Functionalization Of Quantum Dots And Their Application In Biosensor And Bioimaging

Due to the unique optical and chemical properties,quantum dots?QDs?have been widely used in biological application.However,the properties of QDs still need be improved.The optimization of surface ligands is a simple and effective method to improve the stability of QDs.Besides,the biological functionalization of QDs is an important section for the biological application.Scores of researchers have established a series of methods for the biological functionalization of QDs,however,there are some problems with these methods,such as,stability and luminescence efficiency reduction caused in the modification process,precise biological functionalization of QDs,and so on.In addition,the application of functionalization of QDs in biolabeling and bioimaging remains to be further explored.In this paper,two types of QDs were prepared by using dithiol as stabilizers,S sources or reducing agents,which improved the optical and chemical stability of QDs.Then,the Rox-DNA functionalized CdZnTeS QDs,DBCO/N₃-DNA functionalized CdTe:Zn²⁺QDs and BHQ1-DNA functionalized CdTe:Zn²⁺QDs were prepared using bi-modification of both ends of DNA.We used Rox-DNA functionalized CdZnTeS QDs to construct ratiometric fluorescence probes and applied it to enzyme analysis.DBCO/N₃-DNA functionalization CdTe:Zn²⁺QDs were used for the labeling of protease and cancer cells.BHQ1-DNA functionalized CdTe:Zn²⁺QDs were used for imaging and labeling of single viral RNA.The main research contents are as follows:?1?CdTe/CdS QDs modified by various ligands were synthesized by introducing dithiol as ligand and releasing S^2-at high temperature.The pH,chemical and optical stability of QDs and core/shell QDs modified with different ligands were investigated.It was found that CdTe/CdS QDs modified by 2,3-dimercaptopropanesulfonic acid possessed the best stability and highest quantum dot yield?up to 80%?.Due to the strong absorption of CdTe/CdS QDs in the ultraviolet region,combined with the catalytic reaction based on alkaline phosphatase and the inner filtration effect of p-nitrophenol on QDs,the high-sensitive detection of alkaline phosphatase is realized.Then,the 2,3-dimercaptosuccinic acid was used as sulfur source,stabilizer and reducing agent to synthesize CdZnTeS QDs.The pH,optical,chemical stability and cytotoxicity of CdTe:Zn²⁺QDs and CdZnTeS QDs were investigated.It was found that the CdZnTeS QDs have better stability and lower cytotoxicity.A rapid and sensitive method for the detection of Cu²⁺and galactose oxidase was achieved due to the strong binding of mercapto and sulfonic group on the surface of CdZnTeS QDs with Cu²⁺.?2?Using CdZnTeS QDs synthesized in the previous chapter,a ratiometric fluorescent probe based on QDs and Rox was prepared via a one-step method.This method does not need any coupling reagent,and avoids the problem of decreasing the luminescence efficiency and stability of QDs caused in the coupling process.The sulfhydryl group and Te^2-on the surface of the QD can be oxidized by hydrogen peroxide,and the fluorescence of QDs was quenched.Therefore,the ratiometric fluorescent probe can be used for the high sensitive detection of hydrogen peroxide and glucose.The ratiometric fluorescent probe can also be used for visual detection of glucose.The diabetic patients can be distinguished from normal people with naked eyes.At the same time,the ratiometric fluorescent probe can be used to accurately determine the blood glucose concentration.In addition,the telomerase concentration in the urine of patients with bladder cancer was accurately determined based on the principle of QDs oxidated by hydrogen peroxide,which providing a new method for the early diagnosis of cancer.The dopamine and tyrosinase can be detected with the ratiometric fluorescent probe based on the principle of photo-induced electron transfer.The protamine and trypsin can be detected based on the principle of quantum dot aggregation and quenching caused by protamine.?3?A novel labeling reagent based on QDs,which one end was QD and the other end was azide or alkyne,was prepared by one-step method.In this method,the click-reaction reagent?DBCO or N₃?can be quickly modified to the surface of QDs,which avoid the problem of decreasing the luminescence efficiency and stability of QDs in the covalent coupling.This method can provide a new technical method for the preparation of QDs-labeling reagent based on click chemistry.The alkyne group was modified on the surface of glucose oxidase?GOx?through coupling reaction to produce the DBCO-GOx.The glucose oxidase-quantum dot complex?GOx-QDs?was prepared by the reaction of DBCO-GOx and azide-DNA functionalized QDs,which can be used for high sensitive detection of glucose and accurate determination of the blood glucose concentration.It was found that the GOx modified with QDs possess higher enzyme activity,and the QDs modified with glucose oxidase have better chemical stability.Then,the HeLa cells were rapidly labeled by DBCO-DNA functionalized QDs in the culture medium containing glycosyl compounds modified with azide?Ac₄ManNAz?and DBCO-DNA functionalized QDs.This method has many advantages,such as,high labeling efficiency,no chemical modification and excellent optical properties of the QDs.?4?On the basis of the work in chapter 3 and chapter 4,a type of quantum dot?QD?nanobeacons with controllable valencies was constructed.The QD nanobeacons were prepared via a one-pot hydrothermal method,and the BHQ1 and phosphorothioate co-modified DNA has been precisely conjugated with CdTe:Zn²⁺QDs.The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells.Additionally,single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells.QD nanobeacon-labeled HIV-1genomic RNAs were encapsulated in progeny viral particles,which can be used to track the uncoating process of single viruses.The current study provides a platform for nucleic acid labeling and imaging with high sensitivity,being especially meaningful for tracking of individual RNAs in live cells.