Analytica Chimica Acta 494 (2003) 187–197 Amperometric flow-injection determination of phenolic compounds at self-assembled monolayer-based tyrosinase biosensors Susana Campuzano, Beatriz Serra, Mar´ ıa Pedrero, F. Javier Manuel de Villena, José M. Pingarrón ∗ Dpto. Qu´ ımica Anal´ ıtica, Facultad de CC. Qu´ ımicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain Received 3 March 2003; received in revised form 10 July 2003; accepted 21 July 2003 Abstract The performance of a tyrosinase (Tyr) biosensor, constructed by immobilisation of the enzyme by cross-linking atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a Au disk electrode, is reported for the ampero- metric detection under flow-injection (FI) conditions of several phenolic compounds (phenol, catechol, m-cresol, p-cresol, 4-chloro-3-methylphenol, 3-chlorophenol, 4-chlorophenol, 2,4-dimethylphenol, 3,4-dimethylphenol, and 2-aminophenol). Experimental variables such as the detection potential (-100 mV versus Ag|AgCl|KCl 3 M), flow rate (1.02 ml min -1 ), in- jection volume (350 l), and pH of the carrier solution (0.05 M phosphate buffer of pH 7.0) were optimised. Under these conditions, the Tyr biosensor exhibited a good reproducibility of the FI measurements, with no need to apply a cleaning or pre-treatment procedure. The useful lifetime of one single biosensor was 5 days. The kinetic parameters of the Tyr reaction were calculated for the 10 phenolic compounds. The analytical performance of the Tyr–MPA–Au electrode under flow through conditions was compared with that of other Tyr electrochemical biosensors reported in the literature. The usefulness of the biosensor for the analysis of real samples was proved by performing the estimation of the content of phenolic compounds in waste waters from a refinery at three different stages in the waste purification process. © 2003 Published by Elsevier B.V. Keywords: Self-assembled monolayers; Tyrosinase; Flowing systems; Gold disk electrode 1. Introduction Self-assembled monolayers (SAMs) have demon- strated to be powerful and versatile tools to generate monomolecular films of biological molecules on a va- riety of substrates [1]. Several advantages, such as the ∗ Corresponding author. Tel.: +34-91-3944-331; fax: +34-91-3944-329. E-mail address: pingarro@quim.ucm.es (J.M. Pingarr´ on). possibility of mimicking naturally occurring recog- nition processes [2], and the high degree of control over the molecular architecture of the biorecognition surface make SAMs to play an important role in the development of biosensors. Concerning electrochem- ical biosensors, SAM-modified electrodes have been recently used for the construction of biosurfaces be- cause of the dramatic reduction of the double-layer charge current, with the subsequent increase in sensi- tivity [3], and the reproducibility and robustness that 0003-2670/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/S0003-2670(03)00919-X