Synthesis Of Carbon Dots And Their Application In Biosensing

As carbon-based zero-dimensional materials,carbon dots（CDs）are new member of carbon based nano fluorescence materials family,typically smaller than 10 nm in size.CDs have the advantages of good water solubility,good biocompatibility,stable fluorescence,photobleaching resistance,chemical stability,easy functionalization,easy preparation,low cost,etc.CDs have been mostly used in sensing,vivo imaging,photoelectric conversion and other fields.CDs have been used as fluorescent materials in the field of biosensing,and they are capable of performing sensitive,simple,and selective sensing of targets.One of the principles of CDs for biosensing is similar to traditional fluorescent materials used in sensing.Based on the high single-particle brightness,CDs were functionalized by combined with other molecules or proteins to provide fluorescent signals in biosensing.Another principle of CDs for biosensing was based on the internal structure and surface groups of CDs,both of which affected the fluorescence performance of CDs,and allow them to selectively respond to different targets.Many researches have been studied on CDs combined other materials including fluorescent organic molecules,aptamers,dye,etc used in sensing.However,the combination of CDs and antibodies has rarely been used for highly selective protein sensing.Owing to the biocompatibility and fluorescence stability of CDs,they can replace dye or fluorescent organic molecules to be used as alternative dyes for protein fluorescence sensing and imaging.Meanwhile,in the sensing mode where the targets directly acted on CDs,the internal structure and surface functional groups of CDs are mainly changed by two ways.They are one-step synthesis of heteroatom-doped CDs or functional modification on the surface of prepared CDs.Heteroatom doping can improve the performance of CDs and is easy to operate,which is more efficient than functional modification on the surface of CDs.N element doping increases the QY of CDs,and few studies of metal element doped CDs beed reported,it is crucial to study metal doping in CDs.In this paper,non-metal-doped CDs combined with antibodies were used for immunosorbent assays to expand application of CDs.Simultaneously,the synthesis and properties of metal element and non-metal element codoped CDs were studied.The prepared codoped CDs were applied to develop a ratiometric fluorescent probe for sensing of H₂O₂ and metabolites involved in H₂O₂-generation reaction.The main researches are summarized as follows:1.Carbon dots based immunosorbent assay for the determination of GFAP in human serum.Glial fibrillary acidic protein（GFAP）is expressed in the central nervous system and the level of GFAP normally rises with brain injury and astroglial tumors.So,serum GFAP is used as a marker for diagnosing various types of brain damage and astroglial tumors.In this study,a new sensor based on CDs-linked with antibodies to specifically detect GFAP in human serum was developed.Anti-GFAP（Ab1）linked with protein A/G agarose resin（PA/G）as capture antibody（PA/G-Ab1）and anti-GFAP（Ab2）labeled with carbon dots as detection antibody（CDs-Ab2）were prepared firstly.Then the CDs-linked antibodies immunosorbent assay method was constructed based on the sandwich conjunction reaction among PA/G-Ab1,GFAP and CDs-Ab2.The developed method using the fluorescence of PA/G-Ab1-GFAP-Ab2-CDs as the direct signal enabled the proposed immunesensor to detect GFAP sensitively with a linear range of 0.10-8.00 ng mL^-11 and a limit of detection（LOD）25 pg mL^-1.This method was applied to the determination of GFAP in human serum by standard addition method,and the results showed high accuracy and precision.Considering the easy synthetic process and excellent performance of this probe,this investigation has a great potential to be used to monitor GFAP in clinic2.Synthesis of a novel N,Cu codoped carbon dots.In this work,a new kind of N,Cu codoped carbon dots（N/Cu-CDs）was prepared via a facile one-pot hydrothermal method by using citric acid monohydrate,copper acetate monohydrate and diethylenetriamine.The structure and fluorescence properties of N/Cu-CDs were characterized by high-resolution transmission electron microscopy（TEM）,fluorescence spectrofluorometer（FL）,FT-IR spectrometer（FT-IR）,UV-Visible spectrophotometer（UV-vis）and X-ray photoelectron spectroscopy（XPS）.The prepared N/Cu-CDs with a high quantum yield（QY,50.1%）showed excitation-independent emission at 460 nm.The surface traps on N/Cu-CDs were effectively passivated by C–O,C=O,COO–,C–N,and N–Cu–N,possibly leaded to the excitation-independent fluorescence behavior.The prepared N/Cu-CDs could be used in biosensor.3.A ratiometric fluorescence universal platform based on N,Cu codoped carbon dots to detect metabolites participating in H2O2-generation reactions.The prepared N/Cu-CDs were applied to establishing a ratiometric fluorescence probe toward H₂O₂ based on the inner filter effect（IFE）between N/Cu-CDs and DAP（2,3-diaminophenazine,the oxidative product of o-phenylenediamine）,and provided a ratiometric fluorescence universal platform for detection of the metabolites participating in H₂O₂-generation reactions（cholesterol and xanthine）.With the increase of H₂O₂ concentrations,the fluorescence intensity of N/Cu-CDs（at 460 nm）decreased gradually while the fluorescence intensity of DAP（at 572 nm）increased.The ratiometric fluorescence intensity(I₅₇₂/I₄₆₀)is linearly correlated with the concentration of H₂O₂.As the proof-of-concept demonstration,the proposed method was demonstrated to be ultrasensitive and highly selective for cholesterol and xanthine assay,respectively.A good linear relationship（r=0.9992）was obtained in the concentration range of 0.05-100μM from cholesterol detection with detection limits of 0.03μM,and the sensor could detect xanthine in a range of 0.25–75.00μM（r=0.9998）with a LOD of 0.1μM.The fluorescence probe built was applied to the determination of cholesterol and xanthine in human serum with satisfactory results.