Biological detection process using polymer-coated electrodes

#Amperometric methods for the detection and quantification of constituents in biological samples are known. These methods comprise the following steps:

#(1) contacting said constituent with an oxidase enzyme for which the constituent is a substrate, thereby producing a change in the concentration of an electroactive substance in said sample;

#(2) contacting said sample containing said electroactive substance with an inert metal indicator electrode;

#The FIGURE is a side elevational view entirely in section of the flow-through reaction chamber which embodies the second aspect of this invention.

#An inert metal indicator electrode in accordance with the first aspect of the present invention is made by dip coating an inert metal, generally in the form of a thin wire, into a solution of a perfluorosulfonic acid polymer in a lower aliphatic alcohol. Preferred metals are noble metals, particularly platinum and gold, the former being most preferred because of its inherent strength and ability to be soldered directly to electrical contact posts or leads. Platinum wire as thin as 38 gauge has been used successfully. Perfluorosulfonic acid polymers having equivalent weights (ew) of at least 900 can be used but equivalent weights of at least 1100 are preferred. Polymer solutions can be prepared by following the procedure described by Martin et al. in Anal. Chem., Volume 54, 1639 (1982). These workers found that both 50:50 propanol-water and 50:50 ethanol-water, when heated to 250.degree. C. in a high pressure reactor, will dissolve both the 1100 and the 1200 ew Nafion.RTM.. A typical procedure for preparation of a 1% (wt/vol) solution of the polymer is as follows. A sheet of the commercial polymer is placed in a beaker containing the solvent, and this is placed in an ultrasonic cleaner for 1 h. This procedure, while not necessary to the dissolution process, appears to rid the polymer of a suspected impurity in that the commercial polymer has a yellow tinge, but is totally colorless after this treatment. The polymer sheet is then placed in a high pressure reactor with 100 mL of the solvent. The reactor is sealed and purged with nitrogen. A stirrer is turned on and the temperature raised to 250.degree. C. The temperature is held at 250.degree. C. for 1 h, after which the heater is turned off and the reactor is allowed to cool. After cooling, the 1% solution is removed from the reactor. The 1% solution can be concentrated by solvent evaporation. The inert metal is then dip coated in the perfluorosulfonic acid polymer solution. The alcohol is allowed to evaporate after which a powdery coating is left on the inert metal surface. Finally, the powdery coating is sintered by heating to 150.degree. C. in an air oven to produce a smooth polymer coating. This process leaves a thin film of polymer, generally less than 0.001". Films as thick as 0.005" can be used, but thicknesses of less than 0.001" are preferred.

#The electrode made as described above can be used as the indicator electrode in conventional amperometric techniques. A preferred use is in measuring the concentration of a nonelectroactive constituent such as glucose, the reaction of which with glucose oxidase yields hydrogen peroxide and consumes oxygen, both of which are electroactive. Other nonelectroactive constituents and suitable enzymes for their catalysis are disclosed in U.S. Pat. No. 3,539,455 which is incorporated herein by reference.

#In one embodiment, the indicator electrode made as described above, and a counter electrode which is, in general, a nonpolarized, large inert conductive surface, are inserted into a vessel containing the test solution containing the constituent of interest and a nonrate-limiting amount of the oxidase enzyme specific for its catalysis. A preselected potential difference is impressed across the two electrodes. This potential difference and its direction are a function of the electroactive species sought to be measured. The potential difference must be such that it is sufficient to cause an electrochemical reaction at the indicator electrode in a manner which is dependent upon the concentration of the electroactive species generated or consumed by the enzymatic catalysis of the constituent of interest. It is preferable to set the potential difference at a magnitude which avoids interference by nonenzymatically generated electroactive species.