基于生物条形码结合杂交链式反应双重信号放大检测蓖麻毒素
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摘要
建立了一种基于生物条形码结合杂交链式反应扩增的高灵敏、特异性检测蓖麻毒素(Ricin toxin,RT)的方法。使用RT多克隆抗体(p Ab)及条形码DNA (Barcode DNA)制备功能性金纳米探针(GNPs),通过与RT单克隆抗体(m Ab)共同识别作用,形成"m Ab-RT-pAb/Au NPs/Barcode DNA"三明治夹心结构,进一步利用条形码DNA作为引发链触发生物素标记的发卡探针进行杂交链式扩增,形成一条具有重复单元的长双链DNA。当使用辣根过氧化物酶标记链霉亲和素与长链DNA上的生物素结合后,通过鲁米诺与过氧化氢的化学发光底物发光进行测定。结果表明,本方法检测RT的线性范围为0.61 ng/m L～5.00μg/m L,检出限为0.52 ng/m L(S/N=3),本方法具有较宽的检测范围及较低的检出限,测定饮用水及脱脂牛奶样品中RT的加标回收率在83.4%～112.4%之间,相对标准偏差在2.2%～5.4%之间,表明本方法实用性良好,在食品和饮用水安全控制及防范生物恐怖袭击方面具有良好的应用前景。
A novel highly sensitive and specific method for detection of ricin toxin( RT) based on bio-barcode combined with hybridization chain reaction( HCR) amplification was developed. Functional gold nanoparticles( GNPs) were coated with anti-ricin polyclonal antibodies( p Ab) and barcode DNA. The " m Ab-RT-pAb/AuNPs/barcode DNA " sandwich structure was formed by co-identification with the anti-ricin monoclonal antibody( m Ab),and HCR was further carried out by using barcode DNA as an initiation chain probe for triggering the chain-triggered biotin label,which resulted in a long double-stranded DNA with repeating units.Immediately,streptavidin labeled horseradish peroxidase( SA-HRP) was combined with biotin on long strand DNA. Finally,the chemiluminescence values were detected through adding substrates of luminol and hydrogen peroxide. The results showed that the linear range of RT was 0. 61 ng/m L-5000 ng/m L,and the limit of detection( LOD) was 0.52 ng/m L( S/N = 3). This method showed a wide detection range and a low detection limit. The recoveries of spiked samples in milk and water samples ranged from 83. 4% to 112. 4%,and the relative standard deviations( RSDs) ranged from 2. 2% to 5. 4%. This method exhibited great application prospects in food drinking water safety control and prevention of bioterrorism attacks.
A novel highly sensitive and specific method for detection of ricin toxin( RT) based on bio-barcode combined with hybridization chain reaction( HCR) amplification was developed. Functional gold nanoparticles( GNPs) were coated with anti-ricin polyclonal antibodies( p Ab) and barcode DNA. The " m Ab-RT-pAb/AuNPs/barcode DNA " sandwich structure was formed by co-identification with the anti-ricin monoclonal antibody( m Ab),and HCR was further carried out by using barcode DNA as an initiation chain probe for triggering the chain-triggered biotin label,which resulted in a long double-stranded DNA with repeating units.Immediately,streptavidin labeled horseradish peroxidase( SA-HRP) was combined with biotin on long strand DNA. Finally,the chemiluminescence values were detected through adding substrates of luminol and hydrogen peroxide. The results showed that the linear range of RT was 0. 61 ng/m L-5000 ng/m L,and the limit of detection( LOD) was 0.52 ng/m L( S/N = 3). This method showed a wide detection range and a low detection limit. The recoveries of spiked samples in milk and water samples ranged from 83. 4% to 112. 4%,and the relative standard deviations( RSDs) ranged from 2. 2% to 5. 4%. This method exhibited great application prospects in food drinking water safety control and prevention of bioterrorism attacks.
引文
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16 Oh B K,Nam J M,Lee S W,Mirkin C A. Small,2006,2(1):103-108
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18 Nam J M,Thaxton S,Mirkin C A. Science,2003,301(5641):1884-1886
19 Nam J M,Stoeva S I,Mirkin C A. J. Am. Chem. Soc.,2004,126(19):5932-5933
20 Yang G X,Zhuang H S,Chen H Y,Ping X Y,Bu D. Sens. Actuators B,2015:214:152-158
21 Li Z B,Miao X M,Cheng Z Y,Wang P. Sens. Actuators B,2017,243:731-737
22 Xu Y Y,Huo B Y,Sun X,Ning B A,Peng Y,Bai J L,Gao Z X. RSC Adv.,2018,8:16024-16031
23 Zhang B,Liu B Q,Tang D Q,Niessner R,Chen G N,Knopp D. Anal. Chem.,2012,84(12):5392-5399
24 Bui H,Miao V,Garg S,Mokhtar R,Song T Q,Reif J. Small,2017,13(12):1602983
25 Dirks R M,Pierce N A. Proc. Natl. Acad. Sci. USA,2004,101(43):15275-15278
26 Bi S,Chen M,Jia X Q,Dong Y,Wang Z H. Angew. Chem. Int. Edit.,2015,54(28):8144-8148
27 Grabar K C,Freeman R G,Hommer M B,Natan M J. Anal. Chem.,1995,67(4):735-743
28 Zhang X,Serous M R,Liu J. J. Am. Chem. Soc.,2012,134(24):9910-9913
29 Sun X L,Zhao X L,Tang J N,Jun Z,Chu F S. Int. J. Food Microbiol.,2004,99(2):185-194
2 Stine R,Pishko M V,Schengrund C L. Anal. Chem.,2005,77(9):2882-2888
3 Audi J,Belson M,Patel M. J. Ann. Rev. Med. Assoc.,2005,294(18):2432-2351
4 Sun J F,Wang C,Shao B,Wang Z H,Xu D S,Liu Y H,Qi K L,Yang Y,Niu Y M. Anal. Chem.,2017,89(22):12209-12216
5 Schep L J,Temple W A,Butt G A,Beasley M D. Environ. Int.,2009,35(8):1267-1271
6 Bozza W P,Tolleson W H,Rosado L A,Zhang B. Biotechnol. Adv.,2015,33(1):117-123
7 Melchior Jr W B,Tolleson W H. Anal. Biochem.,2010,396(2):204-211
8 YANG Yun-Yun,MU De-Hai,TONG Chao-Yang,MU Xi-Hui,HAO Lan-Qun. Chinese J. Anal. Chem.,2007,35(3):439-442杨运云,牟德海,童朝阳,穆唏惠,郝兰群.分析化学,2007,35(3):439-442
9 WANG Xiao-Chen,ZHOU Lei,SUN Chong-Yun,ZHAO Yong,WANG Xin-Rui,ZHANG Ping-Ping,YANG Rui-Fu,MA Xin. Military Medical Sciences,2016,40(8):676-679王晓晨,周蕾,孙崇云,赵勇,王鑫蕊,张平平,杨瑞馥,马馨.军事医学,2016,40(8):676-679
10 Merkley E D,Jenson S C,Arce J S,Melville A M,Leiser O P,Wunschel D S,Wahl K L. Toxicon,2017,14(15):18-31
11 LIANG-LongHUI,LIUShi-Lei,YUHui-Lan,GAO Chuan,ZHOU Shi-Kun. Journal of Instrumental Analysis,2015,34(74):762-767梁龙辉,刘石磊,于惠兰,高川,周世坤.分析测试学报,2015,34(74):762-767
12 Tang J J,Sun J F,Lui R,Zhang Z M,Liu J F,Xie J W. ACS Appl. Mater. Interfaces,2016,8(3):2449-2455
13 Li C H,Xiao X,Tao J,Wang D M,Huang C Z,Zhen S J. Biosens. Bioelectron.,2017,91:149-154
14 Fan J R,Zhu J,Wu W G,Huang Y. Small,2017,13(1):161710
15 Stern D,Pauly D,Zydek M,Müller C,Avondet M A,Worbs S,Lisdat F,Dorner M B,Dorner B G. Biosens.Bioelectron.,2016,78(15):111-117
16 Oh B K,Nam J M,Lee S W,Mirkin C A. Small,2006,2(1):103-108
17 Hill H D,Mirkin C A. Nat. Protoc.,2006,1(1):324-336
18 Nam J M,Thaxton S,Mirkin C A. Science,2003,301(5641):1884-1886
19 Nam J M,Stoeva S I,Mirkin C A. J. Am. Chem. Soc.,2004,126(19):5932-5933
20 Yang G X,Zhuang H S,Chen H Y,Ping X Y,Bu D. Sens. Actuators B,2015:214:152-158
21 Li Z B,Miao X M,Cheng Z Y,Wang P. Sens. Actuators B,2017,243:731-737
22 Xu Y Y,Huo B Y,Sun X,Ning B A,Peng Y,Bai J L,Gao Z X. RSC Adv.,2018,8:16024-16031
23 Zhang B,Liu B Q,Tang D Q,Niessner R,Chen G N,Knopp D. Anal. Chem.,2012,84(12):5392-5399
24 Bui H,Miao V,Garg S,Mokhtar R,Song T Q,Reif J. Small,2017,13(12):1602983
25 Dirks R M,Pierce N A. Proc. Natl. Acad. Sci. USA,2004,101(43):15275-15278
26 Bi S,Chen M,Jia X Q,Dong Y,Wang Z H. Angew. Chem. Int. Edit.,2015,54(28):8144-8148
27 Grabar K C,Freeman R G,Hommer M B,Natan M J. Anal. Chem.,1995,67(4):735-743
28 Zhang X,Serous M R,Liu J. J. Am. Chem. Soc.,2012,134(24):9910-9913
29 Sun X L,Zhao X L,Tang J N,Jun Z,Chu F S. Int. J. Food Microbiol.,2004,99(2):185-194