Sensors and Aciuators, El (1990) 537-541 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Polypyrrole, a New Possibility for Covalent Binding of Oxidoreductases to Electrode Surfaces as a Base for Stable Biosensors W. SCHUHMANN, R. LAMMERT, B. UHE and H.-L. SCHMIDT Lehrstuhl fiir Allgemeine Chemie und Biochemie, Technische Universtiit Mtinchen, D-8050 Freising- Weihenstephan (F.R.G.) 537 Abstract Preparation and functionalization of conduct- ing polypyrrole films on different materials provide surfaces suitable to bind enzymes co- valently after carbodiimide activation. The en- zyme electrodes have been tested in comparison to electrodes obtained by binding of oxidases to graphite surfaces. Covalent immobilization of the glucose oxidase directly to the functionalized polypyrrole surface led to biosensors with a short response time, reaching a steady-state current within 30 s in stationary measurements and about 10 s in an automatic flow-injection system. The enzyme electrodes show good stability using oxy- gen as an electron acceptor, and more than 700 glucose measurements are possible with the new polypyrrole-glucose oxidase electrodes in a flow system. Introduction The most well-known biosensor in the control of fermentation processes [I] or in clinical chem- istry [2] is the amperometric glucose sensor. The approach commonly used is the combination of immobilized glucose oxidase, preferably within membranes, in close contact to the working-elec- trode surface. The thickness of the membrane determines, and normally increases, the linear range of the amperometric biosensor, because substrate transport is limited by diffusion pro- cesses [3]. This is also the reason for a consider- ably prolonged response time. The most common electrochemical measuring method, oxidation of enzymatically produced H,O,, demands high elec- trode potentials, which imply interferences by other substances; however, most glucose elec- trodes use this technique [4]. To overcome this problem, the use of artificial electron mediators, especially of ferrocene derivatives [5,6] and or- ganic conducting salts [7], has found increasing application. To construct enzyme electrodes with fast re- sponse, it seems to be important that the electron 09254005/90/$3.50 transfer between the enzyme and the electrode material is facilitated. We have therefore concen- trated on techniques to immobilize glucose oxi- dase covalently to modified electrode surfaces and compared oxidized graphite and functionalized polypyrrole electrodes. Graphite electrodes have been chosen because suitable functional groups can easily be introduced by oxidation [8]. Direct immobilization of the enzyme onto graphite elec- trode surfaces can be achieved by the activation of thus-produced carboxylic groups by means of water-soluble carbodiimide [9]. A new possibility to attain functional&d surfaces was seen on the basis of the conducting polymer polypyrrole [lo]. Recently, entrapment of glucose oxidase in con- ducting polypyrrole films has been described [ ll- 141; the immobilization of the enzyme is probably realized by electrostatic forces or by physical enclosure. Covalent binding of enzymes to poly- pyrrole in the /?-position of the heterocycles is first reported in this work. We want to evaluate the possibility of the immobilization of glucose oxi- dase to functionalized polypyrrole electrodes in order to develop amperometric biosensors with short response time. Experimental Reagents Glucose oxidase (EC 1.1.3.4, type 2, from As- pergih niger), supplied by Biihringer Mannheim GmbH (F.R.G.), had an activity of 275 U/mg. I,1 -Dimethylferrocene was from Aldrich Chemi- cal Co., Steinheim (F.R.G.); pyrrole and tetra- butylammonium p-toluenesulfonate (TBAPTS) were from Fluka Feinchemikalien GmbH, Neu- Ulm (F.R.G.). Pyrrole was distilled prior to use over a short Vigreux column and stored in the dark under Ar. TBAPTS was dried at 100 “C in uacuo and stored under Ar. Acetonitrile was dried over P,OlO, distilled, chromatographed over an Al,O, column, and stored under Ar. I-Cyclo- hexyl-3-( 2-morpholinoethyl) carbodiimide-metho- p-toluenesulfonate (CCD) from Sigma Chemical Co., Deisenhofen (F.R.G.), was used as supplied. 0 Elsevier Sequoia/Printed in The Netherlands