基于核酸適配體識別-時間分辨熒光納米探針的生物毒素檢測方法研究
發(fā)布時間：2018-07-23 19:38
【摘要】：生物毒素引起的食品中毒事件在全球各地時有發(fā)生,不但威脅人類的健康,而且由于其具有極強的生理毒性,有可能被恐怖分子用于恐怖襲擊和生物戰(zhàn),對社會的食品安全和人們的身體健康造成了巨大威脅。要從根本上解決這類食品安全問題,就必須對從農(nóng)田到餐桌的食品供應(yīng)鏈中各環(huán)節(jié)實施全程管理和監(jiān)控。而目前傳統(tǒng)的生物毒素檢測方法主要包括高效液相色譜、質(zhì)譜和免疫學(xué)檢測法等,大多需要依賴抗體,檢測設(shè)備昂貴、操作繁瑣且非常耗時,很難滿足對現(xiàn)代食品安全檢測技術(shù)快速、靈敏、便捷的要求。因此,本論文立足于食品安全檢測技術(shù)的研究,以金黃色葡萄球菌腸毒素和蓖麻毒素為檢測對象,結(jié)合核酸適配體技術(shù)和時間分辨熒光分析技術(shù),建立了一系列快速、準確、靈敏、便捷的新型分析方法用于生物毒素定量檢測,并對其相關(guān)機理進行了探討。一方面,通過一步溶劑熱法合成表面氨基化的Fe3O4磁性納米顆粒(即磁珠),采用X射線衍射(XRD)、透射電鏡(TEM)和傅里葉轉(zhuǎn)換-紅外光譜(FT-IR)分別對其晶型成分、形貌和表面基團進行表征,證實該材料具備表面化學(xué)修飾的條件。首先,進行了基于磁性分離的特異性識別金黃色葡萄球菌腸毒素A(SEA)核酸適配體篩選技術(shù)的研究。將SEA進行磁珠固定化,并以此為篩選靶標,以空白磁珠為負篩靶標、金黃色葡萄球菌腸毒素B、C1(SEB、SEC1)磁珠為反篩靶標,采用負篩、反篩相結(jié)合的FLu Mag-SELEX技術(shù)進行十四輪體外篩選。通過熒光分析法檢驗了SEA適配體對靶標結(jié)合的親和力和特異性,確定最佳適配體A15,其解離常數(shù)為48.57±6.52nmol/L,具有良好的特異性識別SEA能力。并將篩選獲得的適配體用于實際樣品牛奶中SEA的測定,方法檢測限為8.7 ng/m L。其次,基于與上述相似的原理和方法,進行了特異性識別SEC1核酸適配體篩選技術(shù)的研究。將SEC1進行磁珠固定化作為篩選靶標,以空白磁珠為負篩靶標、SEA和SEB磁珠為反篩靶標,采用負篩、反篩相結(jié)合的FLu Mag-SELEX技術(shù)進行十四輪體外篩選,獲得了SEC1的特異性結(jié)合適配體,解離常數(shù)為49.43±11.76 nmol/L,并以此建立了SEC1的熒光檢測方法,該法檢測限為6 ng/m L。另一方面,利用一步溶劑熱法合成多種鑭系摻雜的時間分辨熒光納米探針。通過研究反應(yīng)條件,獲得熒光壽命長、發(fā)光性能好、水分散性好的納米級熒光顆粒,并含有生物官能團,為用于生物探針標記提供了條件。經(jīng)過TEM、時間分辨熒光光譜、XRD、FT-IR、紫外吸收光譜(UV)等表征手段證實成功制備新型鑭系摻雜的時間分辨熒光納米探針。在新型鑭系摻雜時間分辨熒光納米探針的應(yīng)用中,首先將蓖麻毒素適配體為識別元件,構(gòu)建一種生物毒素的高靈敏檢測方法。采用一步溶劑熱法制備Eu3+摻雜的KGd F4納米顆粒標記適配體生成熒光探針,利用氧化石墨烯(GO)良好的水分散性和高效的猝滅性能,建立了一種基于適配體的新型時間分辨熒光分析法檢測了均相飲用水中蓖麻毒素含量。在最優(yōu)條件下,該方法的檢測線性范圍為0.05~50 ng/m L(R2=0.9975),檢測限為0.008 ng/m L(3S/N),方法的精密度好。進行了蓖麻毒素(0.075~12.5 ng/m L)在飲用水中的加標試驗,該方法的回收率為89.42%~107.1%。將本方法與商業(yè)化的ELISA試劑盒進行對比試驗,比較兩種方法測定同批蓖麻溶液的濃度,結(jié)果表明二者一致性良好,證實所建立的方法可應(yīng)用于實際分析檢測。在上述研究的基礎(chǔ)上,將基于適配體特異性識別-時間分辨熒光納米探針的技術(shù)應(yīng)用于多組分生物毒素的同時高靈敏檢測。以SEA、SEB和SEC1適配體為識別元件,選擇具有特征熒光發(fā)射互不干擾的多色鑭系摻雜納米顆粒為檢測信號、GO作為高效寬頻熒光猝滅劑,建立了一種基于多色時間分辨熒光共振能量轉(zhuǎn)移(TR-FRET)的同時檢測三種金黃色葡萄球菌腸毒素的分析方法。確定了建立該檢測方法的關(guān)鍵條件為多色熒光生物探針的比例為Eu3+:Tb3+:Dy3+=2:1:3.5、氧化石墨烯濃度為0.25 mg/m L,并在該條件下實現(xiàn)了SEA、SEB和SEC1的同時檢測,檢測線性范圍分別為0.08~10 ng/m L、0.10~9 ng/m L和0.05~5 ng/m L,檢測限分別為0.062 ng/m L、0.069 ng/m L、0.040 ng/m L(3S/N)。將三種腸毒素以三個濃度(0.5 ng/m L、1.0 ng/m L、5.0 ng/m L)加入未稀釋的牛奶樣品中進行加標實驗,回收率的結(jié)果在92.76%~114.58%之間。結(jié)果表明,該方法具有特異性良好、靈敏度高和高通量分析的特點,尤其提高了對食品復(fù)雜基質(zhì)中多組分檢測的分析性能。
[Abstract]:Food poisoning caused by biological toxins occurs all over the world, not only threatens human health, but also because of its strong physiological toxicity, it is likely to be used by terrorists for terrorist attacks and biological warfare, which poses a great threat to food safety and health of the people. The whole problem must be managed and monitored throughout the chain of food supply chain from farmland to table. And the traditional methods of detection of biotoxin mainly include high performance liquid chromatography, mass spectrometry and immunoassay. Most of them need to rely on antibodies, which are expensive, complicated and time-consuming, so it is difficult to meet the modern food. The product safety detection technology is fast, sensitive and convenient. Therefore, this paper is based on the research of food safety detection technology, based on Staphylococcus aureus enterotoxin and ricin as the detection object, combined with nucleic acid aptamer technology and time resolved fluorescence analysis technology, a series of fast, accurate, sensitive and convenient analytical formulas are established. The method is used for quantitative detection of biotoxin and its mechanism is discussed. On one hand, one step solvothermal method is used to synthesize the surface aminated Fe3O4 magnetic nanoparticles (magnetic beads). X ray diffraction (XRD), transmission electron microscopy (TEM) and Fu Liye conversion - infrared spectroscopy (FT-IR) are used to determine the crystalline components, morphology and surface groups of the surface groups respectively. It is confirmed that the material has the condition of surface chemical modification. First, a magnetic separation specific identification of Staphylococcus aureus enterotoxin A (SEA) aptamer screening technology was carried out. SEA was immobilized on magnetic beads and used as a screening target, blank beads as negative screening targets, Staphylococcus aureus enterotoxin B, C1 (SE). B, SEC1), the magnetic bead is the screening target, using the negative sieve and the FLu Mag-SELEX technique of the screen phase combined with the FLu Mag-SELEX. The affinity and specificity of the SEA aptamers to the target binding are tested by fluorescence analysis, and the best aptamer A15 is determined, and its dissociation constant is 48.57 + 6.52nmol/L. It has a good specific identification of SEA ability. And the sieves will be screened. The selected aptamers are used for the determination of SEA in the actual sample milk. The detection limit is 8.7 ng/m L.. Based on the similar principles and methods, the specific identification of SEC1 nucleic acid aptamer screening technology is carried out. SEC1 is used as a magnetic bead immobilization target, blank beads as negative sieve target, SEA and SEB magnetic beads. The screening target is screened by negative sieves and FLu Mag-SELEX combined with screen. The specific binding ligand of SEC1 is obtained, and the dissociation constant is 49.43 + 11.76 nmol/L, and the fluorescence detection method of SEC1 is established. The detection limit of the method is 6 ng/m L. on the other hand, one step solvothermal method is used to synthesize a variety of lanthanide doping. By studying the reaction conditions, the nanofluorescence particles with long fluorescence lifetime, good luminescence performance and good water dispersibility are obtained by studying the reaction conditions, and the biological functional groups are contained, which provide the conditions for the biological probe labeling. After TEM, the time resolved fluorescence spectra, XRD, FT-IR, UV and other characterization methods are proved to be successful. A novel lanthanide doped time resolved fluorescent nano probe was prepared. In the application of a new lanthanide doped time resolved fluorescent nano probe, a highly sensitive detection method for a biological toxin was constructed by using ricin aptamers as identification elements. One step solvothermal method was used to prepare Eu3+ doped KGd F4 nanoparticles. A novel time resolved fluorimetric method based on aptamers was established to detect the content of ricin in homogeneous drinking water by a novel time resolved fluorescence analysis method based on the good dispersibility and high efficiency of GO. Under the optimal conditions, the linear range of the method was 0.05~50 ng/m L (R2=0.9975), and the detection limit was 0.008 ng/m L. 3S/N), the precision of the method is good. The addition test of ricin (0.075~12.5 ng/m L) in drinking water was carried out. The recovery rate of the method was 89.42%~107.1%., the method was compared with the commercial ELISA kit, and the two methods were compared to determine the concentration of the same batch of castor solution. The results showed that the consistency of the two kinds of castor solution was good and proved to be established. The method can be applied to practical analysis and detection. On the basis of the above research, the technology based on the aptamer specific identification time resolved fluorescent nano probe is applied to the simultaneous high sensitivity detection of multi component biotoxin. SEA, SEB and SEC1 aptamers are used as identification elements to select the polychromatic lanthanide doped with characteristic fluorescence emission without interference. A method for the simultaneous detection of three Staphylococcus aureus enterotoxin based on polychromatic time resolved fluorescence resonance energy transfer (TR-FRET) was established by GO as a high efficiency and wide frequency fluorescence quenching agent. The key condition for establishing this detection method was that the proportion of polychromatic fluorescence probe was Eu3+: Tb3+ Dy3+=2:1:3.5, the concentration of graphene oxide is 0.25 mg/m L, and the simultaneous detection of SEA, SEB and SEC1 is realized under this condition. The detection linear range is 0.08~10 ng/m L, 0.10~9 ng/m L and SEA, respectively. The detection limit is 0.069. Three concentrations (0.5, 1, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5 .0 ng/m L) was added to the samples of non diluted milk, and the results of recovery were between 92.76%~114.58%. The results showed that the method had the characteristics of good specificity, high sensitivity and high throughput analysis, and especially improved the analysis performance of multi component detection in the food complex matrix.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：O657.3;TS207.3
本文編號：2140443
[Abstract]:Food poisoning caused by biological toxins occurs all over the world, not only threatens human health, but also because of its strong physiological toxicity, it is likely to be used by terrorists for terrorist attacks and biological warfare, which poses a great threat to food safety and health of the people. The whole problem must be managed and monitored throughout the chain of food supply chain from farmland to table. And the traditional methods of detection of biotoxin mainly include high performance liquid chromatography, mass spectrometry and immunoassay. Most of them need to rely on antibodies, which are expensive, complicated and time-consuming, so it is difficult to meet the modern food. The product safety detection technology is fast, sensitive and convenient. Therefore, this paper is based on the research of food safety detection technology, based on Staphylococcus aureus enterotoxin and ricin as the detection object, combined with nucleic acid aptamer technology and time resolved fluorescence analysis technology, a series of fast, accurate, sensitive and convenient analytical formulas are established. The method is used for quantitative detection of biotoxin and its mechanism is discussed. On one hand, one step solvothermal method is used to synthesize the surface aminated Fe3O4 magnetic nanoparticles (magnetic beads). X ray diffraction (XRD), transmission electron microscopy (TEM) and Fu Liye conversion - infrared spectroscopy (FT-IR) are used to determine the crystalline components, morphology and surface groups of the surface groups respectively. It is confirmed that the material has the condition of surface chemical modification. First, a magnetic separation specific identification of Staphylococcus aureus enterotoxin A (SEA) aptamer screening technology was carried out. SEA was immobilized on magnetic beads and used as a screening target, blank beads as negative screening targets, Staphylococcus aureus enterotoxin B, C1 (SE). B, SEC1), the magnetic bead is the screening target, using the negative sieve and the FLu Mag-SELEX technique of the screen phase combined with the FLu Mag-SELEX. The affinity and specificity of the SEA aptamers to the target binding are tested by fluorescence analysis, and the best aptamer A15 is determined, and its dissociation constant is 48.57 + 6.52nmol/L. It has a good specific identification of SEA ability. And the sieves will be screened. The selected aptamers are used for the determination of SEA in the actual sample milk. The detection limit is 8.7 ng/m L.. Based on the similar principles and methods, the specific identification of SEC1 nucleic acid aptamer screening technology is carried out. SEC1 is used as a magnetic bead immobilization target, blank beads as negative sieve target, SEA and SEB magnetic beads. The screening target is screened by negative sieves and FLu Mag-SELEX combined with screen. The specific binding ligand of SEC1 is obtained, and the dissociation constant is 49.43 + 11.76 nmol/L, and the fluorescence detection method of SEC1 is established. The detection limit of the method is 6 ng/m L. on the other hand, one step solvothermal method is used to synthesize a variety of lanthanide doping. By studying the reaction conditions, the nanofluorescence particles with long fluorescence lifetime, good luminescence performance and good water dispersibility are obtained by studying the reaction conditions, and the biological functional groups are contained, which provide the conditions for the biological probe labeling. After TEM, the time resolved fluorescence spectra, XRD, FT-IR, UV and other characterization methods are proved to be successful. A novel lanthanide doped time resolved fluorescent nano probe was prepared. In the application of a new lanthanide doped time resolved fluorescent nano probe, a highly sensitive detection method for a biological toxin was constructed by using ricin aptamers as identification elements. One step solvothermal method was used to prepare Eu3+ doped KGd F4 nanoparticles. A novel time resolved fluorimetric method based on aptamers was established to detect the content of ricin in homogeneous drinking water by a novel time resolved fluorescence analysis method based on the good dispersibility and high efficiency of GO. Under the optimal conditions, the linear range of the method was 0.05~50 ng/m L (R2=0.9975), and the detection limit was 0.008 ng/m L. 3S/N), the precision of the method is good. The addition test of ricin (0.075~12.5 ng/m L) in drinking water was carried out. The recovery rate of the method was 89.42%~107.1%., the method was compared with the commercial ELISA kit, and the two methods were compared to determine the concentration of the same batch of castor solution. The results showed that the consistency of the two kinds of castor solution was good and proved to be established. The method can be applied to practical analysis and detection. On the basis of the above research, the technology based on the aptamer specific identification time resolved fluorescent nano probe is applied to the simultaneous high sensitivity detection of multi component biotoxin. SEA, SEB and SEC1 aptamers are used as identification elements to select the polychromatic lanthanide doped with characteristic fluorescence emission without interference. A method for the simultaneous detection of three Staphylococcus aureus enterotoxin based on polychromatic time resolved fluorescence resonance energy transfer (TR-FRET) was established by GO as a high efficiency and wide frequency fluorescence quenching agent. The key condition for establishing this detection method was that the proportion of polychromatic fluorescence probe was Eu3+: Tb3+ Dy3+=2:1:3.5, the concentration of graphene oxide is 0.25 mg/m L, and the simultaneous detection of SEA, SEB and SEC1 is realized under this condition. The detection linear range is 0.08~10 ng/m L, 0.10~9 ng/m L and SEA, respectively. The detection limit is 0.069. Three concentrations (0.5, 1, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5 .0 ng/m L) was added to the samples of non diluted milk, and the results of recovery were between 92.76%~114.58%. The results showed that the method had the characteristics of good specificity, high sensitivity and high throughput analysis, and especially improved the analysis performance of multi component detection in the food complex matrix.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：O657.3;TS207.3
【參考文獻】
相關(guān)期刊論文 前10條
1 李東旭;許瀟;李娜;李克安;;時間分辨熒光技術(shù)與熒光壽命測量[J];大學(xué)化學(xué);2008年04期
2 王巍;賈凌云;;適配體篩選方法研究進展[J];分析化學(xué);2009年03期
3 季穎;王清清;付潔;高新;宋海峰;;激光誘導(dǎo)熒光毛細管電泳法優(yōu)化隨機寡核苷酸文庫聚合酶鏈反應(yīng)擴增的條件[J];分析化學(xué);2010年05期
4 徐敦明;吳敏;鄒遠;張強;吳崔晨;周昱;劉賢進;;核酸適體技術(shù)在食品安全分析中的應(yīng)用[J];分析化學(xué);2011年06期
5 梁淼;劉銳;蘇榮欣;齊崴;王利兵;何志敏;;面向食品安全分析的核酸適配體傳感技術(shù)[J];化學(xué)進展;2012年07期
6 魏華;姜永強;趙永凱;畢玉晶;郭兆彪;翟俊輝;宋亞軍;黃惠杰;楊瑞馥;;用光纖生物傳感器檢測炭疽桿菌、鼠疫桿菌及葡萄球菌腸毒素B[J];生物技術(shù)通訊;2006年03期
7 云盛;索曉敏;徐正挺;楊俊平;李宇星;;金黃色葡萄球菌腸毒素檢測的研究進展[J];現(xiàn)代生物醫(yī)學(xué)進展;2012年19期
8 蔡昌學(xué),薛昭華,楊泗,張訓(xùn)恒;反向間接血凝試驗對120株金黃色葡萄球菌產(chǎn)腸毒素分型的實驗研究[J];同濟醫(yī)科大學(xué)學(xué)報;1990年02期
9 黃紹重;秦振華;;生物毒素研究進展[J];毒理學(xué)雜志;2006年04期
10 甄蓓,楊瑞馥,俞守義;用SELEX技術(shù)篩選核酸適配子的原理及應(yīng)用[J];微生物學(xué)免疫學(xué)進展;2001年02期
相關(guān)博士學(xué)位論文 前3條
1 譚明乾;新型稀土熒光探針及時間分辨熒光生化分析法研究[D];中國科學(xué)院研究生院（大連化學(xué)物理研究所）;2005年
2 葉志強;納米稀土熒光材料的制備及在時間分辨熒光免疫分析中的應(yīng)用[D];中國科學(xué)院研究生院（大連化學(xué)物理研究所）;2004年
3 王麗;四種有機磷農(nóng)藥核酸適體的篩選、鑒定及其活性研究[D];南京農(nóng)業(yè)大學(xué);2012年
相關(guān)碩士學(xué)位論文 前2條
1 馬文靜;適體篩選方法及分離方法的探討[D];西南大學(xué);2010年
2 張鮮惠;金黃色葡萄球菌腸毒素A（SEA）適配子的篩選及應(yīng)用[D];福建醫(yī)科大學(xué);2012年
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