The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitiv ely inhibited by pentachlorophenol (PCP) and a modified assay produced a de tection limit of 1 muM (270 mug l(-1)). This work used spectrophotometric r ate-determination but in order to move towards biosensor development the se lected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This c ould be monitored using a screen-printed carbon electrode (SPCE) incorporat ing the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/ AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhi bition, possibly by combining with PCP. A cellulose acetate membrane remove d this effect. Inhibition of the system was greatest at enzyme activities o f 5 U ml(-1) LDH and 0.8 U ml(-1) LOD in reactions containing 246 muM pyruv ate and 7.5 muM NADPH. PCP detection limits were an EC10 of 800 nM (213 mug l(-1)) and a minimum inhibition detectable (MID) limit of 650 nM (173 mug l(-1)). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provi ded cofactor recycling to enable low concentrations of NADPH to be incorpor ated within the assay. NADPH was reduced from 7.5 to 2 muM. PCP detection l imits were obtained for an assay containing 5 U ml(-1) LDH, 0.8 U ml(-1) LO D and 0.1 U ml(-1) GDH with 246 muM pyruvate, 400 mM glucose and 2 muM NADP H. The EC10 limit was 150 nM (39.9 mug l(-1)) and the MID was 100 nM (26.6 mug l(-1)). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electr ochemical biosensor array for pollution monitoring, (C) 2001 Elsevier Scien ce B.V. All rights reserved.