【作者】 郭一琼；

【导师】 甘一如；

【作者基本信息】 天津大学 ， 生物化工， 2007， 硕士

【摘要】 本文首先用Frens法即柠檬酸三钠还原氯金酸的方法制备胶体金,系统地研究了胶体金制备过程中的最佳工艺条件。最终确定的胶体金制备工艺条件为:电热套加热;还原剂快速一次性加入;制备20 nm胶体金采用1 ml还原剂,制备40 nm胶体金采用0.7 ml还原剂;采用玻璃搅拌浆搅拌充分快速;保持氯金酸沸腾2 min后再加入还原剂;还原剂加入前需预热到80℃。随后用全波长扫描和TEM考察了胶体金的质量并研究了金标的制备和纯化,确定了胶体金和hCG抗体结合的最佳pH和最小结合蛋白量。采用荧光光谱测定蛋白,显示抗体的Fab片段没有被胶体金封闭。金标经过低温高速离心的方法纯化,紫外可见光谱扫描显示体系中的游离抗体已经全部除去。在试纸条的制备过程中,通过对样品垫玻璃纤维处理液中表面活性剂、蔗糖、稳定剂各自作用的研究和NC膜划线蛋白浓度、离子强度、金标pH等对灵敏度影响的研究,最终确定灵敏度为25 mIU/ml的条件:胶体金粒径为40 nm,金标OD520 nm为5.0,T线蛋白浓度为1.0 mg/ml,T线NaCl浓度为8.5 g/L,金标结合pH为9.0;灵敏度为1500 mIU/ml的条件为:胶体金粒径为20 nm,金标OD520 nm为2.0,T线蛋白浓度为0.5 mg/ml,T线NaCl浓度为9.5 g/L,金标结合pH为9.2。试纸条在5 min之内显色。制备好的试纸条经过高温稳定性实验,时间稳定性实验,特异性实验,批内重复性实验和批间重复性实验,结果表明,本研究制备的试纸条质量可靠,可以进行进一步的临床实验考察。更多还原

【Abstract】 In this thesis, monodispersional nanogold collid was synthesized firstly according to Frens Method. The optimal conditions were established as follows: The electrothermal box was used as heater; reducing reagent must enter quickly and once-off; for colloidal gold of 20 nm, we used reducing reagent with 1ml as colloidal gold of 40 nm we used reducing reagent with 0.7 ml; mixed acutely; reducing reagent was added after HAuCl4 keeped boiling for 2 min; use glass stirrer; reducing reagent was warmed up 80℃before adding. Then using UV-visible light measurements and transmission electron microscopy (TEM) to assess colloidal gold’s quality. Preparation of the antibody-colloidal gold probe (conjugate) specific to hCG and its purified method was also presented in this paper. Nanogold particles combined with antibodies were characterized by UV-visible light absorption spectra and fluorescence spectroscopy.In addition, the effect of Tween-20, sucrose, PEG and BSA in treating reagent and proteide’s concentration, ion strength on NC membrane are confirmed. With visual observation, we ensured two test limit for hCG. For 25mIU/ml limit of hCG: the diameter of colloidal gold is 20nm, conjugate’s OD520 nm is 5.0, test line proteide’s concentration is 1.0mg/ml, test line’s NaCl concentration is 8.5g/L, conjugate’s pH is 9.0. And for 1500mIU/ml limir of hCG: the diameter of colloidal gold is 40nm, conjugate’s OD520 nm is 2.0, test line proteide’s concentration is 0.5mg/ml, test line’s NaCl concentration is 9.5g/L, conjugate’s pH is 9.2. The stabilization of the strip is reliable after high temperature and three months. The cross-reaction exprement and the repeation exprement of the stips are available for clinic test. Also the strip’s result showed the NC membrane made in China was as good as import NC membrane.更多还原