A novel mediatorless biosensor based on flavocytochrome b_2 immobilized onto gold nanoclusters for non-invasive L-lactate analysis of human liquids

摘要

Lactic acid (L-lactate) is among the most important analytes since it is a universal metabolite of nearly all the living organisms. The determination of the L-lactate level is used in clinical diagnostics of hypoxia, lactic acidosis, some acute heart diseases, in drug toxicity tests as well as in sports medicine, while monitoring the athletic performance to evaluate the best training equipment and regimes. The measurement of metabolites in fluids other than blood is becoming increasingly significant because of major advantages of non-invasive analysis (safety, rapidity, and accuracy). In sensor technology, the most efficient means for the non-invasive analysis of human fluids seems to be the third generation (mediatorless) amperometric biosensors, as they do not require any exogenous cofactors, toxic electron transfer mediators or the use of high working potential. The aim of this work is the construction of a novel non-invasive electrochemical biosensor of the third generation for l-lactate based on yeast L-lactate:cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b_2, FC b_2) immobilized onto gold nanoclusters (nAu). FC b_2 is a tetramer with four identical subunits, each consisting of FMN- and heme-binding domains. The main properties of FC b_2 of thermotol-erant yeast Ogataea (Hansenula) polymorpha are the selectivity for L-lactate, its high stability and ability to direct electron transfer. The developed FC b_2-nAu modified bioelectrodes are characterized by improved parameters compared to the enzyme electrodes, obtained without the use of nAu. The constructed bioelectrodes were adapted for direct non-invasive analysis of human saliva and sweat samples using the amperometric microcomputer-based analyzer system. The obtained results of the real samples analysis have a high correlation (0.7 < R < 1) with the reference approaches.