Flow Injection Biosensor System for 2,4-Dichlorophenoxyacetate Based on a Microbial Reactor and Tyrosinase-modified Electrode

Synthetic chlorinated organic compounds have been used extensively as herbicides and pesticides and the contamination of ecosystems with these compounds has stimulated great interest in investigation of these frequently toxic or bioaccumulatable compounds. 2,4-Dichlorophenoxyacetate (2,4-D), one of these compounds, is a synthetic phytohormone that has been widely used as a herbicide for controlling broadleaf weeds, and huge amount of 2,4-D has been released into the environment. 2,4-D is known, on the other hand, to be susceptible to rapid biological degradation in natural environments, and has been used as a model for genetic and biochemical study on the chloroaromatic degradation. A number of its degrading bacteria have been isolated worldwide from a variety of environments and extensively examined on a molecular basis (Amy et al., 1985; Sinton et al., 1986; Perkins et al., 1990; Fulthorpe et al., 1992, 1995; Ka et al., 1994; Tonso et al., 1995; Top et al., 1995; Maltseva et al., 1996; Suwa et al., 1996; Vallaeys et al., 1996; Kamagata et al., 1997; Cavalca et al., 1999; Laemmli et al., 2000; Itoh et al., 2002). One of the most extensively studied strain is Ralstonia eutrophus JMP134, which carries a 2,4-D-degrading gene cluster on the transmissible plasmid pJP4 (Don & Pemberton, 1981; Neilson et al., 1992; Fukumori & Hausinger, 1993a; Laemmli et al., 2000). On the other hand, reliable determination of 2,4-D is indispensable to investigate its biological degradation. A biosensor is a device utilizing a biological sensing element, and a variety of biosensors for 2,4-D detection have been developed (Table 1). Most of sensors reported so far, however, can be classified into either immunoassay or immunoenzymatic assay. They are based on the specific interaction between 2,4-D (antigen) and its antibody. These biosensors have been demonstrated to show very high sensitivities (e.g. the lower detection limit of less than 1 μg/L), while these assays are generally said to be somewhat cumbersome to perform and require considerably expensive reagents. A biosensor based on the inhibition of catalytic activity of enzyme alkaline phosphatase in the presence of 2,4-D has been also proposed and a detection limit of 0.5-6 μg/L has been obtained. The enzyme inhibition effect has been also observed for another pesticide, indicating the analyte selectivity of the sensor to be not expected. On the other hand, very few attempts have been made on the biosensor employing microorganisms as the sensing component.