本文選題：量子點(diǎn) + 生物素-酪胺��； 參考：《安徽醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的熒光檢測(cè)是生物芯片的常規(guī)檢測(cè)方法。該方法最大的缺點(diǎn)是熒光掃描儀器價(jià)格昂貴，而且體積笨重，限制了生物芯片技術(shù)在中小型醫(yī)療機(jī)構(gòu)以及現(xiàn)場(chǎng)檢測(cè)中的應(yīng)用。近年發(fā)展起來(lái)的一些納米材料如納米金顆粒、量子點(diǎn)、納米銀顆粒由于表現(xiàn)出各種獨(dú)特的光電性質(zhì)，在分子檢測(cè)領(lǐng)域引起了廣泛關(guān)注。本實(shí)驗(yàn)的目的是基于納米顆粒建立高檢測(cè)靈敏度可視化生物芯片檢測(cè)技術(shù)，使生物芯片技術(shù)能夠廣泛地應(yīng)用于分子檢測(cè)。方法通過(guò)對(duì)現(xiàn)有HRP底物的修飾改造，合成一系列新型HRP底物。利用生物偶聯(lián)方法制備了一種新的納米金復(fù)合底物，實(shí)現(xiàn)了HRP直接催化納米金顆粒的沉積，評(píng)價(jià)了該底物用于生物芯片可視化檢測(cè)的靈敏度,并將納米金底物用于基因芯片和蛋白芯片的可視化檢測(cè)。以基因芯片可視化檢測(cè)為例，確定檢測(cè)流程、優(yōu)化檢測(cè)條件、比較納米金復(fù)合底物T-Au、TSA-GLSS、GLSS檢測(cè)靈敏度。 量子點(diǎn)是一種新型納米顆粒，本研究以基因芯片分析技術(shù)為平臺(tái)，將TSA結(jié)合量子點(diǎn)標(biāo)記銀染增強(qiáng)技術(shù)，建立一種基于量子點(diǎn)標(biāo)記銀染增強(qiáng)可視化檢測(cè)方法。量子點(diǎn)標(biāo)記銀染增強(qiáng)可視化檢測(cè)方法：待測(cè)靶基因與固定在玻片上的探針雜交，依次加入鏈霉親和素標(biāo)記的辣根過(guò)氧化物酶、生物素標(biāo)記的酪胺以及鏈霉親和素標(biāo)記的量子點(diǎn)，37℃孵育，然后加入銀增強(qiáng)試劑顯色；最后使用可視化生物芯片掃描儀掃描并記錄結(jié)果。以牛布魯氏菌為檢測(cè)對(duì)象,比較TSA-Cy3和TSA-QDS兩種檢測(cè)法的檢測(cè)靈敏度。 結(jié)果納米金新底物制備條件：T-Au復(fù)合底物的制備，是將納米金的琥珀酰亞胺酯衍生物（Nanogold-NHS）與酪胺在50℃下避光反應(yīng)1.5h即可生成。所制備的納米金復(fù)合底物T-Au基因芯片可視化法檢測(cè)流程為：固定在玻片上的探針與待測(cè)靶標(biāo)雜交后依次加入streptavidin-HRP和T-Au的稀釋液、溫育。加入銀增強(qiáng)試劑顯色并掃描分析。檢測(cè)條件為：T-Au稀釋比例1:200，，37℃孵育時(shí)間25min，銀染增強(qiáng)時(shí)間4-5min。T-Au與TSA-GLSS檢測(cè)靈敏度均為103CFU"mL。T-Au檢測(cè)與TSA-GLSS檢測(cè)靈敏度相同。 確定了基因芯片量子點(diǎn)標(biāo)記銀染增強(qiáng)可視化檢測(cè)方法的檢測(cè)流程，優(yōu)化了檢測(cè)條件并考察了檢測(cè)靈敏度。檢測(cè)條件為：酪胺-生物素稀釋比例1:4000，鏈酶親和素標(biāo)記的量子點(diǎn)稀釋比例1:50，37℃孵育時(shí)間25-30min，銀染增強(qiáng)時(shí)間6-7min。TSA-Cy3和TSA-QDS兩種檢測(cè)法檢測(cè)布魯氏菌,結(jié)果TSA-Cy3和TSA-QDS檢測(cè)靈敏度均為103CFU/mL。 結(jié)論制備了一種納米金復(fù)合底物，實(shí)現(xiàn)了HRP直接催化納米金顆粒的沉積。以所制備的納米金復(fù)合底物基礎(chǔ)，建立了T-Au可視化檢測(cè)方法。該檢測(cè)方法操作簡(jiǎn)單，結(jié)果可視化，實(shí)現(xiàn)了生物芯片高靈敏度可視化檢測(cè)，可以取代TSA-GLSS可視化檢測(cè)方法。建立了一種基于量子點(diǎn)標(biāo)記銀染增強(qiáng)的基因芯片的可視化檢測(cè)方法。本方法具備儀器要求低、結(jié)果可視化等優(yōu)勢(shì)，為量子點(diǎn)在分子檢測(cè)領(lǐng)域的應(yīng)用提供了參考。
[Abstract]:Objective fluorescence detection is a routine method for biochip detection. The biggest drawback of this method is that the fluorescent scanner is expensive and bulky, which limits the application of biochip technology in small and medium-sized medical institutions and field detection. Some nanomaterials, such as gold nanoparticles, quantum dots and silver nanoparticles developed in recent years, have attracted wide attention in the field of molecular detection because of their unique optoelectronic properties. The purpose of this experiment is to establish a high sensitivity visual biochip detection technology based on nanoparticles, so that biochip technology can be widely used in molecular detection. Methods A series of new HRP substrates were synthesized by modifying the existing HRP substrates. A new nano-gold composite substrate was prepared by biological coupling method. The deposition of gold nanoparticles directly catalyzed by HRP was realized. The sensitivity of the substrate for visual detection of biochip was evaluated. The nano-gold substrate was used to visualize the detection of gene chip and protein chip. Taking the visualization detection of gene chip as an example, the detection process was determined and the detection conditions were optimized to compare the sensitivity of T-Au-Au-TSA-GLSSS-GLSS. Quantum dots (QDs) are a new type of nanoparticles. Based on the technology of gene chip analysis, a visual detection method based on QDs was established by combining TSA with QDs. Quantum Dot labeling silver staining enhanced visual detection method: the target gene to be tested was hybridized with the probe fixed on the slide, and the horseradish peroxidase labeled by Streptomycin was added in turn. Biotin labeled tyramine and streptavidin labeled quantum dots were incubated at 37 鈩

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