Facile Fluorescence Monitoring of Gut Microbial Metabolite Trimethylamine N-oxide via Molecular Recognition of Guanidinium-Modified Calixarene

Abstract

Detection and quantification of trimethylamine N-oxide (TMAO), a metabolite from gut microbial, is important for the disease diagnosis such as atherosclerosis, thrombosis and colorectal cancer. In this study, a novel method was established for the sensing and quantitative detection of TMAO via molecular recognition of guanidinium-modified calixarene from complex matrix.

Methods: Various macrocycles were tested for their abilities to serve as an artificial TMAO receptor. Using the optimized receptor, we developed an indicator displacement assay (IDA) for the facile fluorescence detection of TMAO. The quantification of TMAO was accomplished by the established calibration line after excluding the interference from the various interfering substances in artificial urine.

Results: Among various macrocycles, water-soluble guanidinium-modified calix[5]arene (GC5A), which binds TMAO in submicromolar-level, was identified as the optimal artificial receptor for TMAO. With the aid of the GC5A•Fl (fluorescein) reporter pair, TMAO fluorescence “switch-on” sensing was achieved by IDA. The fluorescence intensity increased linearly with the elevated TMAO concentration. The detection was not significantly interfered by the various interfering substances. TMAO concentration in artificial urine was quantified using a calibration line with a detection limit of 28.88 ± 1.59 µM, within the biologically relevant low µM range. Furthermore, the GC5A•Fl reporter pair was successfully applied in analyzing human urine samples, by which a significant difference in fluorescence response was observed between the [normal + TMAO] and normal group.

Conclusion: The proposed supramolecular approach provides a facile, low-cost and sensitive method for TMAO detection, which shows promise for tracking TMAO excretion in urine and studying chronic disease progression in humans.