【作者】 王伟；

【导师】 曹霞；

【作者基本信息】 江苏大学 ， 药学， 2020， 硕士

【摘要】 原发性胆汁性胆管炎(PBC)在发病初期无明显症状,不易发现,患者确诊时常常已进入PBC晚期,此时病情已经严重到肝硬化或者肝衰竭的地步,治疗手段有限。因此,迫切需要开发一种有效用于PBC等早期无明显症状疾病的预防及诊断方法。荧光免疫层析技术是一种重要的体外疾病诊断方法,该方法简单快速,无需专业人员,在家即可实现自检,在疾病早期诊断应用中潜力巨大。目前应用较多的荧光免疫层析是胶体金免疫层析,但胶体金灵敏度有限无法满足荧光免疫层析定量检测和多种物质同时检测的需求。作为一种新型荧光标记物,量子点耐光漂白性能好,激发光谱宽而发射光谱窄,其灵敏度要高于胶体金,因而成为荧光免疫层析中荧光标记物的热门选择。葡萄球菌蛋白A(SPA)可与人血清中的抗体生物分子结合而不影响该抗体与抗原的结合活性,同时还能结合荧光标记物。SPA的这种独特生物活性为荧光免疫层析同时检测多种物质提供了一种思路。基于此,本文制备出荧光性能优异的碲化镉量子点-SPA偶联物用于荧光免疫层析同时检测多种物质,为荧光免疫层析在疾病早期诊断中的进一步应用提供基础。第一章综述本章首先介绍了原发性胆汁性胆管炎不同地区的发病情况以及诊断方法。然后,介绍了量子点的分类、制备方法、表面修饰方法以及量子点与蛋白生物大分子偶联的方法。最后介绍了荧光免疫层析技术的原理、量子点荧光免疫层析技术的优点以及荧光免疫层析技术的发展方向。第二章碲化镉量子点的制备及其表征本章制备了荧光性能较好的碲化镉量子点并采用多种方法对其进行表征。首先,采用水相制备法,以巯基丙酸(MPA)作为修饰剂,制备出羧基修饰的水溶性碲化镉量子点,并对其最佳反应时间进行探究,然后对碲化镉量子点的粒径、紫外吸收光谱、荧光发射光谱以及稳定性等进行表征。结果显示,随着反应时间的增加,碲化镉量子点的最大发射波长呈现红移趋势,荧光强度呈现先增加后降低的趋势,最佳反应时间为3 h,粒径为9 nm,电位为-20 mV,荧光量子产率为33%,在酸性环境中稳定性较差。第三章碲化镉量子点和葡萄球菌蛋白A偶联条件的优化本章将自制的荧光性能优良的碲化镉量子点与葡萄球菌蛋白A进行偶联。采用单因素考察法逐步考察了EDC用量、NHS用量、反应时间以及SPA用量对偶联效果的影响,筛选出最佳反应条件,制备出荧光性能较好的碲化镉量子点-SPA偶联物。对该偶联物的粒径、电位、紫外吸收光谱、荧光发射光谱以及生物活性等进行了表征。结果显示,偶联物粒径为55 nm,电位为-26.9 mV,紫外吸收和荧光发射光谱相比偶联前发生红移,说明偶联物粒径增大,间接证明偶联物制备成功,随后采用斑点验证证明该偶联物仍具有生物活性,可与SPA抗体结合。第四章量子点荧光免疫层析试纸条的制备本章旨在将自制的碲化镉量子点-SPA偶联物用于荧光免疫层析同时检测PBC特征抗体(AMA-M2抗体和gp210抗体),为基于SPA偶联物的荧光免疫层析实现同时检测多种物质提供基础。更多还原

【Abstract】 Primary biliary cholangitis(primary biliary cholangitis,PBC)is an autoimmune disease that has no obvious symptoms in the early stages and is difficult to find.When the patient was diagnosed with PBC,the condition had already progressed to cirrhosis or liver failure,and it was difficult to cure.Therefore,we need a disease early diagnosis method for the prevention of early asymptomatic diseases such as PBC.Fluorescence immunochromatography is an important in-vitro diagnostic method for diseases.It is simple and fast,and it can be self-checked at home without professionals,which has great potential for early diagnosis of diseases.At present,the most widely used fluorescent immunochromatography is colloidal gold immunochromatography,but the sensitivity of colloidal gold cannot meet the needs of quantitative detection of fluorescent immunochromatography and simultaneous detection of multiple substances,which limits the further application of fluorescent immunochromatography.Quantum dots,a new type of fluorescent semiconductor nanomaterials,have better fluorescent properties than organic fluorescent dyes and colloidal gold.Quantum dots have good photobleaching resistance,wide excitation spectrum and narrow emission spectrum.As a fluorescent marker,its sensitivity is higher than that of colloidal gold,so it has become a popular choice for fluorescent markers in fluorescent immunochromatography.Staphylococcal protein A(SPA)can combine to antibody biomolecules in human serum without affecting the binding activity of the antibody to antigen,and at the same time it can also be combined to fluorescent markers.This unique biological activity of SPA provides an idea for the simultaneous detection of multiple substances by fluorescence immunochromatography.Based on this,quantum dot-SPA conjugates with excellent fluorescence properties were prepared for fluorescent immunochromatography to simultaneously detect multiple substances,which provided a basis for the further application of fluorescent immunochromatography in the early diagnosis of diseases.Chapter 1 ReviewThis chapter first introduces the incidence and diagnosis of primary biliary cholangitis in different regions.Then,the classification of QDs,preparation method of QDs,surface modification method of QDs and coupling method of protein biomacromolecule with QDs are introduced.Finally,the principle of fluorescence immunochromatography,the advantages of quantum dot fluorescence immunochromatography and the development direction of fluorescence immunochromatography are introduced.Chapter 2 Preparation and Characterization of CdTe Quantum DotsIn this chapter,cadmium telluride quantum dots with good fluorescence properties are prepared and characterized by various methods.First,the aqueous phase preparation method was used to prepare mercaptopropionic acid as a modifier to prepare carboxylmodified water-soluble cadmium telluride quantum dots.The reaction time of quantum dot preparation was investigated,and various methods such as particle size,ultraviolet absorption and fluorescence emission spectroscopy were used for characterization.The results show that with the extension of the reaction time,the maximum emission wavelength of cadmium telluride quantum dots shows a red-shifted trend,and the fluorescence intensity tends to increase first and then decrease.The optimal reaction time is 3 h,the particle size is 9 nm,the potential is-20 mV,the fluorescence quantum yield is 33%,and the stability is poor in an acid environment.Chapter 3 Optimization of coupling conditions of CdTe quantum dots and staphylococcal protein AIn this chapter,CdTe quantum dots with excellent fluorescence properties prepared in Chapter 2 are used to couple with staphylococcal protein A.The influence of the amount of EDC,NHS,SPA and the reaction time on the coupling effect was investigated using a single factor inspection method.The optimal reaction conditions were screened out to prepare the CdTe quantum dot-SPA conjugates with better fluorescence performance.The particle size,potential,ultraviolet absorption spectrum,fluorescence emission spectrum and biological activity of the conjugates were characterized.The results showed that the particle size of the conjugates was 55 nm and the potential was-26.9 mV.The ultraviolet absorption and fluorescence emission spectra were red-shifted,indicating that the particle size of the conjugates increased,which indirectly proved that the conjugates was successfully prepared.Subsequently,spot verification was used to prove that the conjugates was still biologically active and could combine to SPA antibodies.Chapter 4 Preparation of quantum dots fluorescence immunochromatography test stripThe purpose of this chapter is to use the CdTe quantum dots-SPA conjugates prepared in Chapter 3 for fluorescent immunochromatography to detect PBC characteristic antibodies(AMA-M2 antibody and gp210 antibody).It provides the basis for fluorescent immunochromatography based on SPA to detect multiple substances simultaneously.更多还原