Preparation Of Water-soluble CdSe/ZnS Quantum Dots And Their Highly Sensitive Detection Of Infectious Biomarkers

Semiconductor quantum dots are becoming important fluorescent labeling materials in the biological field because of their excellent optical properties,such as continuously tunable emission spectra with size and component,high fluorescence quantum yields,and good photochemical stability.Especially in the field of in vitro diagnostics,the high fluorescence and stability of quantum dots can not only extend their lifetime as fluorescent probes,but also improve the sensitivity of the assay results.The high quality quantum dots synthesized by high temperature organic synthesis with hydrophobic ligands on their surfaces,making it difficult directly perform biorelevant applications,so surface modification of quantum dots to obtain biocompatibility becomes the key to their biomedical applications.There are many types of hydrophobic ligands on the surface of quantum dots,and the surface modification methods of quantum dots based on oleic acid/oleic amine ligands are relatively mature.However,researchers will choose thiol ligands to improve the quantum yield and stability of quantum dots during the synthesis process,due to the strong coordination force between thiol molecules and Cd²⁺on the surface of quantum dots,while the common ligand exchange method and silane coating method are not applicable to the water transfer of quantum dots based on thiol ligands due to the need to replace the original ligands on the surface.Therefore,a wide range of applicable and effective surface modification method is important.Based on this,in this thesis,we used the amphiphilic polymer coating method and the microemulsion and silica double protection method to modify the surface of hydrophobic quantum dots based on thiol ligands,and applied the synthesis scheme of the microemulsion method to prepare quantum dot nanospheres with different ligands to prepare monochromatic quantum dot nanospheres and bicolor nanospheres,respectively.Based on the quantum dot fluorescence immunosorbent assay（QD-FLISA）,the detection of two infectious biomarkers C-reactive protein（CRP）and serum amyloid A（SAA）was achieved with high sensitivity,respectively,and the simultaneous detection of the two biomarkers was realized.The details include:（1）Preparation of polymer-coated-mononuclear water-soluble quantum dots and quantitative detection of the infectious biomarker C-reactive protein（CRP）.Based on the previous experimental basis,We coated green oil-soluble Cd Se/Zn S quantum dots（517 nm）based on thiol ligands using amphiphilic polymers（PMAH）with different mass ratios,with good dispersion and uniform size.After the investigation of their stability,it was found that the best stability was achieved when QD:PMAH=1:1.5.The linear range was5-1000 ng/m L,and the limit of detection（LOD）was calculated to be 4.67 ng/m L with good reproducibility.（2）Preparation of quantum dot fluorescent nanospheres and quantitative detection of infectious biomarkers CRP.The microemulsion and silica double protection method was used to prepare quantum dot nanospheres.Firstly,the hydrophobic green quantum dots with thiol ligands were entrapped in micelles formed by dodecyltrimethylammonium bromide（DTAB）under the action of a stirrer,and the nanospheres were wound by polymer polyvinylpyrrolidone（PVP）to make them more homogeneous.Next,the nanospheres were coated with silicon dioxide and a hydrophilic layer to further improve their stability.Under the dual protection conditions of PVP and SiO₂,the prepared nanospheres not only have higher fluorescence intensity,but also exhibited greater stability than mononuclear water-soluble quantum dots under different conditions.The QD-FLISA was used as a fluorescent probe for the quantification of CRP antigen in the range of 1-1,000 ng/m L with a limit of detection（LOD）of 0.45 ng/m L.The sensitivity was improved by a factor of 10 fold compared with the mononuclear water-soluble quantum dots in the second chapter,and the quantum dot nanospheres FLISA was also very reproducible and stable.（3）Preparation of two-color quantum dot fluorescent nanospheres and their simultaneous detection of CRP and SAA.In this section,to verify the generality of the double-protection method of microemulsion and silica for the preparation of quantum dot nanospheres,we used this scheme to prepare monochromatic（red）quantum dot nanospheres based on oleic acid/oleylamine ligands and tried to prepare mixed-color quantum dot nanomicrospheres using green（517 nm）as well as red（628 nm）oil-soluble Cd Se/Zn S quantum dots at different mass feeding ratios.The red quantum dot fluorescent nanospheres also showed good stability under different conditions,and the quantitative detection of SAA was performed in the range of 1-1,000 ng/m L with the lowest limit of detection（LOD）of 0.36 ng/m L after biofunctionalization.And then,the green and red quantum dot nanospheres were coupled with CRP and SAA antibodies respectively,to achieve simultaneous detection of CRP and SAA in one well,with a detection range of 1-1,000 ng/m L,and the limits of detection（LOD）of 0.69 ng/m L and 0.83 ng/m L,respectively.In addition,we used the prepared two-color quantum dot nanospheres as probes for Hela cell imaging experiments,and compared with the traditional fluorescent probes,this nanospheres exhibited extremely high fluorescence intensity,which also showed great advantages in the field of cell imaging.