Preparation Of Aqueous CuInS₂/ZnS Quantum Dots And Their Application For The Quantitative Detection Of Cardiovascular Disease Biomarkers

Quantum dots（QDs）were widely used in biomedical fields such as cell labeling,in vivo imaging and in vitro detection due to their wide excitation and narrow emission,strong luminescence,good stability and adjustable size.However,most studies of quantum dots in vitro detection were based on cadmium-containing QDs,and the toxicity of Cd²⁺in vivo and in vitro limited their practical application.As a result,non-toxic and environmentally friendly Cd-free QDs have become the focus of people’s research.Among non-cadmium quantum dots,CuInS₂/ZnS have attracted a lot of attention from researchers by being considered non-toxic and with a high quantum yield of 60%-80%.In order to obtain high quantum yield and good stability of CuInS₂/ZnS QDs,1-dodecyl mercaptan（DDT）was usually used as sulfur source and ligand in the preparation of organic phase.Therefore,the surface of the resulting QDs contained thiol ligands in addition to oleic acid ligands.Due to the strong coordination force between the thiol molecule and the quantum dots,the CuInS₂/ZnS quantum dots containing the thiol ligand were often subject to a sharp decrease in quantum yield and quantum dot aggregation and precipitation by using the silica coating method,the amphiphilic polymer modification and the ligand exchange for surface modification,and it is difficult to obtain non-cadmium CuInS₂/ZnS quantum dots suitable for highly sensitive in vitro detection.Therefore,in this paper,we focued on the preparation of highly fluorescent and stable water-soluble CuInS₂/ZnS QDs.Based the obtained CuInS₂/ZnS QDs,combining the fluorescent immunosorbent assay（FLISA）,the cardiovascular disease-related biomarkers such as cardioid fatty acid binding protein（H-FABP）and C-reactive protein（CRP）were used as the targets to verify their applicability in vitro diagnostic.The main contents were as follows:1.The preparation of aqueous CuInS₂/ZnS@Si O₂ QDs and their application in QD-FLISA for H-FABP and CRP.The surface ligands of oil-phase CuInS₂/ZnS QDs were oleic acid and thiols.Due to the strong coordination force between the thiol molecules and the surface of the quantum dots,thiols ligands could not be replaced by hydrolyzed TEOS during silica coating.To this end,we have improved the conventional silica coating method,and the water-soluble CuInS₂/ZnS@Si O₂ quantum dots were successfully prepared by pre-coating silicon and then step-by-step silicon wrapping by reversed-phase microemulsion,without replacing thiol ligands.First,during pre-silicon coating,the TEOS monomer first replaced the oleic acid ligands on the surface of the quantum dots to form a discontinuous thin layer of silica at the sites.Then,a dense silica shell layer was gradually formed on the discontinuous silica thin layer by adding TEOS step by step.This surface modification method did not involve the substitution of thiol ligands,so the synthesized CuInS₂/ZnS@Si O₂QDs had good acid and alkali resistance,photobleaching resistance and thermal stability.Under the optimal conditions,the quantitative detection of H-FABP and CRP was realized by CuInS₂/ZnS@Si O₂-FLISA,respectively.The detection range of H-FABP was 2-200 ng/m L with the linear correlation coefficient R² of0.996.The detection of limit（LOD）was calculated to 1.41 ng/m L,and the recoveries ranged from 98.8%to105.1%.The detection range for CRP was 2-800 ng/m L with the linear correlation coefficient R² of 0.996.The LOD for CRP was 1.08 ng/m L,and the recoveries were between 98.5%and 106.5%.The CuInS₂/ZnS@Si O₂-FLISA also showed good specificity.The above rusults showed that the aqueous CuInS₂/ZnS@Si O₂ quantum dots prepared based on the improved silica coating method can be used as excellent lable materials for in vitro diagnostics.2.The preparation of aqueous CuInS₂/ZnS fluorescent microspheres and their application for the sensitive detection of H-FABP.In order to further improve the detection sensitivity and expand the application range,we prepared quantum dot fluorescence microspheres（QBs）containing a large number of CuInS₂/ZnS quantum dots,which improved the fluorescence intensity of a single microsphere and thus improve the detection sensitivity.Firstly,oil-soluble CuInS₂/ZnS QDs were confined in the microemulsion droplets formed by cationic surfactantsurfactant DTAB based on the oil-in-water（O/W）microemulsion system.At the same time,in order to increase the stability and water solubility of the microspheres,the DTAB on the surface of the microspheres was replaced by the non-ionic surfactant CO-520.Then the microspheres were coated with silica shell and carboxylated by winding PAA,and the high-quality CuInS₂/ZnS QDs fluorescent microspheres（QBs）were successfully prepared.In order to eliminate the fluorescence resonance energy transfer between QDs,the oil-phase CuInS₂/ZnS QDs were 13 nm in size.Since the microspheres were stabilized by the hydrophobic van der Waals interactions between the hydrophobic ligands（oleic acid and thiol）on the surface of the quantum dots and the hydrophobic chains of DTAB,this method was suitable for QDs with various ligands.The QBs had good dispersibility with an average particle size of 208±10 nm and a silicon layer thickness of 11±2 nm.The stability test showed that the microspheres have good acid and alkali resistance and thermal stability.Under the optimal conditions,the quantitative detection of H-FABP was realized by QBs-FLISA.The detection range of H-FABP was 1-200 ng/m L with the linear correlation coefficient R² of 0.991.The LOD was 0.48 ng/m L and the sensitivity was nearly 3 times higher than that of CuInS₂/ZnS@Si O₂-FLISA,indicating the prepared QBs could effictively impove the detection sensitivity.In addition,the recovery and precision experiments also showed that the detection results have good accuracy and stability.