.-1]. The performances of the impedance-based enzyme immunoassays were compared directly with identical assays employing spectrophotometric detection.