Preparation,Characterization And Preliminary Application Of Bio-carbon Dots

Background:Carbon dots?CDs?,have the particular advantages in several aspects such as almost non-toxicity,stable characterization,small particle size and with little diversity.Meanwhile,it has good luminous performance as equal as traditional quantum dots,and better biological compatibility.So,CDs have been regarded as the potential alternatives to traditional quantum dots in biochemical tests.In general,the preparation of the carbon dots synthesized by different carbon sources have the same or similar characteristics,such as extraordinary for electron and light bleaching and the advantages of small size,but maybe different preparation of carbon source determined the surface functional groups and the content elements,different size and particle size distribution with different fluorescence excitation condition and quenching condition,these attracted many researchers'attention and sparked a fluorescent carbon dots on photoluminescence properties and practical application of research upsurge,such as biological imaging,chemical testing analysis,medicine-loading diagnosis and treatment research fields such as the common research achievements.However,we need to construct more convenient,more environmental and more controllable preparation of carbon preparation method,to further expand its actual applications,especially progress in the biochemical analysis aspects.Hyaluronic acid is a kind of acid mucopolysaccharide,with its unique molecular structure and the physical and chemical properties attain a variety of important physiological functions in the body,such as lubricating joints,regulating the permeability of blood vessel walls,regulating protein,diffusion and operating of the electrolyte to promote wound healing.It is particularly important that hyaluronic acid has special water retention,which is the best moisturizing substance found in nature,which is called the ideal natural moisturizing factor.Hyaluronic acid by?1->3?-2-acetyl amino-2-deoxidation-beta-D-glucose-?1-4?-O-beta-D-glucuronic acid disaccharides repeating units of a polymer and hydrolysis generated when a hexose amine?such as glucosamine?and a kind of uronic acid?e.g.,glucuronic acid?.As a biochemical reagent,it has become a biochemical drug with high clinical value.Hyaluronidase?HAase?belongs to the enzyme family that catalyzes the degradation of hyaluronic acid.Hyaluronic acid is the essential component of extracellular matrix.Hyaluronic acid can reduce the activity and viscosity of hyaluronic acid and improve the permeability of liquid in tissues.Candida Albicans is also called the white candida,widely exists in the nature,also exist in normal oral cavity,the upper respiratory tract,intestinal tract and the vagina,generally don't cause disease in normal body.It is pathogenic fungus for the special condition.Cryptococcus neoformans is Cryptococcus belongs to a kind of?Cryptococcus?,there are at least 38 species of genus Cryptococcus,including plain Cryptococcus?Cryptococcus albidus?and Laurent Cryptococcus?Cryptococcus laurentii?several kinds,such as in immunocompromised patients can also cause cryptococcosis.The morphology of the new Cryptococcus is round or oval in the lesion tissue,with a diameter of 5-10,and a thick polysaccharide capsule is surrounded by a layer of thick polysaccharide capsule,which is the main virulence factor.It is necessary to combine the advantages of fluorescence carbon dots with practical application of bio-sensing,imaging,detection and other fields to solve practical problems of basic scientific research.This topic reported before in the fluorescence of some preparation methods of carbon source and carbon source on the basis of,through the introduction of new natural carbon source,respectively with mushrooms,soybean and sesame seeds for preparation of precursor preparation of success,to broaden the scope of the choice of carbon source,from two aspects,further goal is time consuming and economic perspective actually reduce the cost of the preparation of fluorescent carbon point.And through to the preparation of carbon points respectively used for biological macromolecular compounds,hyaluronic acid and sensing analysis of hyaluronidase and candida albicans and selective detection of cryptococcus neoformans,aims to study the fluorescent carbon point the possibility of practical application in the field of biology and stability.Objective:The selection of natural carbon source mushrooms,beans,sesame seeds by turn,fluorescent carbon dots is obtained by step in the preparation of hydrothermal method,aims to explore extensively in the synthesis of precursor preparation of carbon dots,reduce the synthetic cost,simplify the preparation process,at the same time make it more simple and controllable.The photoluminescence and selectivity of the fluorescence carbon dots are analyzed as far as possible.Will mushroom as carbon source dots obtained from the preparation of carbon used for hyaluronic acid and hyaluronidase sensing analysis,to build a two-way to detect the contents of hyaluronic acid and hyaluronidase method;With soybean and sesame seeds for the preparation of carbon carbon source point for Cryptococcus neoformans and Candida albicans selectivity of bioprobe marker,aims to explore whether it can replace traditional quantum dots as a new type of selective fluorescent probes for specific pathogens.Methods:1.Preparation by hydrothermal method,will be given to natural carbon source for dry grinding processing in advance,according to certain proportion ultrapure water after becoming a member of high pressure reaction kettle,temperature,time,PH conditions can adjust itself according to different sort of carbon source,in order to achieve the optimal preparation conditions and stored after natural cooling and purification separation and lyophilization.2.The use of fluorescence spectrophotometer,UV-Vis,TEM,XRD,fluorescent carbon points obtained by DLS on characterization,using the absolute amount of fluorescence spectrometer when quantum dots yield rate to calculate the amount of fluorescence when quantum dots yield rate,using LSCM time observing fluorescent carbon point in biological imaging,actual effect in use the CKK-8 reagent and SH-hy5y,CATH-a cell test preparation carbon damage to cells.3.Using fluorescence spectrophotometer were measured by the mushroom of fluorescent carbon points under the environment of different ions and biological molecules fluorescence intensity,calculate the hyaluronic acid and hyaluronidase fluorescence quenching of carbon dots caused by specific concentration and recovery,and building linear relationship,people take a urine sample for actual application.4.Through injection and intranasal structure of cryptococcus neoformans infection mouse model,test the fluorescent carbon point in complex organisms environment whether still have the ability to selective detection of specific pathogenic fungi.Results:1.Using mushroom,soybean and sesame as carbon source,the fluorescence carbon point was successfully prepared,and the selection range of precursors of fluorescence carbon point was broadened.The preparation process of natural carbon source is more rapid and controllable,and the preparation cost is reduced.The temperature,time and PH in the preparation process were optimized,and the preparation method was improved and the preparation efficiency was improved.2.Get the each corresponding fluorescence intensity,fluorescent carbon dots maximum incidence and excitation wavelength,XPS and FTIR spectra and according to the functional groups on the surface of the peaks were deduced and calculated the amount of each material preparation of fluorescence rate;By LSCM photos were found in soybean and sesame seeds for the preparation of carbon carbon source point of cryptococcus neoformans and candida albicans selective adhesion and bacteria in the body for a long time to keep fluorescence;The MTT experimental results support the almost nontoxic fluorescence carbon dots.3.Preparation of fluorescent carbon dots with mushrooms as carbon source of photoluminescence performance is not affected by DNase?,papain,CO2+,Mn2+,Zn2+,Pb2+and Ba2+?concentration of 5 mg mL-1?,only to maintain a high level of selectivity and sensitivity of hyaluronic acid,once joined would immediately cause the fluorescence quenching,and it can be added under the condition of appropriate doses of hyaluronidase fluorescence recovery immediately,fluorescence recovery items without interference DNase?,RNase?,EXO?,papain,CO²⁺,Mn²⁺,Zn²⁺and Ba²⁺5 mg mL^-1for?all?.Finally,the linear relationship between hyaluronic acid and hyaluronic acid on fluorescence quenching and recovery was determined by trial and error.Fluorescence quenching and recovery mechanisms can be obtained by DLS measurement of particle size data profile inference and proof,finally in human urine samples have accurate hyaluronidase proved the validity and practicability of this method.4.The success of Cryptococcal infection mouse model is constructed,and the injection point of fluorescent carbon 2 hours after sterilization after slaughter in lung,frozen in mice lung slice of LSCM detected fluorescence in the photo,and fluorescence intensity is proportional to the degree of Cryptococcal infection in mice.Conclusion:These results are of carbon point of preparation,characterization and application in compound detection and pathogenic fungi to explore,through the analysis of the research process and result,for our further understanding the nature of the carbon point and broaden its in the field of biological macromolecular sensing analysis and selective marking pathogens to specific applications,it is concluded that the fluorescent carbon has excellent photoluminescence performance and for some specific biological molecules or microorganisms has the ability of selective adsorption,and the selectivity of a highly intelligent strong anti-interference ability,can in a complicated physiological and biochemical environment is not affected by other interference ions or compounds and expressed correctly,this feature in the analysis and testing in the field of fluorescent sensor has positive significance in practical application,the hope can give other research direction in the field of human health related to provide some reference.