【摘要】：惡性腫瘤是目前乃至未來病死率較高的病種之一,據(jù)世界衛(wèi)生組織發(fā)布的《2014世界癌癥報告》顯示,2012年全球有820萬人死于癌癥。對腫瘤標(biāo)志物的檢測有利于實現(xiàn)癌癥的早期診斷,從而提高治療效率,降低死亡率。腫瘤標(biāo)志物的檢測方法多種多樣,以免疫層析法最為受到關(guān)注。免疫層析技術(shù)因其簡便、經(jīng)濟、低樣品量、無需專業(yè)人員操作、可實現(xiàn)床旁快速檢測等特點而迅速發(fā)展。但以膠體金為代表的免疫層析試紙條靈敏度不高,進行多組分分析時需要多個檢測帶,且以定性檢測為主,無法滿足對多種腫瘤標(biāo)志物的早期同步定量檢測需求。免疫層析法所采用的標(biāo)記材料對免疫傳感器的性能起決定性的作用。熒光量子點(QDs)具有熒光強度高、光化學(xué)穩(wěn)定性好、吸收光譜連續(xù)、發(fā)射光譜窄而對等特性,可實現(xiàn)“一元激發(fā),多元發(fā)射”�；赒Ds的優(yōu)勢,針對目前免疫層析產(chǎn)品的不足,我們提出以多色QDs作熒光標(biāo)記物,開發(fā)一種基于多色QDs標(biāo)記的生物傳感膜,以實現(xiàn)對多種腫瘤標(biāo)志物的快速、同步、定量檢測。本文以1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽(EDC)和N-羥基琥珀酰亞胺(NHS)為偶聯(lián)劑,制備546nm CdSe/ZnS QDs-抗甲胎蛋白單抗(QDs546-anti-AFP McAb)和620nm CdSe/ZnS QDs-抗癌胚抗原單抗(QDs620-anti-CEA McAb)偶聯(lián)物。將兩種偶聯(lián)物按一定比例混合噴涂于結(jié)合墊上,鼠源甲胎蛋白單抗(anti-AFP McAb)和鼠源癌胚抗原單抗(anti-CEA McAb)混合物以及羊抗鼠IgG分別包被于硝酸纖維素膜(NC膜)上作為檢測帶(T帶)和質(zhì)控帶(C帶),在此基礎(chǔ)上制作多色QDs生物傳感膜。通過熒光閱讀儀檢測傳感膜上T帶和C帶的熒光強度,計算出T帶和C帶的比值(T/C),對應(yīng)相應(yīng)的樣品濃度繪制出標(biāo)準(zhǔn)曲線,即可達到定量檢測的目的。在最優(yōu)實驗條件下,本課題組所開發(fā)的多色QDs生物傳感膜對AFP和CEA的檢測限分別為2 ng?ml和1 ng?ml,且無明顯交叉反應(yīng),靈敏度、特異性高,樣品恢復(fù)率好,批內(nèi)穩(wěn)定性好,批間差異小。本文將多色QDs與免疫層析技術(shù)相結(jié)合開發(fā)出的生物傳感膜,NC膜上只需一條T帶和一條C帶便可實現(xiàn)對多種腫瘤標(biāo)志物同步定量檢測。其使用方便簡單,無需專業(yè)人員操作,可在社區(qū)、家庭使用,并有利于將來檢測儀器的小型化和智能化。
[Abstract]:Malignant tumor is one of the diseases with high mortality at present and even in the future. According to the World Cancer report 2014 published by the World Health Organization, 8.2 million people died of cancer in 2012. The detection of tumor markers is beneficial to the early diagnosis of cancer, so as to improve the therapeutic efficiency and reduce the mortality. There are many methods to detect tumor markers, so the method of epidemic chromatography is the most concerned. Because of its simplicity, economy, low sample size and no need of professional operation, immune chromatography technology can realize rapid detection on the bed side and so on. However, the immunochromatographic assay strip represented by colloidal gold is not sensitive and requires multiple detection bands for multi-component analysis, and qualitative detection is the main method, which can not meet the need of simultaneous and quantitative detection of multiple tumor markers in the early stage. The material used in immunochromatography plays a decisive role in the performance of the immunosensor. The fluorescence quantum dot (QDs) has the characteristics of high fluorescence intensity, good photochemical stability, continuous absorption spectrum, narrow emission spectrum and so on. It can realize "mono-excitation and multi-element emission". Based on the advantage of QDs and aiming at the deficiency of immunochromatographic products at present, we propose to develop a kind of biosensory membrane based on multicolor QDs as fluorescent marker to realize rapid, synchronous and quantitative detection of various tumor markers. In this paper, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) were used as coupling agents. The conjugates of 546nm CdSe/ZnS QDs- anti-alpha-fetoprotein monoclonal antibody (QDs546-anti-AFP McAb) and 620nm CdSe/ZnS QDs- anticancer embryo antigen monoclonal antibody (QDs620-anti-CEA McAb) were prepared. The two coupling materials are mixed and sprayed on the bonding pad in a certain proportion. The mixture of mouse-derived alpha-fetoprotein monoclonal antibody (anti-AFP McAb) and mouse-derived carcinoembryonic monoclonal antibody (anti-CEA McAb) and sheep anti-mouse IgG were coated on the nitrocellulose membrane (NC membrane) as a detection band (T-band) and a quality control band (C-band), respectively. On this basis, multicolor QDs biosensory membrane was prepared. The fluorescence intensity of T-band and C-band on the sensing membrane was measured by fluorescence reader, the ratio of T-band to C-band (T / C) was calculated, and the standard curve was drawn corresponding to the concentration of the sample, which could be used for the quantitative detection of T-band and C-band. Under the optimal experimental conditions, the detection limits of the multicolor QDs biosensor membrane for AFP and CEA were 2 ng?ml and 1 ng?ml, respectively, and there was no obvious cross-reaction, with high sensitivity, high specificity and good sample recovery rate. The intra-batch stability is good, and the difference between batches is small. In this paper, multi-color QDs combined with immuno-chromatography technique was combined to develop biological sensing membrane. Only one T-band and one C-band were needed on NC membrane to realize simultaneous quantitative detection of multiple tumor markers. It is easy to use, no professional operation, can be used in the community, home, and conducive to the future miniaturization and intelligence of testing instruments.
【學(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R730.4

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