Biotechnol. Prog, zyxwvut 1995, zyxwvu 11, 699-703 699 NOTES Chemiluminescence-Based Inhibition Kinetics of Alkaline Phosphatase in the Development of a Pesticide Biosensor Madhu S. AyyagariJto Sanjay Kamtekar? Rajiv PandeJ Kenneth A. Man,*,? Jayant Kumar,* Sukant K. Tripathy? Joseph Akkara,o and David L. Kaplano Centers for Advanced Materials and Intelligent Biomaterials, Departments of Chemistry and Physics, University of Massachusetts Lowell, Lowell, Massachusetts 01854, and Biotechnology Division, US. Army Natick RD&E Center, Natick, Massachusetts 01760 The use and application of the enzyme alkaline phosphatase in a chemiluminescence assay are discussed. The enzyme catalyzes the hydrolysis of a macrocyclic phosphate compound generating a chemiluminescence signal. On the basis of inhibition of this signal, a methodology for the detection and quantitation of organophosphorus-based pesticides has been developed. The methodology is studied with alkaline phosphatase in the bulk aqueous phase, and detection of the signal is accomplished by a simple optical setup. Parts per billion level detection of paraoxon and methyl parathion in bulk solutions is achieved. The technique is rapid and sensitive and is applicable to the detection of most organophosphorus-based pesticides. The results from kinetic studies indicate a mixed type of inhibition of the enzyme by paraoxon and methyl parathion. The detection methodology forms an integral part of a biosensor under development and is adaptable to incorporating optical fibers for remote detection of pesticides. Introduction The persistence of chlorinated hydrocarbon pesticides such as DDT, endrin, and lindane in the biosphere and the resultant ecological impact have turned the attention of chemists to the development of environmentally com- patible pesticides. These chlorinated compounds are not easily degraded, and their environmental mobility in soil makes them ubiquitous (Coats, 1993). zyxwvut As an alternative, organophosphorus (0P)-based pesticides have been widely used since the 1950s. Typically, OP-based pesticides are more soluble in water in comparison to the chlorinated pesticides and pose a threat to aquatic life. It is therefore essential to monitor the levels of these pesticides in various environments to determine compliance with EPA regulations as well as efficacy of treatments (EPA report EP 1.29/2,1980). Among a number of methods employed to separate and/or detect the pesticides (Martinez et al., 1992; Sanchez et al., 1991; Bier et al., 1992; Palleschi et al., 1992), biological methods can afford rapid, specific, and sensitive detection as well as compatibility with miniaturized and portable devices. In this article, we discuss the action of the enzyme alkaline phosphatase on a 1,2-dioxetane phenyl phos- phate compound. These compounds release chemical energy without the addition of oxidants. Dephosphory- lation produces an unstable phenolate anion (Tizard et al., 1990). The weak 0-0 bonds in the highly strained + Department of Chemistry, University of Massachusetts Lowell. Department of Physics, University of Massachusetts Lowell. U.S. Army Natick RD&E Center. * Address correspondence to Prof. Kenneth A. Marx, Center for Intelligent Biomaterials, Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854. Tel: (508) 934 3658. Fax: (508) 458 9571. ring break, and the decomposition of the phenolate anion emits light [Ayyagari et al., 1994al. The half-life of the anion varies from a few minutes to a few hours depending on its surrounding environment. In the presence of OP- based pesticides, the enzyme activity is inhibited, which leads to the generation of a weaker chemiluminescence signal. Thus the signal intensity, which is inversely proportional to the inhibitor concentration, is related to the amount of the pesticide in solution. The zy in-situ generation of light in the reaction mixture is easily detected and processed using a simple optical setup. Unlike the case of a fluorescence signal, there is no need for an external excitation light source and a monochro- mator for the detection of a chemiluminescence signal. Although this article discusses the inhibition kinetics of alkaline phosphatase, we underscore the importance of the technique for biosensor applications in the detec- tion of OP-based pesticides. A number of approaches based on amperometry,chromatography, fluorescence, or a simple color change for the detection of pesticides have been reported in the literature (Martinez et al., 1992; Sanchez et al., 1991; Bier et al., 1992; Palleschi et al., 1992; Trettnak et al., 1993). Although sensitive, some of these techniques involve either extensive sample preparation or lengthy analytical procedures or both. In this article, we describe a methodology by which a parts per billion level detection is possible within a few seconds, and at least 2 orders of magnitude faster than the literature values. This detection methodology, in con- junction with a generic molecular assembly of enzymes and conjugated polymers developed for optical fibers (Ayyagari et al., 19951, forms the sensing element of a biosensor under study for the detection of environmental pollutants. 8756-7938/95/3011-0699$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers