Suspension Array Technology Based On Quantum Dot Encoded Microspheres And Their Applications

Cancer is one of the deadliest diseases in the world,especially advanced cancer,which has a high mortality rate.Early diagnosis and treatment of cancer can effectively inhibit the appearance of advanced cancer.However,early cancers lack obvious symptoms and are difficult to diagnose effectively by imaging.With the development of bioanalysis techniques,some small biomolecules from blood or tissues can be used as tumor markers to diagnose early cancer.In addition,in order to diagnose cancer efficiently and accurately,it is often necessary to detect multiple tumor markers simultaneously.Suspension array technology based on fluorescent coded microspheres has attracted wide attention due to its advantages such as high sensitivity,small sample size and strong multivariate analysis ability.This technique has a broad application prospect in the multivariate detection of tumor markers.Fluorescent microspheres are the core components of suspension array technology.The preparation of high-quality fluorescent microspheres with uniform particle size,high fluorescence intensity,uniform fluorescence distribution,good dispersion and rich functional groups on the surface is the focus of research and development of suspension array technology.It is very important to reduce the background signal of fluorescent coding microsphere-based barcodes to improve the detection sensitivity of suspension array technology.Efficient construction of high-capacity coding library based on high quality fluorescent microspheres is the key to improve the multi-detection capability of suspension array technology.This dissertation mainly focuses on the construction of fluorescence coding microsphere-based suspension array technology system and its highly sensitive multivariate detection for tumor markers.The main research content includes the following parts:（1）Firstly,Cd Se/Zn S quantum dots with two emission wavelengths were synthesized by single step synthesis method.Then,Cd Se/Zn S quantum dot fluorescence coding microspheres with high fluorescence intensity,uniform fluorescence distribution,good dispersion and stability and abundant surface groups were prepared by membrane emulsification method.The matrix material of microsphere is polystyrene maleic anhydride copolymer.Next,the boundary conditions for the absence of aggregation quenching effect and fluorescence resonance energy transfer between bicolor quantum dots within microspheres were determined by exploring the influence of different quantum dot concentrations on the fluorescence intensity of microspheres.A two-color fluorescence model was obtained by simplifying multicolour model according to the experimental boundary conditions.The barcode simulation strategy based on two-color model can effectively predict the influence of microsphere size distribution on barcode signal.After adopting barcode simulation strategy,the barcode library can be constructed without the iterative trial and error experiment of flow cytometer reading and decoding fluorescence encoding microspheres.This strategy simplifies iterative trial and error experiments and improves the efficiency of barcode library construction.Finally,four microsphere-based barcodes are designed and constructed efficiently under the guidance of the simulation strategy.Furthermore,suspension array technology system was constructed based on microsphere based barcodes and used to detect three tumor markers CEA,CA125,and CA199,with detection limits of 0.028 ng/m L,1.5 KU/L,and 0.8 KU/L,respectively.The suspension array technology has good multivariate analysis ability for tumor markers,and it is expected to further develop the suspension array technology with stronger multivariate detection ability,and finally realize the large-scale screening of cancer.（2）The combination strategy of scattered light signal and fluorescence signal was used to prepare double-signal fluorescence coded microspheres.Firstly,polystyrene microspheres（PS-S microspheres）with small particle size were prepared by suspension polymerization and coated with silica.The PS-S microspheres coated with silica were carboxylated and bioantibody was conjugated on their surfaces.Similarly,the surface of PS-L microspheres and Si O₂-L microspheres with large sizes purchased were carboxylated and coupled with biological antibodies.Then,Cd Te quantum dots were coupled to the surface of the microsphere by constructing sandwich immune structures on the surface of the microsphere.PS-S microspheres,PS-L microspheres and Si O₂-L microspheres with sandwich immune structure were excited by the flow cytometer laser,and the scattered light signals were obtained in the FSC and SSC channels of flow cytometer.Fluorescence signals for encoding microspheres and quantifying targets were obtained in FL1-H and FL2-H channels of flow cytometry.The scattering light coding signal effectively solves the problem of interference of common fluorescence coding signal to detection signal and fluorescent coding microspheres with low background signal are obtained.Next,the coupling amount of the captured antibody on the surface of the microsphere was optimized.Specific and non-specific binding phenomena were verified and identified through individual incubation experiments and pairwise combination incubation experiments of reagents（microspheres,captured antibodies,antigens,and detection antibodies）.Finally,a highly sensitive suspension array technology system was constructed based on double-signal fluorescence coded microspheres.A highly sensitive multivariate analysis was conducted on six tumor markers CEA,CA125,SCCA,AFP,NSE,and CA724,with detection limits of 0.05 ng/m L,0.92 KU/L,0.28 ng/m L,0.09 ng/m L,and 0.16 ng/m L,respectively.The dual-signal coding strategy provides a new approach to improving the detection sensitivity of sensitive suspension array technology,and will effectively promote the widespread application of sensitive suspension array technology in the efficient and accurate diagnosis of early cancer.（3）Firstly,magnetic Fe₃O₄/TiO₂ nanoparticles were prepared by coating TiO₂ shells on the surface of magnetic Fe₃O₄ nanoparticles.Then,visible light Cd Se/Zn S quantum dots and Fe₃O₄/TiO₂nanoparticles were simultaneously embedded into polystyrene maleic anhydride copolymer（PSMA）microspheres by membrane emulsion technique.Magnetic fluorescent coded microspheres with strong fluorescence intensity in visible region were prepared,and the microspheres were further screened by gravity sedimentation.The prepared magnetic fluorescence coded microspheres have the advantages of uniform particle size,high fluorescence intensity,uniform fluorescence distribution,good dispersion and abundant surface groups.Compared with Fe₃O₄ nanoparticles,Fe₃O₄/TiO₂nanoparticles have weaker absorption of visible light and weaker fluorescence quenching effect on Cd Se/Zn S quantum dots in solution and microspheres,resulting in stronger fluorescence intensity of the obtained magnetic fluorescent microspheres.Next,three monochromatic magnetic quantum dot barcode libraries were constructed using three colored Cd Se/Zn S quantum dots and Fe₃O₄/TiO₂nanoparticles,each containing six barcodes;A dual color magnetic quantum dot barcode library containing 30 barcodes was constructed using two colors of Cd Se/Zn S quantum dots and Fe₃O₄/TiO₂nanoparticles;A dual color magnetic quantum dot barcode library containing 21 barcodes was constructed using two colors of Cd Se/Zn S quantum dots and Fe₃O₄ nanoparticles.The results indicate that Fe₃O₄/TiO₂ nanoparticles can effectively improve the encoding ability of magnetic quantum dot encoded microspheres.Finally,a highly sensitive suspension array technology system was constructed based on magnetic fluorescence encoded microspheres.By comparing the highly sensitive multivariate detection performance of suspension array technology based on magnetic fluorescence encoded microspheres and non-magnetic fluorescence encoded microspheres for four tumor markers,it was found that suspension array technology with immunomagnetic separation has lower detection limits,which is feasible and practical in the early diagnosis and treatment of cancer.