本文選題：免標(biāo)記 + 熒光傳感器��； 參考：《福建醫(yī)科大學(xué)》2015年碩士論文

【摘要】：現(xiàn)代化的檢測技術(shù)正朝著快速、靈敏、高通量的方向發(fā)展,與此同時,熒光檢測因其所具有的操作簡便、省時、靈敏等特點受到越來越多的關(guān)注,新型熒光檢測技術(shù)有望成為下一代高通量生物分析技術(shù)�，F(xiàn)在的熒光檢測技術(shù)主要通過以下兩種技術(shù)路線:一:熒光染料標(biāo)記的熒光生物傳感器用于成像與檢測,常見的熒光染料有FITC及FAM等;二:應(yīng)用半導(dǎo)體量子點、金屬簇等熒光納米材料發(fā)展熒光檢測方法用于DNA、蛋白質(zhì)和其它小分子等物質(zhì)的檢測�，F(xiàn)有的熒光生物傳感器通常需要標(biāo)記處理,造成檢測方法費時費力、高成本等不足。因此,開發(fā)高靈敏、高特異檢測目標(biāo)物的免標(biāo)記熒光檢測新方法具有廣闊的應(yīng)用前景。基于以上考慮,本課題建立了三種新型的免標(biāo)記熒光傳感體系:第一章基于石墨烯量子點的免標(biāo)記熒光傳感器用于多巴胺檢測本章節(jié)設(shè)計了一種基于石墨烯量子點的免標(biāo)記熒光傳感體系檢測多巴胺。通過檸檬酸(Ascorbic Acid,CA)熱解法制備石墨烯量子點(Graphene Quantum Dots,GQDs)并作為熒光探針,多巴胺(Dopamine,DA)在堿性條件下自聚合形成聚多巴胺(Polymerization of Dopamine,pDA)并吸附在GQDs表面淬滅熒光,熒光淬滅強(qiáng)度可用于多巴胺檢測。利用紫外可見光譜(Ultravioletand Visible Spectrophotometry,UV-vis)傅里葉變換紅外光譜(Fourier Transform Infrared Spectroscopy,FTIR)、原子力顯微鏡(Atomic Force Microscope,AFM)、透射電鏡(Transmission Electron Microscopy,TEM)、zeta電勢、光電子能譜(X-ray Photoelectron Spectroscopy,XPS)、對本實驗體系中GQDs、pDA的形貌以及FRET的反應(yīng)過程進(jìn)行表征。在優(yōu)化的條件下測得多巴胺檢測體系的線性范圍為0.01-60.0μM,檢測限(Limit of Detection,LOD)為0.008μM。本方法可實現(xiàn)多巴胺注射液及血清樣品中多巴胺的加標(biāo)檢測,實際樣品結(jié)果表明,本方法具有良好的選擇性,且能夠準(zhǔn)確檢測復(fù)雜體系下多巴胺的濃度,為臨床便捷檢測多巴胺提供新的選擇。第二章基于SGI和核酸適配體的ATP檢測的免標(biāo)記熒光傳感器本章節(jié)建立了一種基于雙鏈DNA嵌合熒光染料(SYBR Green I,SGI)和核酸適配體的免標(biāo)記熒光傳感體系用于三磷酸腺苷(Adenosine Triphosphate,ATP)檢測。設(shè)計核酸適配體序列以形成長鏈雙鏈DNA,加入ATP,ATP與適配體雙鏈作用形成G四聯(lián)體使得雙鏈DNA(Double-stranded DNA,dsDNA)解鏈,釋放原本結(jié)合在雙鏈的SGI,使SGI的熒光強(qiáng)度減弱,并且降低的熒光強(qiáng)度與ATP濃度呈正相關(guān),以此實現(xiàn)ATP的檢測。本章利用電泳成像技術(shù)驗證該檢測機(jī)理。在優(yōu)化的條件下,ATP檢測體系的線性范圍為10.0-5000.0μM,LOD為6.1μM,并具有良好的特異性。同時,通過與ATP化學(xué)發(fā)光試劑盒檢測腫瘤細(xì)胞中ATP結(jié)果作相對誤差分析,考察本方法用于實際樣品檢測的準(zhǔn)確性。實驗結(jié)果表明,本方法可準(zhǔn)確檢測實際樣品的ATP濃度,可為檢測復(fù)雜體系中ATP含量提供新的方法選擇。第三章基于SGI和GO的新型免標(biāo)記熒光傳感器檢測汞離子本章節(jié)設(shè)計了基于SGI和GO的檢測汞離子的免標(biāo)記熒光傳感體系。設(shè)計T堿基錯配探針,與Hg以T-Hg-T反應(yīng)形成發(fā)夾結(jié)構(gòu)DNA并結(jié)合SGI產(chǎn)生熒光作為信號實現(xiàn)對汞離子的檢測。檢測體系中加入GO可降低未嵌入雙鏈DNA的SGI所產(chǎn)生的背景信號以提高信噪比。優(yōu)化反應(yīng)條件后,SGI信號與汞離子在10.0-2000.0nM范圍內(nèi)線性相關(guān),LOD為2.2 nM。本方法可實現(xiàn)實際水樣中汞離子的準(zhǔn)確測定,可推廣應(yīng)用于環(huán)境監(jiān)測、農(nóng)殘分析、食品藥品工業(yè)等領(lǐng)域中汞離子的檢測。
[Abstract]:Modern detection technology is developing towards fast, sensitive and high throughput direction. At the same time, fluorescence detection has attracted more and more attention because of its simple operation, time saving and sensitivity. The new fluorescence detection technology is expected to become the next generation high throughput biosegregation technology. The current fluorescence detection technology is mainly through the following two Technology routes: 1: fluorescent dye labeled fluorescent biosensors for imaging and detection, common fluorescent dyes including FITC and FAM; two: fluorescence detection methods used for the development of fluorescent nanomaterials, such as semiconductor quantum dots, metal clusters, etc., for the detection of DNA, protein and other small components. Therefore, the new method of developing high sensitive and highly specific detection target has broad application prospects. Based on the above considerations, three new type of free fluorescent sensing systems are established in this subject: the first chapter is based on the free standard of graphene quantum dots. The fluorescence sensor is used for dopamine detection in this chapter, a free tagged fluorescence sensing system based on graphene quantum dots is designed to detect dopamine. The graphene quantum dots (Graphene Quantum Dots, GQDs) are prepared by the pyrolysis of Ascorbic Acid (CA) and as a fluorescent probe, and the dopamine (Dopamine, DA) is self polymerized under alkaline conditions. Polymerization of Dopamine (pDA) is formed and adsorbed on the surface of GQDs to quenching fluorescence. The intensity of fluorescence quenching can be used for dopamine detection. The ultraviolet spectrum (Ultravioletand Visible Spectrophotometry, UV-vis) Fu Liye transform infrared spectroscopy (Fourier Transform Infrared), atomic force microscope (atomic force microscope) C Force Microscope, AFM), transmission electron microscopy (Transmission Electron Microscopy, TEM), zeta potential, photoelectron spectroscopy (X-ray Photoelectron Spectroscopy), characterization of the morphology and reaction process of this experimental system. The test limit (Limit of Detection, LOD) is 0.008 mu M. this method can realize dopamine addition detection in dopamine injection and serum samples. The actual sample results show that this method has good selectivity and can accurately detect dopamine concentration under complex system, which provides a new choice for convenient detection of dopamine in the bed. The second chapter is based on this method. The SGI and ATP detection of nucleic acid aptamers free labeling fluorescence sensor in this chapter established a free labeling fluorescence sensing system based on double stranded DNA chimeric fluorescent dye (SYBR Green I, SGI) and nucleic acid aptamers for adenosine triphosphate (Adenosine Triphosphate, ATP) detection. P, ATP and aptamer double chain action formed G four coupling to make double stranded DNA (Double-stranded DNA, dsDNA) dissolve chain, release the original binding of SGI in double chain, weaken the fluorescence intensity of SGI, and reduce the fluorescence intensity with ATP concentration, so as to achieve ATP detection. This chapter uses electrophoretic imaging to verify the detection mechanism. The optimization is optimized. Under the condition, the linear range of ATP detection system is 10.0-5000.0 mu M, LOD is 6.1 mu M and has good specificity. At the same time, the accuracy of this method is used to detect the actual sample by relative error analysis with the ATP chemiluminescence kit for detecting the ATP results in tumor cells. The experimental results show that this method can detect the actual sample accurately. The concentration of ATP can provide a new method for detecting the content of ATP in complex systems. In the third chapter, a new free labeling fluorescence sensor based on SGI and GO is used to detect mercury ions in this chapter. A fluorescent sensing system for detecting mercury ions based on SGI and GO is designed. A T base mismatch probe is designed to form a hairpin structure DNA with Hg with T-Hg-T. SGI produces fluorescence as a signal to detect the mercury ion. Adding GO in the detection system can reduce the background signal produced by the SGI of unembedded double strand DNA to improve the signal to noise ratio. After optimizing the reaction conditions, the SGI signal is linearly related to the mercury ion within the 10.0-2000.0nM range, and the LOD is the 2.2 nM. method for the realization of the mercury ion in the actual water sample. It can be widely applied to the detection of Hg in environmental monitoring, pesticide residue analysis, food and pharmaceutical industry, etc.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.6

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相關(guān)期刊論文 前3條

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本文編號：1897336