RESEARCH ARTICLE N.W. Beyene, H. Moderegger and K. Kalcher, 1 S. Afr. J. Chem., 2004, 57, 1–7, <http://journals.sabinet.co.za/sajchem/>. Simple and Effective Procedure for Immobilization of Oxidases onto MnO2-bulk-modified, Screen-printed Carbon Electrodes Negussie W. Beyene*, Helmut Moderegger and Kurt Kalcher Institute of Chemistry-Analytical Chemistry, Karl-Franzens University of Graz, A-8010 Graz, Austria. Received 25 June 2003; revised 7 September 2003; accepted 20 January 2004. ABSTRACT ® Nafion film entrapment of oxidases onto the surface of MnO2-(5%, m:m) bulk-modified, screen-printed carbon electrodes was studied taking the inexpensive glucose oxidase as a model enzyme. The Nafion®-enzyme film was formed by drop-coating the suspension onto the surface of the electrode. Comparisons were made among biosensors developed by using as-received, neutral- ized, and diluted (to 1% in methanol, ethanol or phosphate buffer) Nafion® in a flow injection (FI) mode. The amperometric re- sponse to injections of standard glucose solution was the highest for biosensors produced using neutralized Nafion®. The effect of enzyme load per electrode (10–1190 µg) was investigated and 50 µg enzyme in the film per electrode was found to produce very good sensitivity as well as to be economical. Good reproducibility was achieved for three different biosensors (RSD of the means = 5.7%). The operational parameters for the glucose biosensor produced with this immobilization procedure were as- sessed and figures of merit obtained at the selected parameters. It has been observed that the effect of Nafion® to repel interfering ions like ascorbate and urate was lacking in this system. A preliminary investigation incorporating a hydrogen peroxide permselective membrane layer prior to enzyme immobilization gave promising result with polyurethane. KEYWORDS Amperometric glucose biosensor, flow injection analysis, Nafion® film entrapment, immobilization, screen-printed electrodes, MnO2-modified electrodes. 1. Introduction hence the name chemically modified electrodes was coined.5–8 Solid electrodes based on heterogeneous carbon materials are Heterogeneous carbon electrodes were among the first to be used more frequently as electrochemical sensors and biosensors chemically modified. because of their availability in a variety of forms, low cost, broad The advantages of modified electrodes are reducing the exploitable potential window, low background current, chemi- overvoltage and hence the interference from other species, cal inertness, ease of chemical derivatization and modification, promoting electron transfer reactions that may increase the and suitability for various applications.1,2 Various carbon-based selectivity, specificity and reproducibility of the electrode electrodes have been designed from glassy carbon, reticulated surface, and improving the detection limit.1–8 vitreous carbon, graphite epoxy composites, pyrolytic graphite, Modified electrodes can be prepared by chemisorption, carbon pastes, carbon inks and carbon fibres. Among these covalent bond formation between specific functional group on materials, carbon paste electrodes (CPEs) and screen-printed the electrode surface and the reagent, coating the electrode carbon electrodes (SPCEs) have gained widespread popularity with polymeric films (including electropolymerization of in the development of electrochemical sensors and biosensors monomers), and forming heterogeneous layers (e.g. thorough owing to their ease of preparation and modification, ease of mixing of the modifier with carbon paste or carbon ink).1,2 surface renewal and reproducibility in case of CPEs, and mass- Among these, the simplest, most reliable and frequently used production of highly reproducible electrodes for SPCEs.2–4 modification technique is admixing the modifier with conduct- In electroanalysis employing amperometric techniques there ing pastes and inks prior to putting the electrode in the desired is an inherent problem of the dependence of the electrochemical geometry. In this context, numerous transition metal species reaction on the type of electrode. Many electroactive species (oxides and complexes) have been used as modifiers. Ferrocene undergo redox reactions at a slower rate than expected, requir- derivatives, organometallics like ruthenium and osmium ing application of a higher potential. But at this over-potential, complexes, hexacyanoferrate, Methylene Blue and Methyl other species present in the sample may interfere, affecting the Viologen are most often employed.8 A very recent example is the analytical reaction significantly. Sometimes, thermodynamically use of manganese dioxide. MnO2 has been employed to modify possible redox reactions may not be observed due to electrode CPEs and SPCEs in the production of sensors and biosensors for fouling caused by adsorption or by chemical reactions involving different analytes. Thus, the development and characterization the electrode surface. These phenomena can be controlled by of MnO2 film/bulk-modified carbon paste and screen-printed deliberately attaching chemical reagents to the electrode surface 9–12 13 14 amperometric sensors for H2O2, uric acid and ascorbic acid so that manipulating the nature of the surface is possible and have been successfully achieved. Moreover, glucose biosensors * To whom correspondence should be addressed. Present address: Department of have been designed based on CPEs bulk-modified with MnO2 Chemistry, University of Pretoria, 002 Pretoria, South Africa; 15,16 E-mail: [email protected] and glucose oxidase (GOx) as well as based on screen-printed RESEARCH ARTICLE N.W. Beyene, H. Moderegger and K. Kalcher, 2 S. Afr. J. Chem., 2004, 57, 1–7, <http://journals.sabinet.co.za/sajchem/>. Scheme 1 16,17 Catalytic redox cycle of manganese dioxide and chemical oxidation of H2O2. amperometric sensors.17,18 At an operational potential of 400– in the working buffer solution and stored at 4°C when not in use. 500 mV vs Ag/AgCl heterogeneous carbon electrodes bulk-modi- Solutions of lower concentrations were prepared immediately fied with 3.8–5% MnO2 responded to H2O2 (either directly before use. By the same token, stock solutions (4395 and 5000 mg present in the sample or as a product of the enzymatic oxida- L–1 respectively) of uric acid (sodium salt) and ascorbic acid tion). The tetravalent manganese is reduced to lower oxidation were prepared by dissolving 0.5 g of each in 100 mL of the states by hydrogen peroxide and is reoxidized again electro- corresponding working buffer solution. Solutions of lower chemically (Scheme 1). concentrations were prepared immediately before use. In the aforementioned works that deal with biosensors based on MnO2-modified electrodes, the biological recognition 2.3. Electrode Preparation element has been introduced in the bulk of the electrodes. The same electrode preparation protocol reported elsewhere However, introducing expensive enzymes such as L-glutamate was employed.21 oxidase and L-amino acid oxidase in the bulk is not economically feasible since relatively large quantities of these enzymes are 2.4. Enzyme Immobilization required. To alleviate this problem, Nafion® film entrapment of Nafion® (5% solution) was diluted to 1% either in phosphate –1 oxidases onto the surface of MnO2- (5%, m:m) bulk-modified buffer (0.1 mol L , pH 7.0), absolute ethanol, or absolute metha- SPCEs was studied using the inexpensive glucose oxidase as a nol. It was also neutralized by ammonia solution or used as- model enzyme. received. Adding a drop of ammonia solution to 1.5 mL Nafion® solution effected the neutralization and the pH adjustment was 2. Experimental monitored by using litmus paper (Macherey-Nagel, Dueren, Germany). A weighed amount (in mg) of GOx was dissolved in 2.1. Materials phosphate buffer (0.1 mol L–1, pH 7.0) and mixed with an equal Glucose oxidase, GOx (EC 1.1.3.4 from Aspergillus niger, 210 amount (as the phosphate buffer) of Nafion® (diluted, neutralized U/mg solid) and D-glucose, anhydrous, were obtained from or as-received) solution. Five µL (unless otherwise specified) of Sigma Chemicals Co., (St. Louis, MO, USA). Nafion®, the resulting mixture were directly applied onto the active area 2 perfluorinated ion-exchange resin, 5% (w/w) solution in lower of the SPCE (MnO2-bulk-modified) surface (~0.40 cm area), air- aliphatic alcohols and water was from Aldrich (Aldrich-Chemie dried, another 5 µL aliquot applied, air-dried and inserted in GmbH & Co KG, Steinheim, Germany). Polyurethane was the FIA system. The final enzyme load onto the electrode was synthesized according to Yun et al.19 from poly-(tetramethylene estimated from the starting amount, assuming complete dissolu- ether glycol) and 4,4’-methylenebis(phenyl isocyanate). All tion. No attempt was made to measure the enzyme activity other chemicals used were analytical reagent grade. retained after immobilization. The final concentration of Nafion® applied to the electrode was 0.5% in all diluted cases but 2.2. Reagents and Solutions 2.5% in the neutralized and as-received applications. The water used was double-distilled in a quartz still and deionized with an ion exchange System (Nanopure, Barnstead). 2.5. Flow Injection System Phosphate buffer (0.1 mol L–1) was prepared by mixing aqueous The flow injection system employed was basically the same as solutions (0.1 mol L–1) of sodium di-hydrogen phosphate (Fluka) reported earlier.21 and di-sodium hydrogen phosphate (Fluka) to produce solutions of the required pH. A stock solution