【作者】 张博；

【导师】 丁收年； 张庆；

【作者基本信息】 东南大学 ， 先进制造（专业学位）， 2023， 博士

【摘要】 癌症是世界上最致命的疾病之一,特别是死亡率很高的晚期癌症。癌症的早期诊断和治疗可以有效抑制晚期癌症的出现。然而,早期癌症缺乏明显的症状,难以通过影像学进行有效诊断。随着生物分析技术的发展,一些来自血液或组织中的小生物分子可以作为肿瘤标志物用于诊断早期癌症。此外,为了高效、准确地诊断癌症,往往需要同时检测多种肿瘤标志物。基于荧光编码微球的液相芯片技术因其检测灵敏度高、所需样品量少、多元分析能力强等优点引起了广泛关注,该技术在肿瘤标志物的多元检测方面具有广阔的应用前景。荧光微球是液相芯片技术的核心元件,制备粒径均一、荧光强度高、荧光分布均匀、分散性良好、表面富含功能基团的高质量荧光微球是液相芯片技术的研发重点;降低荧光编码微球基条形码的背景信号对提高液相芯片技术检测灵敏度至关重要;基于高质量荧光微球高效构建高容量编码库是提高液相芯片技术多元检测能力的关键。本文主要围绕荧光编码微球基液相芯片技术体系的构建及其对肿瘤标志物的高灵敏多元检测开展研究,主要研究内容有以下几个部分:（1）首先,通过“单步合成法”合成了两种发射波长的Cd Se/Zn S量子点。然后,通过膜乳化法制备了荧光强度高、荧光分布均匀、分散性和稳定性良好、表面基团丰富的Cd Se/Zn S量子点荧光编码微球,微球的基体材料为聚苯乙烯马来酸酐共聚物。接着,通过探究不同量子点浓度对微球荧光强度的影响确定了微球内双色量子点之间不发生聚集猝灭效应和荧光共振能量转移的浓度边界条件。根据实验所得的浓度边界条件简化多色荧光模型后得到了双色荧光模型,基于双色模型的条形码模拟策略能够有效预测微球尺寸分布对条形码信号的影响。采用条形码模拟策略后无需进行流式细胞仪读取并解码荧光编码微球的迭代试错实验即可构建条码库,该策略简化了迭代试错实验、提高了条码库的构建效率。最后,在模拟策略的指导下精确设计并高效构建了四个微球基条形码,进而基于微球基条形码构建了液相芯片技术体系并用其检测了三种肿瘤标志物CEA、CA125和CA199,其检测限分别为0.028 ng/m L、1.5 KU/L和0.8 KU/L。该液相芯片技术对肿瘤标志物具有良好的多元分析能力,有望进一步开发出多元检测能力更强的液相芯片技术,最终实现癌症的大规模筛查。（2）采用散射光信号与荧光信号的组合策略制备双信号荧光编码微球。首先,通过悬浮聚合法制备了较小粒径的聚苯乙烯微球（PS-S微球）并用二氧化硅包覆PS-S微球,随即将二氧化硅包覆后的PS-S微球羧基化并且在其表面偶联生物抗体。同理,对所购商品化较大粒径的PS-L微球和Si O₂-L微球表面进行羧基化并偶联生物抗体。然后,通过在上述微球表面构建的三明治免疫结构将Cd Te量子点偶联于微球表面,具有三明治免疫结构的PS-S微球、PS-L微球和Si O₂-L微球被流式细胞仪的激光激发后,在流式细胞仪的FSC和SSC通道中获得了仅用于微球编码的散射光信号,同时在流式细胞仪的FL1-H和FL2-H通道中获得了用于编码微球和量化分析目标物的荧光信号。散射光编码信号有效解决了常见的荧光码编码信号对检测信号的干扰问题,获得了具有较低背景信号的荧光编码微球。接着,优化了微球表面捕获抗体的偶联量,通过试剂（微球、捕获抗体、抗原、检测抗体）的单独孵育实验和两两组合孵育实验验证并识别了特异性结合和非特异性结合现象。最后,基于双信号荧光编码微球构建了高灵敏液相芯片技术体系,并对六种肿瘤标志物CEA、CA125、SCCA、AFP、NSE和CA724进行了高灵敏多元分析,其检测限分别为0.05 ng/m L、0.92 KU/L、0.28 ng/m L、0.09ng/m L和0.16 ng/m L。双信号编码策略为提高液相芯片技术的检测灵敏度提供了新思路,将有力促进液相芯片技术在高效、准确诊断早期癌症方面的广泛应用。（3）首先,通过在磁性Fe₃O₄纳米粒子表面包覆TiO₂壳层制得磁性Fe₃O₄/TiO₂纳米粒子。然后,利用膜乳化技术将可见光Cd Se/Zn S量子点和Fe₃O₄/TiO₂纳米粒子同时包埋进聚苯乙烯马来酸酐共聚物（PSMA）微球,制备了在可见光区具有较强荧光强度的磁性荧光编码微球,并且通过重力沉降法进一步筛分了微球。所制备磁性荧光编码微球具有粒径均一、荧光强度高、荧光分布均匀、分散性良好、表面基团丰富的优点。与Fe₃O₄纳米粒子相比,Fe₃O₄/TiO₂纳米粒子对可见光的吸收作用较弱以及在溶液和微球中对Cd Se/Zn S量子点的荧光猝灭效应较弱,所得磁性荧光微球的荧光强度更强。接着,用三种颜色的Cd Se/Zn S量子点和Fe₃O₄/TiO₂纳米粒子构建了三种单色磁性量子点条码库,每种条码库含有6个微球基条形码;用两种颜色的Cd Se/Zn S量子点和Fe₃O₄/TiO₂纳米粒子构建了含有30个微球基条形码的双色磁性量子点条码库;用两种颜色的Cd Se/Zn S量子点和Fe₃O₄纳米粒子构建了含有21个微球基条形码的双色磁性量子点条码库。结果表明Fe₃O₄/TiO₂纳米粒子能够有效提高磁性量子点编码微球的编码能力。最后,基于磁性荧光编码微球构建了高灵敏液相芯片技术体系。通过比较基于磁性荧光编码微球和非磁性荧光编码微球的液相芯片技术对四种肿瘤标志物的高灵敏多元检测性能,发现具有免疫磁分离的液相芯片技术的检测限更低,在癌症的早期诊疗中具有可行性和实用性。更多还原

【Abstract】 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.更多还原