Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols Seung Cheol Chang a , Keith Rawson b , Calum J. McNeil a, * a School of Clinical and Laboratory Sciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK b Cambridge Life Sciences plc, Cambridgeshire Business Park, Angel Drove, Ely CB7 4DT, UK Received 17 October 2001; accepted 13 March 2002 Abstract A new disposable amperometric bi-enzyme sensor system for detecting phenols has been developed. The phenol sensor developed uses horseradish peroxidase modified screen-printed carbon electrodes (HRP-SPCEs) coupled with immobilized tyrosinase prepared using poly(carbamoylsulfonate) (PCS) hydrogels or a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) matrix. Optimization of the experimental parameters has been performed with regard to buffer composition, pH, operating potential and storage stability. A co-operative reaction involving tyrosinase and HRP occurs at a potential of  /50 mV versus Ag/AgCl without the requirement for addition of extraneous H 2 O 2 , thus, resulting in a very simple and efficient system. Comparison of the electrode responses with the 4-aminoantipyrine standard method for phenol sample analysis indicated the feasibility of the disposable sensor system for sensitive ‘in-field’ determination of phenols. The most sensitive system was the tyrosinase immobilized HRP-SPCE using PCS, which displayed detection limits for phenolic compounds in the lower nanomolar range e.g. 2.5 nM phenol, 10 nM catechol and 5 nM p -cresol. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Disposable amperometric sensor; Horseradish peroxidase (HRP); Tyrosinase; Screen-printed electrode; Phenol detection 1. Introduction Phenols are among the top 50 chemicals produced. Although they may have hazardous adverse effects, they are most commonly used in the manufacture of resins, polymers and pharmaceutical products (Manahan, 1991). However, the high cost and slow turnaround times of conventional photometric and chromato- graphic methods (Janda and Krijt, 1984) indicate a need for more sophisticated analytical techniques. To meet this need, a sensor-based system which is simple to use, inexpensive, disposable and highly sensitive to phenols, is becoming increasingly important in environ- mental analysis (Kotte et al., 1995). Amperometric biosensors for the detection of pheno- lic compounds have been introduced based on a number of approaches. Most amperometric biosensors for the detection of phenolic compounds have been introduced as a mono-enzyme system using either tyrosinase or horseradish peroxidase (HRP) (Ortega et al. , 1993; O ¨ nnerfjord et al., 1995; Lindgren et al., 1997). In order to develop enzymatic phenol sensors, various sensor systems have been introduced based on a number of approaches; oxygen consumption during the enzymatic reaction (Campanella et al., 1993), substrate recycling system (Makower et al., 1996) and redox mediators (Gru ¨ndig et al., 1992). In the presence of tyrosinase, phenol is oxidized to o - quinone via catechol by oxygen (Toussaint and Lerch, 1987; Casella et al., 1996). Classically, the enzymatic product, o -quinone, is reduced at low potential (Kulys and Schmid, 1990) and the schematic diagram is shown in Fig. 1a. As can be seen, the dashed line represents the fact that the electrochemical reduction of quinones is incomplete. This is because quinones are highly unstable * Corresponding author. Fax:  /44-191-222-6227 E-mail address: calum.mcneil@ncl.ac.uk (C.J. McNeil). Biosensors and Bioelectronics 17 (2002) 1015 /1023 www.elsevier.com/locate/bios 0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0956-5663(02)00094-5