Synthesis Of Novel Small Molecular Fluorescent Probes And Imaging Of The Gut Microbiota

A large and diverse group of microorganisms live in the gastrointestinal tract of animals,collectively known as the gut microbiota.The gut microbiota has been shown to be closely related to host metabolism,nutrient absorption,immune regulation and other activities.Currently,our studies on the gut microbiota highly rely on the high-throughput DNA sequencing technology to analyze the genomes of gut microbes and compare the abundance and diversity levels of species.It accelerated the process of the gut microbiota research.However,there are still many complications to be solved urgently in the research of gut microbiota,such as the localization and tracking of the microbiota,dynamic monitoring of the microbiota,etc.The existing methods cannot meet our needs,hindering the progress of our research on the gut microbiota.New approaches to gut microbiota research are needed.In recent years,in vivo imaging technology based on organic small molecule fluorescent probes has developed rapidly and has the advantages of high resolution,low cost,high efficiency,and low toxicity.This may be an effective way to break through the bottleneck of gut microbiota research.In this study,two types of novel small molecule fluorescence probes were synthesized,one is the positive charge-based small molecule fluorescence probes,and the other is the NIR-II small molecule metabolic fluorescence probe,both of which were used for in vitro and in vivo bacterial fluorescence imaging.This study provides a novel probe selection for gut microbiota fluorescence imaging and a new research strategy for the in-depth study of gut microbiota imaging.Based on the previous research,the famous mitochondrial targeting probe F16 was modified and transformed to improve the fluorescence properties and reduce the toxicity.The novel positive charge-based small molecule fluorescence probes were named EF and 6F,respectively.EF and 6F have simple chemical structures,easy synthetic routes and cheap cost,which are suitable for large-scale production.EF and6 F have similar fluorescence properties,the absorption spectrum ranges from 320 nm to 500 nm,and the emission spectrum ranges from 470 nm to 620 nm.They have excellent fluorescence stability and nearly no fluorescence quenching was found in the simulated gastrointestinal tract environment or under sunlight.In vitro bacterial fluorescence imaging tests,EF and 6F can fast label Gram-positive bacteria: S.aureus and L.reuteri;Gram-negative bacteria: E.coli and S.pullorum.EF and 6F have little bacterial toxicity and are used at a maximum concentration of 200 ?M.In vivo fluorescence imaging test,the mice were taken EF and 6F by intragastric administration.On the IVIS small animal in vivo imaging system,EF and 6F were found that mainly accumulated in the distal ileum,cecum and colon where the intestinal flora was most abundant,and were not found in other major organs.fluorescence signal.On the Optiscan small animal in vivo confocal imaging system,colonizing flora was found that adhered to the surface of the intestinal villi of the distal ileum and the surface of the colonic mucosa.The labeled intestinal colonizing flora was also found that adhered to the ileal intestinal villi epithelial cells by confocal microscopy.Meanwhile,EF and 6F did not cause damage to intestinal tissue at the maximum working concentrations.A D-amino acid was conjugated to the side chain of the NIR-II small molecule fluorescence probe CQ to synthesize a new NIR-II small molecule bacterial fluorescence probe CQDB.The absorption and emission spectra of CQDB was similar to the CQ,which were 732 nm at the maximum absorption and 1042 nm at the fluorescence wavelength.L.reuteri was selected as the supplementary bacteria,and CQDB at the working concentration did not affect the growth performance of L.reuteri.In the NIR-II in vitro fluorescence imaging test,CQDB could label L.reuteri and perform fluorescence imaging.In the NIR-II in vivo fluorescence imaging test,it was observed that L.reuteri as a supplementary probiotic,as expected,could colonize the stomach in large quantities for a long time.At the same time,images of a small amount of uncolonized L.reuteri moving with the peristalsis of the mouse gut were also monitored,and their location in the gut could be recorded in real time.Uncolonized L.reuteri began to enter the cecum after 3 h of movement in the small intestine,and then slowed down;after 5 h,the bacteria entered the colon;after 6 h,the uncolonized bacteria were gradually excreted with the feces.In conclusion,the novel positive charge-based small molecule fluorescence probes designed and synthesized in this study could effectively label bacteria in vitro and in vivo,and could localize the colonizing flora in vivo.The designed and synthesized novel NIR-II small molecule metabolic fluorescence probe had the advantages of high penetration and high signal-to-noise ratio.By in vivo imaging system MARS,the labeled bacteria can be detected in the mouse intestine.The visual imaging of the labeled bacteria could be located and tracked in real time.Neither of the above two types of probes had adverse effects on bacterial activity or host health.This study provides a novel probe selection for gut microbiota imaging research and a new strategy for in-depth study of gut microbiota.