Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors

Abstract

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2 (Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb2 to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2 and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M−1·m−2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2 and the hexacyanoferrate of gold, having a sensitivity of 253 A·M−1·m−2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

Recent Advances in Electrochemical Enzyme-Based Biosensors for Food and Beverage Analysis

“…Lactic acid is a universal organic acid that when present reflects several aspects of food and beverages, including quality, stability, and organoleptic characteristics. Several recent studies [ 79 , 80 , 81 , 82 ] indicate that the dairy and wine industries would benefit significantly if the evaluation of lactate level can be performed with more sensitive lactate biosensors. For instance, a highly sensitive biosensor for L-lactate determination based on direct electron transfer enzymes was developed [ 81 ].…”

“…Several recent studies [ 79 , 80 , 81 , 82 ] indicate that the dairy and wine industries would benefit significantly if the evaluation of lactate level can be performed with more sensitive lactate biosensors. For instance, a highly sensitive biosensor for L-lactate determination based on direct electron transfer enzymes was developed [ 81 ]. These authors improved existing biosensors by enhancing the electron transfer between the electrode surface and the enzyme cytochrome c oxidoreductase (flavocytochrome b 2 , EC 1.1.2.3) by applying bimetallic and trimetallic nanoparticles, and verified the best electrode set-up with yoghurt samples.…”

“…Briefly, PtZn, NiPtPd, and hexacyanoferrates of Au (AuHCF) served as active redox mediators and were safely co-immobilized with L-lactic specific cytochrome c oxidoreductase. A modification of the working electrode surface with AuHCF led to a 3.5-fold increased sensitivity and a 2.7-fold decreased LOD compared to a control biosensor composed of bare L-lactate-cytochrome c oxidoreductase immobilised on the graphite electrode [ 81 ]. Ozoglu and co-workers studied the detection of lactate produced by lactic acid bacteria isolated from cheese samples.…”