基于核酸适配体的食品中OTA和Hg（Ⅱ）的生物传感器检测方法研究
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摘要
特异性强、灵敏度高的重金属、毒素的快速检测方法,对于食品安全有着重大的意义。通过指数富集配体系统进化技术(SELEX)筛选得到的核酸适体,在毒素、重金属的检测等很多领域有重要作用。本论文选取了代表性的生物毒素(赭曲霉毒素)和重金属离子(Hg~(2+))作为检测对象,以核酸适配体为基础,构建了检测Hg~(2+)的核磁共振方法,以及胶体金和量子点试纸条检测赭曲霉毒素(OTA)的方法。
本文以这几个方面为主要内容:
1、制备了核酸适配体功能化的胶体金试纸条,采用竞争法检测OTA,主要优化了金标核酸适配体的偶联比、重悬液体系、硝酸纤维素膜和结合垫上点样量等条件。在金:核酸=1:10,0.01M pH7.4 PB(含0.5%PEG,1%蔗糖,0.1%Tween20,0.02% MgSO4,0.05% (NH4)2SO4)的溶液体系下,采用CN140硝酸纤维素膜,试纸条能够在15min内实现对OTA的定性及半定量测。肉眼检测限2.5ng/mL,标准曲线得到的线性范围0.2-2.5ng/mL,检测限0.18ng/mL。
2、构建了核酸适配体的量子点荧光试纸条,采用竞争法,量子点与适配体偶联后,优化重悬液体系、硝酸纤维素膜和结合垫上点样量等条件。发现在0.01M pH7.4 PB(含1.5%PVP,1%蔗糖,0.1%Tween20、0.02% MgSO4,0.05% (NH4)2SO4)体系中,采用M135硝酸纤维素膜,试纸条能够在15min内实现对OTA的定性及半定量测,在最优条件下,肉眼检测限5ng/mL,标准曲线得到的线性范围2.2-10ng/mL,检测限1.9ng/mL。
3、优化磁粒子标记适配体的偶联比以后,探针与Hg~(2+)反应,形成T-Hg~(2+)-T错配结构,磁粒子发生聚集,水中质子状态变化。利用核磁共振成像仪器,得到T2加权图像,T2造影图像。实验结果中,随着Hg~(2+)浓度的提高,水中的质子环境改变越多,MRI图像强度减弱,即T2加权图像强度变暗,T2弛豫时间逐渐减小,线性范围0.25pg/mL-10pg/mL,检测限0.15pg/mL。
Specificity, high sensitivity and rapid detection of heavy metals and toxins have great significance for food safety. Aptamer can get through systematic evolution of ligands by exponential enrichment (SELEX). Aptamer have played an important pole in many areas of the toxin and heavy metals’detection. Based on aptamer, we have build the NMR method to detect Hg~(2+) ion, as well as colloidal gold and quantum dot strip for the detection of ochratoxin A method.
In this paper, these aspects as the main content:
1. Aptamer of OTA marked gold nanoparticles, prepared by this complex, we developed a quick and on-site sensor for detection of OTA in the competion format. The best ratio between gold nanoparticles and DNA is 1:10 , the best buffer is 0.01M pH7.4 PB(0.5%PEG,1%sucrose,0.1%Tween20,0.02% MgSO4,0.05% (NH4)2SO4)and take the CN140 nitrocellulose membrane. The test strip took the qualitative and semi-quantitative measurement of OTA within 15min .We got a good linear relationship between OTA and the response signal. The limit of detection with naked eyes is 2.5ng/mL, and according to the standard curve, the linear range is 0.2-2.5ng/mL and LOD is 0.18ng/mL.
2. Aptamer of OTA marked quantum dots, prepared by this complex, we developed a quick and on-site sensor for detection of OTA in the competion format. The best buffer is 0.01M pH7.4 PB(1.5%PVP,1%sucrose,0.1%Tween20、0.02% MgSO4,0.05% (NH4)2SO4) and take the M135 nitrocellulose membrane. The test strip took the qualitative and semi-quantitative measurement of OTA within 15min. The limit of detection with naked eyes is 5ng/mL, and according to the standard curve, the linear range is 2.2-10ng/mL and LOD is 1.9ng/mL.
3. We conjugated the T-rich nucleic acid to magnetic nanoparticles which would form the T- Hg~(2+)-T structure with the accession of Hg~(2+). Then magnetic nanoparticles began to aggregate, hence lead to a change in the distribution of water proton. Using the magnetic resonance imaging apparatus, we got T2–weighted images and T2 contrast image. The more Hg~(2+) concentration increased, the more water proton environment changed. Accordingly T2 contrast image became dimmed and T2 relaxation time gradually decreased. We got a good linear relationship between the analyte concentration and the response signal. The linear range is 0.25-10pg/mL, and the limit of detection is 0.15pg/mL.
本文以这几个方面为主要内容:
1、制备了核酸适配体功能化的胶体金试纸条,采用竞争法检测OTA,主要优化了金标核酸适配体的偶联比、重悬液体系、硝酸纤维素膜和结合垫上点样量等条件。在金:核酸=1:10,0.01M pH7.4 PB(含0.5%PEG,1%蔗糖,0.1%Tween20,0.02% MgSO4,0.05% (NH4)2SO4)的溶液体系下,采用CN140硝酸纤维素膜,试纸条能够在15min内实现对OTA的定性及半定量测。肉眼检测限2.5ng/mL,标准曲线得到的线性范围0.2-2.5ng/mL,检测限0.18ng/mL。
2、构建了核酸适配体的量子点荧光试纸条,采用竞争法,量子点与适配体偶联后,优化重悬液体系、硝酸纤维素膜和结合垫上点样量等条件。发现在0.01M pH7.4 PB(含1.5%PVP,1%蔗糖,0.1%Tween20、0.02% MgSO4,0.05% (NH4)2SO4)体系中,采用M135硝酸纤维素膜,试纸条能够在15min内实现对OTA的定性及半定量测,在最优条件下,肉眼检测限5ng/mL,标准曲线得到的线性范围2.2-10ng/mL,检测限1.9ng/mL。
3、优化磁粒子标记适配体的偶联比以后,探针与Hg~(2+)反应,形成T-Hg~(2+)-T错配结构,磁粒子发生聚集,水中质子状态变化。利用核磁共振成像仪器,得到T2加权图像,T2造影图像。实验结果中,随着Hg~(2+)浓度的提高,水中的质子环境改变越多,MRI图像强度减弱,即T2加权图像强度变暗,T2弛豫时间逐渐减小,线性范围0.25pg/mL-10pg/mL,检测限0.15pg/mL。
Specificity, high sensitivity and rapid detection of heavy metals and toxins have great significance for food safety. Aptamer can get through systematic evolution of ligands by exponential enrichment (SELEX). Aptamer have played an important pole in many areas of the toxin and heavy metals’detection. Based on aptamer, we have build the NMR method to detect Hg~(2+) ion, as well as colloidal gold and quantum dot strip for the detection of ochratoxin A method.
In this paper, these aspects as the main content:
1. Aptamer of OTA marked gold nanoparticles, prepared by this complex, we developed a quick and on-site sensor for detection of OTA in the competion format. The best ratio between gold nanoparticles and DNA is 1:10 , the best buffer is 0.01M pH7.4 PB(0.5%PEG,1%sucrose,0.1%Tween20,0.02% MgSO4,0.05% (NH4)2SO4)and take the CN140 nitrocellulose membrane. The test strip took the qualitative and semi-quantitative measurement of OTA within 15min .We got a good linear relationship between OTA and the response signal. The limit of detection with naked eyes is 2.5ng/mL, and according to the standard curve, the linear range is 0.2-2.5ng/mL and LOD is 0.18ng/mL.
2. Aptamer of OTA marked quantum dots, prepared by this complex, we developed a quick and on-site sensor for detection of OTA in the competion format. The best buffer is 0.01M pH7.4 PB(1.5%PVP,1%sucrose,0.1%Tween20、0.02% MgSO4,0.05% (NH4)2SO4) and take the M135 nitrocellulose membrane. The test strip took the qualitative and semi-quantitative measurement of OTA within 15min. The limit of detection with naked eyes is 5ng/mL, and according to the standard curve, the linear range is 2.2-10ng/mL and LOD is 1.9ng/mL.
3. We conjugated the T-rich nucleic acid to magnetic nanoparticles which would form the T- Hg~(2+)-T structure with the accession of Hg~(2+). Then magnetic nanoparticles began to aggregate, hence lead to a change in the distribution of water proton. Using the magnetic resonance imaging apparatus, we got T2–weighted images and T2 contrast image. The more Hg~(2+) concentration increased, the more water proton environment changed. Accordingly T2 contrast image became dimmed and T2 relaxation time gradually decreased. We got a good linear relationship between the analyte concentration and the response signal. The linear range is 0.25-10pg/mL, and the limit of detection is 0.15pg/mL.
引文
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2 Tuerk C, Bold L. Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase [J]. Science, 1 990, 249(4968):505-510
3 Ellington A D, Szostak J W. In vitro selection of RNA molecules that bind specific ligands [J]. Nature, 1990, 346(6287):818-822
4 Jayasena S D. Aptamers: an emerging class of molecules that rival antibodies in diagnostics [J]. Clin. Chem., 1999, 45(9):1 628-1 650
5 Nieuwlandt D, Wecker M, Gold L, et al. Invitro selection of RNA ligands to substance [J]. Biochemistry, 1995, 249(16):5651-5659
6 Schurer H, Stembera K, Knoll D, et a1. Aptamers that bind to the antibiotic moenomycin A [J]. Bioorg. Med. Chem., 2001,92(1 0):2557-2563
7付玉荣,邱宗荫. SELEX技术及其应用研究进展[J].国外医学病毒学分册, 2005, 12(3), 70-72
8 Jeremy R B, Stephen R A, Roger Y T, et a1.Aptamers Switch Off Fluorescence of Triphenylmethane Dyes [J]. J.Am.Chem.Soc., 2003,125(48):14716-1471
9 Kensch O, Connolly B A, Steinhoff H J, et a1. HIV-1 reverse transcriptase pseudoknot RNA aptamer interaction has a binding affinityin the low picomolar range coupled with higll specificity [J].J. Bi01. Chem., 2000, 275(4):18271-18278
10 Clark S L, Remcho V T. Aptamers as analytical reagents [J]. Electrophoresis, 2002, 23(9):1335-1340
11 Jenison R D, Gill S C, Pardi A, et a1. High-resolution molecular discrimination by RNA [J]. Science, 1994, 263(5152) :1425-1429
12 Schneider D J, Feigon J, Hostomsky Z, et a1. High-affinity ssDNA inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus [J]. Biochemistry, 1995, 34(29):9599-9610
13 Shang D H, Li Y, TangZ W, et a1. Aptamers evolved from live cells as effective molecular probes for Cancer study [J]. Proc. Natl. Acad. Sci. U.S.A., 2006, 103:11838-1 1843
14游勇,鞠荣.重金属对食品的污染及其危害[J].学术交流, 2007, (2):102-105
15蔡云升.同液萃取及其在食品工业中的应用[J].上海轻工业高等专科学校学报, 1997, 12(2): 1-9
16 Yang H, Zhou Z G, Huang K W, et a1. Multisignaling optical-electrochemical sensor for Hg~(2+) based on a rhodamine derivative with a ferrocene unit [J]. Org lett, 2007, 9(23): 4729-4732
17 Wegner S V, Okesli A, Chen P, et a1. Design of an emission ratiometric biosensor from MerR family proteins: a sensitive and selective sensor for Hg~(2+) [J]. J Am Chem Soc, 2007, 129(12): 3474-3475
18 Xu H, Mao H, Zeng Q X, et a1. Aptamer-functionalized gold nanoparticles as probes in adry-reagent strip biosensor for protein analysis [J]. Anal Chem, 2009, 81(2): 669-675
19 Wang Z X, Ma L N. Gold nanoparticle probes[J]. Coord Chem Rev, 2009, 253(11-12): 1607-1618
20 Huang C C, Huang Y F, Cao Z H, et a1. Aptamer-modified gold nanoparticles for colorimetric detection of platelet-derived growth factors and their receptors [J]. Anal Chem, 2005, 77(17): 5735-5741
21 Lee J S, Hart M S, Mirkin C A. Colorimetric detection of mercuric ion (Hg~(2+)) in aqueous media using DNA-functionalized gold nanoparticles[J]. Angew Chem Int Ed, 2007, 46(22): 4093-4096
22 Mohri K, Uchiyama T, Panina L V. Recent advances of microm agnetic sensors and sensing application [J]. Sensor Actuat A-Phys, 1997, 59(1-3): 1-8
23 Handley R C. Modem magnetic materials: principles and applications (Chinese Edition), John Wiley & Sons, New York, 2000. 323-386
24 Aslam M, Schultz E A, Sun T, et a1. Synthesis of amine-stabilized aqueous colloidal iron oxide nanoparticles [J]. Cryst Growth Des, 2007, 7(3): 471-475.
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