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

Abstract 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 Fe 3+ 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 H 2 O 2 by lactate oxidase enzyme (LOx) after 30 min incubation in a 37 °C water bath. Subsequently, H 2 O 2 reacts with 3,3′,5,5′-tetramethylbenzidine substrate (TMB) catalyzed by Fe 3+ ion utilizing its peroxidase-mimetic activity. Fe 3+ ion has peroxidase-like activity which could rapidly catalyze the oxidation reaction of TMB by H 2 O 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 H 2 O 2 with the assistance of the intrinsic peroxidase-like activity of Fe 3+ 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.