Fluorescent assay for quantitative analysis of trimethylamine N-oxide

Trimethylamine N-oxide (TMAO), a gut microbial metabolite involved in cardiovascular and kidney diseases, has great potential as a biomarker, thus making TMAO quantification of great significance. The current assay methods are mainly established on mass spectrometry. However, the classic enzymatic approach is absent, which may be because there is no appropriate single-enzyme reaction. Here, we prepared TMAO demethylase and formaldehyde dehydrogenase and found that these two bacterial enzymes catalyze an efficient coupled reaction that produces NADH from TMAO conversion. With the participation of another enzyme, diaphorase, the multienzymatic coupling system was constructed, which realizes the output of fluorescence signals from TMAO input using resazurin as a probe, thus laying the foundation for fluorescent assay. Through optimization, the sensitivity and specificity were improved. A pretreatment procedure was developed to eliminate formaldehyde that pre-exists with TMAO to avoid an interference effect. Our assay is suitable for quantifying serum TMAO in the range of 2.05-50 μM, covering actual levels in clinical samples, and exhibits a high degree of accordance with mass spectrometry. Therefore, the established fluorometric microplate assay is facile, sensitive and accurate and may enable low-cost and high-throughput analysis of TMAO in clinical laboratory diagnosis.