Anal. Chem. 1990, 62,1111-'1117
1111
Electropolymerized 1,2=Diaminobenzeneas a Means To Prevent Interferences and Fouling and To Stabilize Immobilized Enzyme in Electrochemical Biosensors Sylvia V. Sasso,' Raymond J. Pierce,I Robert Walla, and Alexander M. Yacynych* Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903
A platlnlzed, reticulated vltreous carbon (RVC) electrode, bnmoblRzed wtth glucose oxldase and electropdymerized with 1,2-dlamlnobenzene, Is used In the construction of a sensor for the determlnatlon of glucose In human serum. A flow injection analysls (PIA) system was used for the determinations. The platinum coating provldes an Increased current response from the oxldatlon of hydrogen peroxide, as compared with a bare RVC electrode. The 1,2-dlamlnobenzene polymer coating drastlcaily reduces the effects of eiectrochemkally actlve interferents, such as L-ascorblc acid, uric acld, and L-cystehe, and vhtuaHy eliminates electrode foullng by proteins in blood serum. There Is no fouling of the sensor after 60 repetitlve InJectlons of serum. The polymer film also increases the thermal stablllty of the Immobilized glucose oxldase. The enzyme remains actlve over one and one-hatl months of dally use, and the 1,2dlamlnobenrene polymer fllm can be redeposited, as needed, durlng the working Metime of the electrode. Thermal studles were done on hnmoblllzed enzyme electrodes using carbodllmlde as a covalent llnklng agent or glutaraldehyde as a cross-linking agent. Results oMalned wtth thk bbsensor showed exceaent correlation with Natlonal Institute for Standards and Technology serum Samples.
INTRODUCTION Two major problems facing electrochemical biosensors are interferences and electrode fouling. Interferences result from species, other than the analyte, present in complex matrices, such as blood serum and fermentation broths. Electrode fouling is caused by the adsorption of high molecular weight species, such as proteins, on the electrode surface, thereby decreasing the electrode response. Glucose oxidase is highly specific for glucose; however, the electrode compromises this specificity by oxidizing any species present in solution that is oxidizableat the applied potential. This results in significant interference with the hydrogen peroxide signal. Several attempts have been made to circumvent this problem. Masoom and Townshend ( I ) have incorporated a dialyzer or a column of copper(I1) diethyldithiocarbamate on controlled porosity glass in front of the enzyme column. Yao et al. (2) place an electrolytic column before the electrode. Conventional enzyme electrodes employ discrete macroscopic membranes to overcome problems associated with interferences, enzyme immobilization, and electrode fouling. While these systems have been commercially developed,there are some limitations with this approach. Three relatively thick membranes are often used, resulting in a slow and complex
* Author to whom correspondence should be addressed. 'Present address: I-Stat Corp., 303 College Farm Rd. East, Princeton, NJ 08540. 0003-2700/90/0362-1111$02.50/0
diffusion path for reactants reaching the enzyme and hydrogen peroxide reaching the electrode. Slow diffusion in this system adversely affects the response time and sampling rate. This construction technique is limited to two-dimensional surfaces and each sensor must be individually constructed. In addition, for sensors that have complex and slow diffusion paths, rates of diffusion must remain constant, otherwise calibration of the biosensor, and more important the maintenance of calibration, are difficult. A variety of factors can influence rates of diffusion and, consequently, the performance of the enzyme layer and the performance of the sensor. These complicated and most often uncharacterizable properties have made the development of most biosensors difficult. In this work, the use of an electropolymerized film of 1,2diaminobenzene has solved the problems with interferents and electrode fouling, while minimizing diffusion. Polymer-filmmodified electrodes are used for a variety of applications, such as electrocatalysis (3-6) and analysis (7,8), and more recently for their permselectivity characteristics (9-18). These polymer films are formed by casting the film on an electrode surface (12),using radio frequency plasma (19),or by electropolymerization (17,20,21).Alternatively, a polymer film is formed as a discrete membrane and subsequently applied to an electrode (22). The uses of both cast and discrete membrane films are essentially limited to two-dimensional electrode surfaces, as it is nearly impossible to control the reproducibility, uniformity, and thickness of the polymer film on an intricately complex surface, such as reticulated vitreous carbon (RVC), which is useful as a flow-through electrode (23,24). With electropolymerizationit is much easier to control the parameters of film thickness, homogeneity, and reproducibility. Dubois et al. (25,26)have showed that the electrochemical oxidation of phenol and its derivatives, on metal surfaces, produced hydrophobic, adherent, and insulating polymer f i i of uniform thickness. Both Yacynych and Mark (21) and Heineman et al. (17) showed the oxidation of 1,2diaminobenzene to be irreversible and, with successive cyclic voltammetric scans, formed an insulating polymer film completely covering the electrode surface. Heineman et al. (17) further showed that 1,2-diaminobenzene forms a polymeric film over a pH range of 4-10, and that platinum electrodes coated with the poly(l,2-diaminobenzene)provided a nearly Nernstian response to pH. Cheek et al. (20) studied the pH response of platinum and vitreous carbon with polymer films of either 1,2-diaminobenzeneor phenol. These polymer films are selective enough to allow the permeation of protons, while limiting access to larger molecules, which could be potential interferents. Oyama et al. (9-1 1) studied electropolymerized films for their permselectivity and ion-exchange properties. Electropolymerization of 1,Qdiaminobenzene offers the advantages of producing a very thin (
