Study On The Determination Method Of Intestinal Flora Metabolite-Trimethylamine N-oxide(TMAO)

Intestinal flora metabolites are the metabolites of microorganisms that are co-born with human beings and exist in large quantities in human intestine.Therefore,the accurate determination of the metabolites of intestinal flora is of great significance in the study of the mutual disease mechanism and prevention and control of intestinal flora and host metabolism.Recently,the trimethylamine N-oxide?TMAO?,a related product of human intestinal metabolism,has gradually attracted the attention of the medical investigator.A large number of research experiments have proved that the substance is closely related to human cardiovascular disease,chronic kidney disease,diabetes,cancer and so on.However,the existing methods for the determination of trimethylamine N-oxide are Ray's salt colorimetric method,picric acid colorimetry,ion chromatography,UHPLC-MS/MS and so on,but these test methods have the problems of low sensitivity,high cost and not applicable,which can not meet the actual demand.Therefore,the main goal of this paper is to develop a test method with low test cost,simple operation and batch sample detection,which can be carried out from the two aspects of headspace GC and the preparation of trimethylamine N-oxide molecular imprinting-fluorescence composite.The details are as follows:Section 1,an indirect method for the determination of TMAO in samples was established.In this method,the Titanium trichloride?TiCl₃?as a reducing agent,TMAO was reduced to trimethylamine?TMA?.And the corresponding TMAO concentration can be calculated according to the stoichiometric relationship,when the concentration of TMA is determined.At the optimized experimental conditions,the aqueous solution of TMAO with the linear concentration range was from 5 to 200?mol/L?the range of TMAO with each sample is 0.005⁰.2?mol?was measured by gas chromatography and the results indicate that the linear correlation coefficient is0.9994 and the detection limit of this assay was 2.16?mol/L,and the standard recovery rate of human serum samples ranged from 87.0%to 110.4%.Repeatability verification and comparative test were carried out on different human serum samples,the relative standard deviation is 2.12⁸.84%,the relative error of the two methods is lesss than 9.27%by compared with the method of UHPLC-MS/MS.Section 2,MIP-TMAO@CdTeQDs was prepared by the reverse microemulsion method with the L-Cys-CdTe as the core,TMAO as the template,?3-aminopropyl?triethoxysilane?APTES?as the functional monomer and tetraethyl orthosilicate?TEOS?as the cross-linking agent,ammonium hydroxide as the initiator,aim to determine the TMAO in human serum.In the experimental process,MIP-TMAO@CdTeQDs and NIP@CdTeQDs imprinted fluorescent probe composites with excellent performance were prepared with TMAO:APTES:TEOS=1:2:15 and the dosage of L-Cys-CdTe was 600?L?5 mg/mL?.Through the analysis of the test results of FT-IR,SEM,Fluorescence performance test,adsorption and selection performance test,it can be seen that:?1?The synthetic products MIP@CdTeQDs are all spherical with an average particle size of about 400 nm.;?2?The adsorption equilibrium time of TMAO imprinted fluorescent probe on the template molecule TMAO was about 35 min,and the saturated adsorption amount was about 64?g/g.?3?Compared with the interfering substances such as TMA and DMA,MIP@CdTeQDs fluorescent probe has the distinctive ability to identify and adsorb TMAO.?4?TMAO molecules have a certain fluorescence quenching effect on MIP@CdTeQDs,and there is a good linear relationship between fluorescence quenching efficiency and TMAO concentration.And the linear correlation coefficient is 0.99334,the minimum detection limit is 1.13?mol/L.