基于磁球放大技术的电化学发光免疫分析研究
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摘要
论文的第一章研究了三联吡啶钌的N-羟基琥珀酰亚胺酯(Ru(bpy)_2(dcbpy)NHS)修饰磁球的构建及其电化学发光(ECL)。首先合成了Ru(bpy)_2(dcbpy)NHS并用红外光谱，紫外光谱，荧光光谱等方法对其进行了表征。然后利用线性扫描伏安法对Ru(bpy)_2(dcbpy)NHS进行了ECL检测，ECL曲线的峰电位为1．25 V。采集到Ru(bpy)_2(dcbpy)NHS ECL的光谱图，峰值在672 nm处。其ECL强度与同浓度的Ru(bpy)_3Cl_2溶液的ECL强度相当。然后构建了Ru(bpy)_2(dcbpy)NHS修饰的生物素包被的磁球(Bio-磁球)及链霉亲和素包被的磁球(SA-磁球)。利用Ru(bpy)_2(dcbpy)NHS上的酰基与链霉亲和素(SA)上的氨基发生偶联发应形成酰胺键，得到SA修饰的Ru(bpy)_2(dcbpy)NHS(Ru(bpy)_2(dcbpy)NHS-SA)。利用生物素与SA的特异性反应，将Bio-磁球与Ru(bpy)_2(dcbpy)NHS-SA连接在一起，得到Ru(bpy)_2(dcbpy)NHS修饰的Bio-磁球。Ru(bpy)_2(dcbpy)NHS修饰SA-磁球的构建利用了磁球上SA的伯胺与Ru(bpy)_2(dcbpy)NHS上的酯基反应形成酰胺键使Ru(bpy)_2(dcbpy)NHS直接与磁球结合。结果表明，Ru(bpy)_2(dcbpy)NHS修饰的这两种磁球可以产生较强的ECL。
论文的第二章提出了一种新的提高电化学发光免疫分析(ECLIA)灵敏度的方法：基于磁球放大技术的ECLIA方法。在此方法中，我们利用小鼠抗人CA125抗体上的氨基与硅烷化基底上的环氧基的反应将小鼠抗人CA125抗体固定在基底表面。经双抗夹心免疫反应，生物素与SA的特异性反应等形成磁球-抗体-目标抗原-抗体夹心复合体，然后将过量Ru(bpy)_2(dcbpy)NHS-SA与固定在基底上的磁球复合体进行反应，形成了Ru(bpy)_2(dcbpy)NHS标记的磁球复合体，然后进行ECL检测。由于一个磁球上结合了大量的Ru(bpy)_2(dcbpy)NHS，提高了电化学发光效率，从而提高了方法的灵敏度。本章还优化了实验条件(如BSA封闭时间，抗原抗体孵育浓度及孵育时间等)。在最佳条件下，实现了对人卵巢癌抗原CA125的定量测定，该方法的线性浓度范围为5．00×10~(-9)mol／L～1．00×10~(-7)mol／L。
In the chapter one, Ru bis(2, 2'-bipyridine) (2, 2'-bipyridine-4, 4'-dicarboxylic acid) N-hydroxysuccinimide ester (Ru(bpy)_2(dcbpy)NHS) was synthesized and characterized using infrared spectroscopy, ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy and electrochemiluminescence (ECL) spectroscopy. A maximum appeared at 672 nm on the ECL spectrum of Ru(bpy)_2(dcbpy)NHS. In order to fabricate the magnetic beads modified with Ru(bpy)_2(dcbpy)NHS, biotin-coated magnetic beads (bio-MB) or streptavidin-coated magnetic beads (SA-MB) was used. The ECL of the magnetic beads modified was investigated.
In the chapter two, we developed a new ECL immunoassay method to detect cancer antigen 125 (CA125) using the magnetic beads modified with Ru(bpy)_2(dcbpy)NHS. In this method, the primary antibody (Abi) was immobilized on the sinalized transparent indium-tin oxide (ITO) electrode. Then, the target antigen (Ag, CA125) was bound to Abi through the immunoreaction, followed by conjugating the biotinylated secondary antibody (Ab_2) to Ag. Subsequently, the streptavidin (SA) was conjugated to the Ab_2, followed by binding bio-MB to SA. Finally. Ru(bpy)_2(dcbpy)NHS was labeled to the surface of the MBs. The ECL spectrum of the system fabricated using different Ag concentrations was recorded. The maximum of the intensity on the ECL spectra was used to quantify Ag. The linear range of the method was 5.00×10~(-9)mol/L-1.00×10~(-7)mol/L for CA 125.
论文的第二章提出了一种新的提高电化学发光免疫分析(ECLIA)灵敏度的方法：基于磁球放大技术的ECLIA方法。在此方法中，我们利用小鼠抗人CA125抗体上的氨基与硅烷化基底上的环氧基的反应将小鼠抗人CA125抗体固定在基底表面。经双抗夹心免疫反应，生物素与SA的特异性反应等形成磁球-抗体-目标抗原-抗体夹心复合体，然后将过量Ru(bpy)_2(dcbpy)NHS-SA与固定在基底上的磁球复合体进行反应，形成了Ru(bpy)_2(dcbpy)NHS标记的磁球复合体，然后进行ECL检测。由于一个磁球上结合了大量的Ru(bpy)_2(dcbpy)NHS，提高了电化学发光效率，从而提高了方法的灵敏度。本章还优化了实验条件(如BSA封闭时间，抗原抗体孵育浓度及孵育时间等)。在最佳条件下，实现了对人卵巢癌抗原CA125的定量测定，该方法的线性浓度范围为5．00×10~(-9)mol／L～1．00×10~(-7)mol／L。
In the chapter one, Ru bis(2, 2'-bipyridine) (2, 2'-bipyridine-4, 4'-dicarboxylic acid) N-hydroxysuccinimide ester (Ru(bpy)_2(dcbpy)NHS) was synthesized and characterized using infrared spectroscopy, ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy and electrochemiluminescence (ECL) spectroscopy. A maximum appeared at 672 nm on the ECL spectrum of Ru(bpy)_2(dcbpy)NHS. In order to fabricate the magnetic beads modified with Ru(bpy)_2(dcbpy)NHS, biotin-coated magnetic beads (bio-MB) or streptavidin-coated magnetic beads (SA-MB) was used. The ECL of the magnetic beads modified was investigated.
In the chapter two, we developed a new ECL immunoassay method to detect cancer antigen 125 (CA125) using the magnetic beads modified with Ru(bpy)_2(dcbpy)NHS. In this method, the primary antibody (Abi) was immobilized on the sinalized transparent indium-tin oxide (ITO) electrode. Then, the target antigen (Ag, CA125) was bound to Abi through the immunoreaction, followed by conjugating the biotinylated secondary antibody (Ab_2) to Ag. Subsequently, the streptavidin (SA) was conjugated to the Ab_2, followed by binding bio-MB to SA. Finally. Ru(bpy)_2(dcbpy)NHS was labeled to the surface of the MBs. The ECL spectrum of the system fabricated using different Ag concentrations was recorded. The maximum of the intensity on the ECL spectra was used to quantify Ag. The linear range of the method was 5.00×10~(-9)mol/L-1.00×10~(-7)mol/L for CA 125.
引文
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