Chemiluminescence‐Based Inhibition Kinetics of Alkaline Phosphatase in the Development of a Pesticide Biosensor

Abstract

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.

A Biotinylated Undecylthiophene Copolymer Bioconjugate for Surface Immobilization: Creating an Alkaline Phosphatase Chemiluminescence-Based Biosensor

“…It can be observed in Figure 6 that the linear fits to these data do not intersect either on the x axis or on the y axis as would be the case for noncompetitive or competitive inhibition, respectively. This behavior, indicative of a mixed type of inhibition, was similar to that observed and reported in solutionbased studies of alkaline phosphatase (18). Figure 7 demonstrated the extent of inhibition of the chemiluminescence signal in the presence of different paraoxon concentrations at fixed enzyme (10 -15 mol) and substrate (0.4 mM CSPD) concentrations.…”

“…The K m for the immobilized enzyme was 0.58 mM -1 . This was close to the K m measured for the free solution enzyme, 0.81 mM -1 (18). An investigation of paraoxon-mediated inhibition kinetics of capillary-immobilized alkaline phosphatase was carried out by varying the CSPD concentration while keeping the inhibitor (paraoxon) concentration at a constant value of either 0.36 or 0.18 mM.…”

Directed Evolution of E. coli Alkaline Phosphatase Towards Higher Catalytic Activity

“…Additionally, EAP has a higher thermostability compared to mammalian APs, which invited many applications in biosensors, despite its lower (Â/20 fold or more) specific activity. Some biosensors with EAP as a recognition element have been reported (Ayyagari et al ., 1995;Brennan et al ., 1995). It is obvious that an EAP with higher activity could increase the dephosphorylation efficiency of oligonucleotides and the detection sensitivity of biosensors.…”

Electrochemical biosensors for rapid determination of pesticide residues in agricultural products

“…(Ayyangari, 1995;Rekha ,2000;Wang, 2002;Anh,2004). [9][10][11][12] Immunoanalytical techniques are based on the specific binding of an analyte to an antibody and they can reach low limits of detection. In many cases no extraction step and no cleanup are necessary.…”