ANALYST FULL PAPER THE www.rsc.org/analyst Mixed enzyme (laccase/tyrosinase)-based remote electrochemical biosensor for monitoring phenolic compounds Renato S. Freire, a Sompong Thongngamdee, b Nelson Durán, ac Joseph Wang* b and Lauro T. Kubota* a a Instituto de Química, Universidade Estadual de Campinas –UNICAMP, CP 6154, CEP 13083-970 Campinas, SP, Brazil. E-mail: kubota@iqm.unicamp.br; Fax: +55 19 7883023; Tel: +55 19 7883127 b Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces NM 88003, USA. E-mail: joewang@nmsu.edu; Fax: +1 505 646 6033; Tel: +1 505 646 2140 c Núcleo de Ciências Ambientais, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil Received 2nd November 2001, Accepted 3rd December 2001 First published as an Advance Article on the web 11th January 2002 An electrochemical biosensor for remote continuous monitoring of phenolic compounds in environmental analysis is described. The probe relies on rapid and sensitive amperometric detection at a submersible biosensor assembly, connected to a 50 ft long shielded cable. The enzymes laccase and tyrosinase were used as individual sensors and also as a bienzymatic sensor; these enzymes were immobilized chemically on the carbon fiber transducer. The analysis was based on the amperometric detection of the enzymatic products at a potential of 20.10 V vs. Ag/AgCl. Operational conditions were optimized to meet the requirements of remote operations. Tests with untreated river water spiked with phenolic compounds gave results similar to those obtained with synthetic buffer solutions. The remote laccase biosensor allowed the convenient quantification of guaiacol and chloroguaiacol at levels down to 22 and 9 nmol L 21 , respectively. The co-immobilization of laccase and tyrosinase allowed the efficient detection of a larger group of phenolic compounds. Introduction Contamination of water resources or waste sites with organic pollutants represents a serious environmental problem. The determination of phenol and its derivative compounds is of great importance, since these species are released into the environment by a large number of industries, e.g., the manufacture of plastics, dyes, drugs, antioxidants and waste waters from pulp and paper production. 1–5 Effluents from Kraft paper mills are one of the most deleterious, since these effluents contain a broad range of organic compounds, especially chlorinated compounds. 6 In addition, some of these compounds, such as guaiacol and chloroguaiacol, have been determined to be recalcitrant, toxic to aquatic species, genotoxic, lipophilic and with a propensity for bioaccumulation. 7–12 Owing to health and ecological risks caused by long- and short-term exposure to phenolic compounds, there is consider- able interest in their measurement in environmental matrices. Continuous measurement of these compounds, effected in natural media, is preferred as it provides an appropriate feedback during the characterization or remediation of contami- nated sites, offers rapid warning in case of sudden contamina- tion and minimizes the huge labor and analytical costs, as well as errors and delays inherent to laboratory-based analy- ses. 13–16 Accordingly, extensive efforts have been devoted to the development of innovative and effective sensors, capable of monitoring phenols both in time and location. 17,18 This paper describes the development and optimization of an electrochemical biosensor for remote on-site monitoring of phenolic compounds. Biosensors can make ideal sensing systems for monitoring the effects of pollution on the environ- ment, owing to their biological base, ability to operate in complex matrices, short response time and small size. 19 Laccase- and tyrosinase-based electrodes have been shown to be useful for the selective determination of phenols in environmental matrices. 1,3,4,20–25 The use of laccase is very important because it is more sensitive for chlorinated organic compounds, which is very significant in environmental re- spects. Thus, laccase can react with phenolic compounds that are not reactive with tyrosinase. Laccase and tyrosinase are both copper-containing oxidases catalyzing the reduction of molec- ular oxygen by different electron donors, e.g., phenolic compounds. One of the most important points in using laccase is the sensitivity for phenolic compounds that are considered very toxic. In these reactions the oxygen is reduced directly to water without the intermediate formation of hydrogen per- oxide. 1 These two enzymes display different substrate specific- ities and mechanisms, hence the co-immobilization of laccase and tyrosinase on the transducer element of an electrochemical sensor allows more phenolic compounds to be detected. 1 However, remote laccase-based and bienzymatic bioelectrodes, capable of making continuous real-time measurements of phenolic compounds at large sample/instrument distances, have not been reported in the literature. The aim of this work was to develop a remote biosensor for field monitoring of phenolic compounds related to paper-mill effluents. This device couples the sensitivity and selectivity capabilities of laccase- and tyrosinase-based biosensors, with the advantages of remote monitoring systems. Operational conditions, optimization and performance of this device are reported for monitoring guaiacol, chloroguaiacol, cresol and phenol. Experimental Reagents All solutions were prepared with de-ionized water. Laccase (from Coriolus hirsutus, EC 1.10.3.2, 416 U mg 21 ) was This journal is © The Royal Society of Chemistry 2002 258 Analyst, 2002, 127, 258–261 DOI: 10.1039/b110011d