本文選題：功能化熒光核酸探針 + 發(fā)夾型探針��； 參考：《湖南大學》2011年博士論文

【摘要】：準確和靈敏地獲取生物樣品中小分子、核酸、蛋白質(zhì)和細胞的相關(guān)信息對生物醫(yī)學研究以及臨床診斷和治療都具有十分重要的意義。功能化熒光核酸探針,其功能超出了核酸傳統(tǒng)的基因角色,受到廣大研究者越來越多的關(guān)注,但是,如何獲得更高的靈敏度,如何提高抗復雜環(huán)境干擾的能力,如何實時、動態(tài)、靈敏、準確地獲取生命活動相關(guān)信息,仍然是分析化學工作者所面臨的重大挑戰(zhàn)。 本論文瞄準上述挑戰(zhàn)進行研究,基于分子識別和信號放大技術(shù)發(fā)展了一系列分別以小分子、核酸、蛋白質(zhì)和癌細胞為檢測對象的新型功能化熒光核酸探針,主要內(nèi)容包括: (1)發(fā)展了一種基于熒光共振能量轉(zhuǎn)移和鏈置換放大技術(shù)的核酸適體探針用于小分子可卡因的高靈敏檢測。在此,含有核酸適體的發(fā)夾探針的3’端標記熒光受體Cy5,而引物的5’端標記熒光供體FAM,當核酸適體與可卡因結(jié)合會導致其構(gòu)型發(fā)生變化從而打開發(fā)夾結(jié)構(gòu),引物便可以與打開的發(fā)夾探針雜交,在聚合酶作用下引物延伸成與發(fā)夾探針序列完全互補,產(chǎn)生新的雙鏈使得供體和受體靠近發(fā)生熒光共振能量轉(zhuǎn)移,同時可卡因被引物延伸的鏈競爭下來變成自由的可卡因分子,又可以與另外一條核酸適體結(jié)合,不斷循環(huán)實現(xiàn)信號放大。該方法可以在16min內(nèi)達到200nM的檢測下限,選擇性良好。 (2)發(fā)展了一種基于芘激發(fā)態(tài)二聚體和雜交鏈式放大技術(shù)的核酸探針用于DNA的高靈敏檢測。首先對兩個發(fā)夾探針兩端分別進行芘分子標記,由于發(fā)夾探針粘性末端的長度使得兩端的芘分子以單體形式存在,當目標DNA觸發(fā)雜交鏈式反應發(fā)生后,會形成一條長的帶缺口的雙鏈,使大量的芘分子以二聚體的形式存在。通過穩(wěn)態(tài)熒光分析和時間分辨熒光測量技術(shù)可以實現(xiàn)緩沖溶液和復雜生物樣品中DNA的高靈敏檢測。利用該方法在緩沖溶液中對DNA的檢測下限可以達到250 fM。 (3)發(fā)展了一種基于芘激發(fā)態(tài)二聚體和競爭反應的核酸適體探針用于人血清中溶菌酶的高靈敏檢測。該體系中含有一條兩端標記芘分子的發(fā)夾探針和一條未標記的核酸適體:當沒有目標分子時,兩條鏈部分雜交可以使發(fā)夾探針打開,芘分子以單體形式存在;而當存在目標分子時,目標分子結(jié)合核酸適體并以競爭的方式將芘分子雙標探針擠開,被擠開的探針呈發(fā)夾構(gòu)型,使得芘分子以二聚體形式存在。我們結(jié)合穩(wěn)態(tài)和時間分辨熒光測量技術(shù)可對緩沖溶液或血清中的溶菌酶進行檢測。該方法在緩沖溶液中對溶菌酶的檢測下限可以達到200 pM。利用該方法還實現(xiàn)了ATP的檢測,說明這是一種通用的檢測方法。 (4)發(fā)展了一種基于分子信標和缺刻酶信號放大技術(shù)的核酸適體探針用于目標細胞的高靈敏檢測。一段能打開分子信標的單鏈DNA與核酸適體雜交形成核酸適體/單鏈DNA復合物。當沒有靶細胞存在時,單鏈DNA不能打開分子信標;當有靶細胞存在時,核酸適體與其結(jié)合形成核酸適體/靶細胞復合物,導致原來的雙鏈結(jié)構(gòu)分散并將單鏈DNA釋放出來。被釋放的單鏈DNA序列含有缺刻酶的識別位點,當其與分子信標結(jié)合以后,缺刻酶會將分子信標切開,單鏈DNA隨即又可以與新的分子信標雜交。以這種方式,每條單鏈DNA可以多次循環(huán)使多個分子信標被切開,從而實現(xiàn)信號放大。利用該方法檢測Ramos細胞可以達到200 cells/mL的檢測下限,且特異性良好。 (5)發(fā)展了一種基于人血管生成素介導進入細胞的核酸適體探針用于增強光動力學治療的效果。合成并表征了光敏劑Ce6標記的血管生成素核酸適體,與血管生成素特異結(jié)合以后,在目標細胞膜表面受體蛋白的介導下進入目標細胞,經(jīng)過特定波長光照后,Ce6激活周圍的氧分子變成單態(tài)氧將目標細胞殺死。結(jié)果顯示該方法具有較高的光動力學的治療效果。
[Abstract]:Accurate and sensitive access to small molecules, nucleic acids, proteins and cells in biological samples is of great significance for biomedical research and clinical diagnosis and treatment. Functional fluorescent nucleic acid probes are beyond the traditional gene roles of nucleic acids. It is still a major challenge for analytical chemistry workers to obtain higher sensitivity, how to improve the ability to resist complex environmental interference, and how to obtain information about life activities in real time, dynamically, sensitively and accurately.
This thesis aims at studying the above challenges. Based on molecular recognition and signal amplification, a series of new functional fluorescent nucleic acid probes are developed with small molecules, nucleic acids, proteins and cancer cells. The main contents are as follows:
(1) a nucleic acid aptamer probe based on fluorescence resonance energy transfer and chain replacement amplification is developed for the high sensitivity detection of small molecular cocaine. Here, the 3 'terminal labeled fluorescent receptor Cy5 with a nucleic acid aptamer probe, while the 5' end of the primer is labeled with the fluorescent donor FAM, and the binding of the aptamer to cocaine will lead to its structure. The primers can hybridize with the open hairpin probe. The primers can be hybridized with the open hairpin probe. The primers are extended to complement the hairpin probe sequence under the polymerase chain reaction. The new double strands make the donor and the receptor close to the fluorescence resonance energy transfer, and cocaine is competing to become free by the chain that is extended by the primer. The cocaine molecule can also be combined with another aptamer to continuously circulate and amplify the signal. This method can reach the lower limit of 200nM in 16min, and the selectivity is good.
(2) a nucleic acid probe based on pyrene excited state two polymer and hybrid chain amplification technology is developed for high sensitivity detection of DNA. First, pyrene molecular markers are carried out on both ends of two hairpin probes. The pyrene molecules at both ends are in the form of monomers due to the length of the sticky ends of the hairpin probes, when the target DNA triggers the crosslinked reaction. After the occurrence, a long, notched double chain will be formed so that a large number of pyrene molecules exist in the form of two polymers. The high sensitivity detection of DNA in buffer solutions and complex biological samples can be achieved by steady fluorescence analysis and time resolved fluorescence measurement. The detection limit of DNA in buffer solution can reach 250 fM. by this method.
(3) a highly sensitive detection of lysozyme in human serum based on an aptamer probe based on pyrene excited state two polymer and competitive reaction is developed. The system contains a hairpin probe with a pyrene molecule at both ends and an unlabeled aptamer: when there is no target molecule, two strand part hybridization can open the hairpin probe and pyrene. The molecule exists in the form of a monomer, and when the target molecule exists, the target molecules combine with the aptamer to squeeze the pyrene molecular double probe in a competitive manner, and the extruded probe is in the hairpin configuration, which makes the pyrene molecule in the form of two polymer. The detection limit of the lysozyme in the buffer solution can be reached to 200 pM. in the buffer solution. This method is also used to detect the ATP, which shows that this method is a common detection method.
(4) a nucleic acid aptamer probe based on molecular beacon and short enzyme signal amplification technology is developed for high sensitivity detection of target cells. A single strand DNA that opens molecular beacons and nucleic acid aptamers can form an aptamer / single strand DNA complex. When there is no target cells, single strand DNA can not open molecular beacons; when there are target cells In existence, the nucleate aptamer combines with the complex of an aptamer / target cell that causes the original double stranded structure to disperse and release the single strand DNA. The released single strand DNA sequence contains the identification site of the absence of the enzyme, and when it is combined with the molecular beacon, the absence of the enzyme will cut the molecular beacon, and the single strand DNA can then be associated with the new molecule. Beacon hybridization. In this way, each single strand DNA can be repeatedly circulated to make multiple molecular beacons cut in order to achieve signal amplification. The method is used to detect Ramos cells to reach a lower limit of 200 cells/mL, and the specificity is good.
(5) a nucleo aptamer probe based on human angiopoietin into cells was developed to enhance the effect of photodynamic therapy. The angiopoietin aptamer was synthesized and characterized by a photosensitizer Ce6 labeled angiopoietin. After specific binding with angiopoietin, a target cell membrane surface receptor protein was introduced into the target cells. After a specific wavelength of light, Ce6 activates the surrounding oxygen molecules to become mono oxygen to kill the target cells. The results show that the method has a high photodynamic therapeutic effect.
【學位授予單位】：湖南大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R341

【共引文獻】

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