分子印迹荧光纳米粒子的制备及其在免疫分析中的应用
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摘要
荧光纳米粒子由于其相对高的荧光强度,不易光漂白,容易标记,低毒和易于制备等特点,使得其在生物医学领域有着广泛的应用前景。目前主要有三类纳米标记物,分别是量子点,核壳型纳米粒子和金属纳米粒子,它们在应用上各有优缺点。在核壳型荧光纳米粒子中,染料泄露一直是难以解决的困难之一。人们采用了各种方法来减少染料的泄露,但都只能在一定程度上缓解这个问题。因此,制备出一种稳定的核壳型荧光纳米粒子是研究方向之一。本文利用分子印迹模板分子和功能单体以氢键方式结合作用力强的优点,制备出一种基于分子印迹原理的新型荧光纳米颗粒,并将其用于荧光免疫分析中。
第1章综述对荧光纳米粒子及其在免疫分析中的应用进行了综述。
第2章分子印迹荧光纳米粒子的制备与表征采用沉淀聚合法,甲基丙烯酸为功能单体,三羟甲基丙烷三甲基丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,乙腈为溶剂,制备了一种基于分子印迹原理的包裹荧光素的新型荧光微球。探讨了交联剂种类及其他反应条件对微球形貌、光稳定性和荧光强度的影响。结果表明,在最佳条件下制得的荧光微球粒径约为100nm,并具有光稳定性好、泄漏率低的特点。
第3章丁基罗丹明B分子印迹型荧光纳米粒子标记免疫分析法测定白介素-4采用分子印迹荧光纳米粒子作为标记物,双抗体夹心法为免疫检测模式,荧光显微成像为分析方法,对白介素-4进行了检测。用Image-pro plus 6.0软件对采集到的图像进行定量分析。检出限0.3ng/mL,对2.0 ng/mL白介素-4测定11次的RSD为3.2%。结果表明,该法具有操作简单,结果直观的特点。
第4章丁基罗丹明B分子印迹型荧光纳米粒子标记免疫分析法测定人血清中甲胎蛋白采用分子印迹荧光纳米粒子作为标记物,抗体—抗原—抗体—标记二抗的免疫反应模式,结合荧光显微成像技术,对甲胎蛋白进行了检测。采用Image-pro plus 6.0软件对采集到的图像进行定量分析。检出限0.2ng/mL。并对血清样品进行了测定。结果表明,该法具有操作简单,灵敏度高,结果直观的特点。
As a member of multitudinous fluorescent labels, dye-doped nanoparticles have widespread prospects of application in the biology and medicine domain because of its excellent characters such as the strong fluorescence intensity, easy surface modification, low toxicity and easy preparation. However, the dye leakage from the matrix is still a problem no matter which kind of matrix was used. In order to solve this problem, lots of methods have been employed in the issued papers. Conjugation of the dye with the precursor of nanoparticles matrix, transforming the solubility of dye, creating the electrostatic attraction between of FNPs matrix and dye, increasing the size of dye, and so on, are the usually used methods. To a certain extent, these methods could decrease the leaking of the dye from the NPs matrix, but most of the methods have their intrinsic disadvantages. For example, it is very difficult to conjugate the dye without active group with the precursor of nanoparticles matrix. In this study, a new type of fluorescent nanoparticles (FNPs) has been developed with precipitation polymerization.
Chapter One We have reviewed nanoparticles and its application in immunoassay. The research section contains three subunits.
Chapter Two A novel kind of fluorescent nanoparticles (FNPs) has been prepared using a precipitation polymerization method. Methacrylic acid, trimethylolpropane trimethacrylate and azobisisobutyronitrile were used as functional-monomer, cross-linker and initiator, respectively. Compared with other fluorescent nanoparticles, the FNPs have the characteristics including low dye leakage and good photostability.
Chapter Three In this chapter, we have developed a detection method of IL-4. The imprinted fluorescent nanoparticles were labeled on anti-IL-4 monoclonal antibody. IL-4 was measured based on the specific interaction between captured IL-4 antigen and fluorescent nanoparticles-labeled anti-IL-4 monoclonal antibody. The detection limits were 0.3ng/mL.The RSD for eleven parallel measurements of 2.0 ng/mL IL-4 was 3.2%. The results demonstrate that the method offers potential advantages of sensitivity, easily labeling to the antibody, optical detection.
Chapter Four A novel method of detectingα-fetoprotein was developed. The method combined the model of Ab-Ag-Ab-Ab* with fluorescence microscopy imaging. The software, Image-pro plus 6.0, was used to gain the data of image. The detection limits were 0.3ng/mL. The results demonstrate that the method offers potential advantages of easily labeling to the antibody, sensitivity, simplicity and reproducibility for the determination of AFP. And the method is applicable to the determination of AFP in serum samples.
第1章综述对荧光纳米粒子及其在免疫分析中的应用进行了综述。
第2章分子印迹荧光纳米粒子的制备与表征采用沉淀聚合法,甲基丙烯酸为功能单体,三羟甲基丙烷三甲基丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,乙腈为溶剂,制备了一种基于分子印迹原理的包裹荧光素的新型荧光微球。探讨了交联剂种类及其他反应条件对微球形貌、光稳定性和荧光强度的影响。结果表明,在最佳条件下制得的荧光微球粒径约为100nm,并具有光稳定性好、泄漏率低的特点。
第3章丁基罗丹明B分子印迹型荧光纳米粒子标记免疫分析法测定白介素-4采用分子印迹荧光纳米粒子作为标记物,双抗体夹心法为免疫检测模式,荧光显微成像为分析方法,对白介素-4进行了检测。用Image-pro plus 6.0软件对采集到的图像进行定量分析。检出限0.3ng/mL,对2.0 ng/mL白介素-4测定11次的RSD为3.2%。结果表明,该法具有操作简单,结果直观的特点。
第4章丁基罗丹明B分子印迹型荧光纳米粒子标记免疫分析法测定人血清中甲胎蛋白采用分子印迹荧光纳米粒子作为标记物,抗体—抗原—抗体—标记二抗的免疫反应模式,结合荧光显微成像技术,对甲胎蛋白进行了检测。采用Image-pro plus 6.0软件对采集到的图像进行定量分析。检出限0.2ng/mL。并对血清样品进行了测定。结果表明,该法具有操作简单,灵敏度高,结果直观的特点。
As a member of multitudinous fluorescent labels, dye-doped nanoparticles have widespread prospects of application in the biology and medicine domain because of its excellent characters such as the strong fluorescence intensity, easy surface modification, low toxicity and easy preparation. However, the dye leakage from the matrix is still a problem no matter which kind of matrix was used. In order to solve this problem, lots of methods have been employed in the issued papers. Conjugation of the dye with the precursor of nanoparticles matrix, transforming the solubility of dye, creating the electrostatic attraction between of FNPs matrix and dye, increasing the size of dye, and so on, are the usually used methods. To a certain extent, these methods could decrease the leaking of the dye from the NPs matrix, but most of the methods have their intrinsic disadvantages. For example, it is very difficult to conjugate the dye without active group with the precursor of nanoparticles matrix. In this study, a new type of fluorescent nanoparticles (FNPs) has been developed with precipitation polymerization.
Chapter One We have reviewed nanoparticles and its application in immunoassay. The research section contains three subunits.
Chapter Two A novel kind of fluorescent nanoparticles (FNPs) has been prepared using a precipitation polymerization method. Methacrylic acid, trimethylolpropane trimethacrylate and azobisisobutyronitrile were used as functional-monomer, cross-linker and initiator, respectively. Compared with other fluorescent nanoparticles, the FNPs have the characteristics including low dye leakage and good photostability.
Chapter Three In this chapter, we have developed a detection method of IL-4. The imprinted fluorescent nanoparticles were labeled on anti-IL-4 monoclonal antibody. IL-4 was measured based on the specific interaction between captured IL-4 antigen and fluorescent nanoparticles-labeled anti-IL-4 monoclonal antibody. The detection limits were 0.3ng/mL.The RSD for eleven parallel measurements of 2.0 ng/mL IL-4 was 3.2%. The results demonstrate that the method offers potential advantages of sensitivity, easily labeling to the antibody, optical detection.
Chapter Four A novel method of detectingα-fetoprotein was developed. The method combined the model of Ab-Ag-Ab-Ab* with fluorescence microscopy imaging. The software, Image-pro plus 6.0, was used to gain the data of image. The detection limits were 0.3ng/mL. The results demonstrate that the method offers potential advantages of easily labeling to the antibody, sensitivity, simplicity and reproducibility for the determination of AFP. And the method is applicable to the determination of AFP in serum samples.
引文
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