Rapid Detection Of Gram-negative Bacteria Signaling Molecules(AHLs) Based On Carbon Dots-molecularly Imprinted Recognition Material

Bacterial food pollution not only lead to serious waste of food resources,but also cause major public health and safety risks.The prevention and control of bacterial food contamination needs to be solved urgently.The discovery of Quorum Sensing?QS?system and its signal molecules can provide a new idea for the prevention and control of bacterial food pollution.N-acyl-homoserine lactones?AHLs?,as the most typical signal molecules regulating the QS system of Gram-negative bacteria,developed an analytical method for rapid detection of AHLs,which can effectively simplify the detection procedure and shorten the analysis time,to clarify the QS system regulation mechanism in the process of food corruption and developing new control strategy is of great significance.Therefore,here combines molecular imprinting technology with fluorescent nanomaterials to develop a new type of fluorescent molecular imprinting recognition material.Based on this material,a fast and efficient analytical method for AHLs detection is constructed.The main contents are summarized as follows:A kind of carbon dots?CDs?,which can be used as fluorescent recognition elements,was prepared and its optical and signal transduction properties were investigated.N,S Co-doped fluorescent CDs were synthesized by one step hydrothermal method using 2,5-diaminobenzenesulfonic acid as a single carbon precursor.At the excitation wavelength of 470?520 nm,CDs showed an excitation independent fluorescence peak at 593 nm.Through the characterization of the morphology,optical properties and structure of CDs,it is found that CDs has uniform particle size and excellent optical properties,and because of the doping of N,S heteroatom,the CDs surface has introduced a wealth of functional groups?amino,hydroxy,mercapto?,which endows it with unique sensing performance.By constructing a“switch-on”fluorescent sensor,the potential value of the prepared CDs as a fluorescent recognition element is further verified.Combining the high selectivity of molecularly imprinting polymers?MIPs?with the sensitive signal transduction characteristics of CDs,a rapid detection method of AHLs based on fluorescent molecularly imprinted recognition materials was developed.The fluorescent molecular imprinting polymer?CD@MIP?was prepared by sol-gel method using 2,5-dimethyl-4-hydroxy-3?2H?-furanone as template molecule.After template elution,the imprinted cavity formed on the surface of CD@MIP can selectively recognize AHLs and show a good fluorescence response.Under the best condition of fluorescence detection,with the increase of template molecular concentration,the fluorescence quenching efficiency F₀/F increases gradually,showing a good linear relationship in the concentration range of 0?2.0?M,and the lowest detection limit?LOD?is 0.0672?M?S/N=3?.This method was used to investigate the AHLs in supernatant of 5 kinds of bacteria.Except for E.coli and S.aureus,the supernatant of other bacteria all showed fluorescence response,which was consistent with the characteristics of the two bacteria that do not produce AHLs.And the spiked recovery experiment was performed on the sterile supernatant,and the recovery range was 95.3%?103.0%,which proved the feasibility of this method.In order to further improve the detection efficiency,the CD@MIP microporous plate for the efficient detection of AHLs was constructed.The successfully prepared CD@MIP was“branched”into the microwells of a 96-well plate.By optimizing the preparation conditions and the adsorption medium,the CD@MIP microporous plate was successfully prepared,and its adsorption performance was evaluated.Then the CD@MIP microporous plate was used to detect four kinds of AHLs.The logarithm values of the concentrations of C4-HSL,C6-HSL,3-O-C6-?L?-HSL and C8-HSL all have a good linear relationship with the fluorescence quenching efficiency F₀/F,the linear range is 0?25?M and the LOD?S/N=3?was 0.0643?M,0.0657?M,0.0729?M,0.0745?M,respectively.The spiked recovery experiment of bacterial supernatant was carried out with the microporous plate,and the recovery range was 96.1%?103.0%,which proved that this method had good accuracy and stability.