本文選題：磁性微球 + 共價(jià)偶聯(lián)�。� 參考：《東南大學(xué)》2016年碩士論文

【摘要】：在信息時(shí)代,人們對(duì)健康的關(guān)注達(dá)到了前所未有的高度,對(duì)健康數(shù)據(jù)的獲得方式和處理過程也提出了新的要求。免疫層析作為體外診斷技術(shù)的一種,因其廉價(jià)、快速、簡易等優(yōu)勢,在互聯(lián)網(wǎng)時(shí)代迎來了發(fā)展的新機(jī)。但是基于膠體金和膠乳微球探針的免疫層析技術(shù)普遍存在制備過程繁瑣,可重復(fù)性差、靈敏度低等問題。因此,開發(fā)出新類型的標(biāo)記探針,對(duì)于免疫層析技術(shù)具有革命性的意義。另一方面,磁性微球憑借其在外加磁場下的磁響應(yīng)特性,廣泛應(yīng)用于生物分子的分離純化和體內(nèi)藥物運(yùn)輸?shù)确矫�。本研究致力于將磁性微球作為�?biāo)記探針,用于免疫層析檢測當(dāng)中,取得了以下結(jié)果：(1)實(shí)驗(yàn)采用溶劑熱法制備了磁性微球,通過場發(fā)射掃描電鏡、紅外光譜儀、振動(dòng)樣品磁強(qiáng)計(jì)、動(dòng)態(tài)光散射測試對(duì)其進(jìn)行了表征,結(jié)果表明制備的磁性微球平均粒徑在250nm,單分散性良好,紅外光譜顯示磁性微球表面帶有羧基,具有良好的水溶性和單分散性。磁性微球呈超順磁性,比飽和磁化強(qiáng)度為56.15emu/g。實(shí)驗(yàn)還測試了反應(yīng)鐵濃度和反應(yīng)時(shí)間對(duì)磁性微球的影響,發(fā)現(xiàn)隨著鐵濃度的增加,磁性微球整體大小隨之增加,隨著反應(yīng)時(shí)間的延長,組成磁性微球的小顆粒尺寸隨之增加。(2)采用1-(3-二甲氨基丙基)-3-乙基碳二亞胺(EDC)/N-羥基琥珀酰亞胺(NHS)法將單克隆抗體蛋白偶聯(lián)在磁性微球表面,制備出免疫磁性微球。采用BCA試劑盒進(jìn)行單因素分析,得到偶聯(lián)的最佳條件是：活化過程中,Fe、EDC、NHS之間的質(zhì)量比為1：0.5：0.3,反應(yīng)體系為純水；偶聯(lián)過程中緩沖體系是離子強(qiáng)度為0.02 M,pH=6.6的硼酸緩沖溶液,偶聯(lián)1h,采用胎牛血清白蛋白封閉1h。通過紅外光譜儀、熱分析儀確定單克隆抗體蛋白已經(jīng)偶聯(lián)在磁性微球表面。動(dòng)態(tài)光散射測試表明,偶聯(lián)過后磁性微球依然維持良好的水溶性和穩(wěn)定性。(3)初步驗(yàn)證了免疫磁性微球作為免疫層析標(biāo)記探針的性能,制備出檢測人絨毛膜促性腺激素(hCG)免疫層析和心肌肌鈣蛋白I (cTnl)試紙條。實(shí)驗(yàn)研究了不同層析速度的硝酸纖維素膜對(duì)試紙條的靈敏度的影響,并且創(chuàng)新性的采用普魯士藍(lán)染色對(duì)檢測結(jié)果進(jìn)行放大。實(shí)驗(yàn)結(jié)果表明,層析速度慢的層析膜靈敏度較高,hCG試紙條檢測線性范圍為0～1000 IU/L, cTnl檢測線性范圍為0～8 ng/mL。并且采用普魯士藍(lán)染色能夠進(jìn)一步放大檢測信號(hào),使試紙條達(dá)到能夠用肉眼觀察的程度。通過以上研究實(shí)驗(yàn)確定,采用溶劑熱法制備的磁性微球能夠作為免疫層析檢測的標(biāo)記探針,并且極大的簡化了試紙條的制備過程,采用普魯士藍(lán)染色能夠有效放大檢測信號(hào),且制備流程和工藝具備一般性和可重復(fù)性。
[Abstract]:In the information age, people pay more attention to health than ever before. Immunochromatography as an in vitro diagnostic technology, because of its cheap, fast, simple and other advantages, in the Internet era ushered in a new development of the machine. However, immunochromatography based on colloidal gold and latex microsphere probes has many problems, such as tedious preparation, poor repeatability and low sensitivity. Therefore, the development of a new type of labeled probes has revolutionary significance for immunochromatographic techniques. On the other hand, magnetic microspheres are widely used in the separation and purification of biomolecules and drug transport in vivo. In this study, magnetic microspheres were used as labeling probes for immunochromatographic detection. The following results were obtained: (1) the magnetic microspheres were prepared by solvothermal method. The magnetic microspheres were prepared by field emission scanning electron microscopy, infrared spectrometer, vibrating sample magnetometer. The results showed that the average diameter of the prepared magnetic microspheres was 250 nm, the monodispersity was good, and the surface of the magnetic microspheres had carboxyl groups and good water solubility and monodispersity. The magnetic microspheres are superparamagnetic with a specific saturation magnetization of 56.15 emu / g. The effects of reaction iron concentration and reaction time on magnetic microspheres were also tested. It was found that with the increase of iron concentration, the overall size of magnetic microspheres increased and the reaction time increased. The size of the small particles of the magnetic microspheres increased with the increase. 2) Immunomagnetic microspheres were prepared by coupling monoclonal antibody proteins to the surface of the magnetic microspheres by the method of 1-D _ 3-dimethylamino-propyl -3-ethylcarbodiimide (EDCN / N-hydroxysuccinimide). Single factor analysis using BCA kit showed that the optimum conditions for coupling were as follows: the mass ratio of EDCHs during activation was 1: 0.5: 0.3, the reaction system was pure water, and the buffer system was boric acid buffer solution with ion strength of 0.02 M0. 2, pH = 6. 6 during the coupling process, and the optimum conditions were as follows: 1: 0. 5: 0. 3 in the activation process, and 1: 0. 5: 0. 3 in the reaction system. Fetal bovine serum albumin (FSA) was used to block the coupling for 1 h. The monoclonal antibody protein was confirmed to be coupled to the surface of magnetic microspheres by IR spectrometer and thermal analyzer. Dynamic light scattering (DLS) test showed that the magnetic microspheres maintained good water solubility and stability after coupling.) the performance of immunomagnetic microspheres as immunochromatographic probe was preliminarily verified. A test strip for detecting human chorionic gonadotropin (hCG) and cardiac troponin I (cTnl) was prepared. The effect of nitrocellulose membrane at different chromatographic speeds on the sensitivity of the test strip was studied, and Prussian blue staining was used to amplify the results. The experimental results show that the sensitivity of the membrane with low chromatography speed is higher than that of the hCG strip, and the linear range of cTnl detection is 0 ~ (8) ng 路mL ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) 渭 m 路L ~ (-1). Prussian blue staining can further amplify the detection signal and make the test strip visible with the naked eye. The results show that the magnetic microspheres prepared by solvothermal method can be used as a labeling probe for immunochromatographic detection, and the preparation process of the test strip is greatly simplified. Prussian blue staining can effectively amplify the detection signal. The preparation process and process are general and reproducible.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R446.6

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