Development of a biosensor for spectrophotometric determination of L-lactate in artificial saliva

Analysis of critical biomarkers like L-lactate is extremely important in clinical practice. Herein, a non-invasive and sensitive colorimetric biosensor for accurate L-lactate determination has been developed. The proposed method demonstrates the ability of Fe³⁺ ions of iron(III) chloride to substitute the traditional horseradish peroxidase enzyme in the colorimetric determination of L-Lactate. The biosensor is based on the release of H₂O₂ by lactate oxidase enzyme (LOx) after 30 min incubation in a 37 °C water bath. Subsequently, H₂O₂ reacts with 3,3',5,5'-tetramethylbenzidine substrate (TMB) catalyzed by Fe³⁺ ion utilizing its peroxidase-mimetic activity. Fe³⁺ ion has peroxidase-like activity which could rapidly catalyze the oxidation reaction of TMB by H₂O_2, producing a characteristic blue colored product at 30 °C water bath for 15 min. Based on the catalytic mechanism of fast electron transfer between TMB and H₂O₂ with the assistance of the intrinsic peroxidase-like activity of Fe³⁺ ion, a colorimetric biosensor for determination of L-lactate was developed. The obtained colored product of oxidized TMB could be measured spectrophotometrically at λmax 652 nm. The biosensor yielded a reproducible response over a linear range of 5 µM-20 µM of L-lactate with a limit of detection of 1.278 µM. Furthermore, satisfactory results were obtained upon application of the method to artificial saliva samples.