Development of an enzymatic assay system of d-lactate using d-lactate dehydrogenase and a UV-LED fluorescent spectrometer

• A new d-lactate sensing system was constructed by UV-LED fluorescent spectrometer.
• The system had good linearity (R2 = 0.9964) in the range from 5 to 150 μM.
• The intra-day and inter-day accuracies were within 109.09% and 104.59%.
• Precisions of the intra-day and inter-day assay were between 4.04% and 12.40%.
• The system could detect 10–15 samples compared to only one sample by HPLC in 90 min.

In this study, we aimed to develop a new enzymatic assay system of d-lactate with good precision, accuracy, and sensitivity for the determination of d-lactate concentrations in rat serum. d-Lactate dehydrogenase (d-LDH) was utilized to catalyze d-lactate and NAD+ to pyruvate and NADH, respectively. The generated NADH was excited by using a 340-nm UV-light-emitting diode (LED), and the fluorescence at 491 nm was detected to determine the concentration of d-lactate in rat serum. The optics, consisting of the sample cuvette, were set on three-dimensional stages to receive the most intensive fluorescence signal into the spectrometer. The optimal conditions of the d-LDH reaction were pH 8.5 and 25 °C for 90 min. The results showed that the new d-lactate assay system had good linearity (R2 = 0.9964) in the calibration range from 5 to 150 μM. Intra-day and inter-day accuracies were in the range of 103.96–109.09% and 102.84–104.59%, respectively, and the intra-day and inter-day precision was 4.28–6.82% and 4.04–12.40%, respectively. Finally, serum d-lactate concentrations determined by the proposed enzymatic assay system were compared with those obtained by a conventional HPLC method. The newly developed d-lactate assay system could detect 10–15 samples in 90 min, whereas the HPLC method could detect only one sample over the same time period.

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