Development of Mlra Enzyme-Based Electrochemical Biosensor for the Direct Detection of MC-LR

Abstract

Microcystins are potent hepatotoxins and carcinogens produced by cyanobacteria (blue-green algae). In particular, MC-LR, one of the most toxic microcystins, tends to accumulate and pollute water reservoirs around the world, exhibiting extraordinary environmental stability. Microcystinase (MlrA) is a key enzyme in the detoxification pathway of MC-LR expressed by aerobic gram-negative bacteria. MlrA hydrolyzes a peptide bond within the cyclic MC-LR structure thus generating a nearly benign linear product.The substrate-specificity and stability of MlrA makes it an ideal candidate for an enzyme-based electrochemical biosensor. In this study, the enzyme kinetics of partially purified, heterologously expressed recombinant Mlr-A was elucidated. Polypyrrole-modified screen-printed electrodes were constructed and MlrA was incorporated via covalent immobilization. The resulting enzyme-modified chips were used to study the electrochemical properties of MC-LR and MlrA. Within a small potential window, both MC-LR substrate and MlrA enzyme are electrochemically inert. However, it was shown that enzymatic hydrolysis of MC-LR significantly affected the capacitive current measured overtime. These concentration-dependent changes in non-faradic current were observed even at relatively low MC-LR concentration of 10mM/L, and provide a proof-of-concept for the use of MlrA in an enzyme-based electrochemical biosensor. This study may pave the way towards designing biosensors for environmental diagnostics that enable both monitoring and remediation. Figure 1