Immunofluorescence Probe Based On Fluorescent Carbon Dots And Its Application In Detection Of Microorganism

Objective:The objective of the research is to produce carbon dots using citric acid as carbon source and modified by passivator on the surface, to achieve the highest fluorescent quantum yield and to study their luminescence mechanism through optimizing the preparation procedures and reaction conditions. With antibody-antigen specific binding properties, the carbon dots are used as the fluorescent markers and are bound with antibodies to fabricate immunofluorescent probe; and the Enterohemorrhagic Escherichiacoli O157:H7 is chosen as the detection target to observe the fluorescence after combining Escherichiacoli sample with the antibodies, and to prove whether the construction of the carbon dots in immunofluorescent probe is successful. The application of carbon dots-based immunofluorescent probe in Escherichiacoli detection is explored.It is expected to produce the carbon dots which are low-toxicity, economical,effective and secure In the meanwhile, the fluorescent markers, as immunofluorescent probe, are able to safely and effectively identify the detection target.Contents and Methods: Several jobs will be developed in this paper from the following aspects:(1)Selecting anhydrous citric acid as the carbon source and PEI as the modifier,the carbon dots of surface amination is directly synthesized by microwave heating,and fluorescence carbon dots of fine fluorescence property are synthesized without surface modification; the influences of the synthetic conditions such as the microwave time and the ratio of reactant on the productivity, structure, composition, microscopic morphology, particle size and distribution of carbon dots are systematically studied in this experiment to reveal their formation rules. In addition, their components,structure and morphological characteristics are analyzed by means of the characterization methods such as TEM, FTIR, FL, UV and so on.(2)Carbon dots are coupled with anti-E.coli antibodies through amidation reaction, and then the immunofluorescent probe is developed; other bacteria similar with Escherichiacoli are selected as the control model so as to analyze thenon-specific adsorptive property and the interactions between the carbon dots and the control model and between the immunofluorescent probe and the control model, and to study the specificity of the immunofluorescent probe.(3)The application of the immunofluorescent probe based on carbon dots in the Escherichiacoli detection is explored, the relationship between the concentration of Escherichiacoli and the intensity of immunofluorescent probe is created.Results:(1)It is proved in the experiment that the intensity of the carbon dots modified by PEI is distinctly higher than the carbon dot without PEI modification, and the number of the amino functional groups on the surface of carbon dots is increased, which lays a foundation for the construction of immunofluorescent probes in the future. The characterization result shows that the carbon dots has a spherical shape and narrow particle size distribution, and their average particle diameter size is 3 nm. The average particle diameter size less than 10 nm can reduce its interference on creatures. Carbon dots have fine fluorescence property, and their fluorescent quantum yield reaches46.7%, which is relatively high and is slightly less than that of the standard substance,quinine sulfate. Besides, they have strong light stability, so they can maintain stable fluorescence intensity for a long time. When the excitation wavelength is 360 nm, their fluorescence emission peak reaches the highest, nearly 440 nm. With the red shift of the excitation wavelength, the emission wavelength of the synthesized carbon dots also has the red shift. It respectively launches blue, green and red fluorescence with the UV emission, blue light emission and green light emission, so they can be used in multi-fluorescence imaging. The experimental conditions such as raw material configuration, p H, microwave time are optimized in the process of carbon dots preparation in the experiment, and their optimum synthesis condition is that the portion of anhydrous citric acid and polyethyleneimine is 3:1. Now the solution has a p H value of 5, weak acidity and microwave time of 10 min. The acquired carbon dots own even particle size, narrow distribution range and fine fluorescence property.(2)By incubating the carbon dots with E.coli O157:H7 and utilizing the advantage of their small particle size and the endocytosis of bacteria, they enter the inside of the bacteria, and the fluorescent label and imaging of E.coli O157:H7 are achieved, showing that the carbon dots possesses fine biocompatibility and can penetrate the cell wall into the inside of the bacteria. In addition, the fluorescent label of the carbon dots on E.coli O157:H7 can produce multi-fluorescence, and under thelaunching of UV, blue and green light, the bacteria takes on fluorescence images of blue, green and red light. The excitation light source with its longer wavelengths can be adopted in the future fluorescence image application to reduce the interference of the autofluorescence of the target marker. The cytotoxicity of carbon dots is measured in the MTT method, and the result reveals that even if the highest concentration we use in our test is 1.2mg/m L, the cells still have a survival rate of over 80%, fully showing their advantage of low cytotoxicity.(3)The carbon dots modified by amino are covalently combined with antibodies with the EDC/NHS coupling technique, thus the immunofluorescent probe based on carbon dots is created and it enables carbon dots, a novel fluorescence nanomaterials,to be applied to the fluorescent antibody technique. The E.coli O157:H7 can be specifically identified in a short time, and it can create multi- fluorescence imaging.The immunofluorescent probe based on carbon dots can quickly detect the E.coli O157:H7. The detecting sensitivity reaches 103 CFU/m L, and it can be finished within1 h and detection can be quickly finished under the wild environment with portable spectrometer.Conclusion:The carbon dots of higher fluorescent quantum yield are successfully produced and are applied to the fluorescent antibody technique, thus constructing the immunofluorescent probes, specifically identifying the E.coli O157:H7 and reaching a higher sensitivity. It is believed that after the solving of the current shortcomings and problems, carbon dots, as a novel fluorescence nanomaterials, can be greatly promoted in the application of the immunofluorescence technique. Therefore, it is expected to be a new low-toxicity biosensor of independent intellectual property rights and to be applied to the detection of viruses and other bacterial microorganisms,which is of great value in theories and practical applications.