Proximity Ligation Assay-induced Structure-switching Hairpin DNA toward Development of Electrochemical Immunosensor
Proximity Ligation Assay-induced Structure-switching Hairpin DNA toward Development of Electrochemical Immunosensor
复制标题
邻近连接分析诱导结构转换发夹 DNA 促进电化学免疫传感器的发展
DOI：
10.1002/elan.201501173
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发表时间：
2016-03
期刊：
影响因子：
3
通讯作者：
Tang Dianping
中科院分区：
文献类型：
--
作者：
Lu Minghua;Tang Dianping
通过将邻近连接分析诱导的发夹DNA构象转换与抗原抗体反应偶联，设计了一种新型电化学免疫传感器，用于灵敏检测真菌毒素（本例中使用的黄曲霉毒素B1，AFB 1）。用抗黄曲霉毒素B1抗体标记的DNA 1（mAb-DNA 1）、黄曲霉毒素B1-牛血清白蛋白标记的DNA 2（AFB 1-DNA 2）和发夹DNA进行检测。每个发夹包含一个6碱基对的茎和一个6核苷酸（nt）的环，在3 '端标记的二茂铁标签通过在5'端的末端巯基部分的自组装固定在电极上。通过mAb-DNA 1和AFB 1-DNA 2之间的特异性抗原抗体反应进行邻位连接试验，以形成omega样DNA连接。之后，该结与发夹DNA杂交以打开探针，从而导致二茂铁标签远离电极以降低氧化还原电流。在引入目标AFB 1后，在分析物和AFB 1-DNA 2之间进行mAb-DNA 1的竞争型免疫测定。在最佳条件下，电化学信号与目标AFB 1浓度成正比，并允许在低至3.2 pg mL−1的浓度下检测目标AFB 1。 我们的策略也表现出高选择性，良好的重现性和精密度。重要的是，该方法的准确性进行了验证，用于分析加标或自然污染的花生样品，得到的结果与商业化的AFB 1 ELISA试剂盒获得的结果匹配良好。
A novel electrochemical immunosensor was designed for sensitive detection of mycotoxins (aflatoxin B1, AFB1, used in this case) by coupling proximity ligation assay-induced conformation switch of hairpin DNA with the antigen-antibody reaction. The assay was carried out by anti-AFB1 antibody-conjugated DNA1 (mAb-DNA1), AFB1-BSA-labeled DNA2 (AFB1-DNA2) and hairpin DNA. Each hairpin included a stem of 6 base pairs and a 6 nucleotide (nt) loop with the labelled ferrocene tag at the 3′ end, which was immobilized on the electrode via self-assembly of the terminal thiol moiety at the 5′ end. The proximity ligation assay was carried out via the specific antigen-antibody reaction between mAb-DNA1 and AFB1-DNA2 to form an omega-like DNA junction. Thereafter, the junction hybridized with hairpin DNA to open the probe, thus resulting in the ferrocene tag far away from the electrode for the decreasing of the redox current. Upon target AFB1 introduction, the competitive-type immunoassay was executed between the analyte and AFB1-DNA2 for mAb-DNA1. Under the optimal conditions, the electrochemical signal was indirectly proportional to target AFB1 concentration, and allowed the detection of target AFB1 at a concentration as low as 3.2 pg mL−1. Our strategy also exhibited high selectivity, good reproducibility and precision. Importantly, the accuracy of this methodology was validated for analysis of spiked or naturally contaminated peanut samples, giving results matched well with those obtained from commercialized available AFB1 ELISA kit.
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DOI：
10.1016/j.cca.2015.03.006
发表时间：
2015-05
期刊：
Clinica chimica acta; international journal of clinical chemistry
影响因子：
--
作者：
V. Nicolaidou;C. Koufaris
通讯作者：
V. Nicolaidou;C. Koufaris
影响因子：
--
作者：
Bing Zhang;D. Tang;I. Goryacheva;R. Niessner;D. Knopp
通讯作者：
Bing Zhang;D. Tang;I. Goryacheva;R. Niessner;D. Knopp
DOI：
10.1515/bc.2006.096
发表时间：
2006-06
期刊：
--
影响因子：
--
作者：
Lei Zhu;H. Koistinen;Ping Wu;A. Närvänen;Edith Schallmeiner;S. Fredriksson;U. Landegren;U. Stenman
通讯作者：
Lei Zhu;H. Koistinen;Ping Wu;A. Närvänen;Edith Schallmeiner;S. Fredriksson;U. Landegren;U. Stenman
DOI：
10.1039/c5an01006c
发表时间：
2015-08
期刊：
The Analyst
影响因子：
--
作者：
Baoting Dou;Cuiyun Yang;Y. Chai;R. Yuan;Yun Xiang
通讯作者：
Baoting Dou;Cuiyun Yang;Y. Chai;R. Yuan;Yun Xiang
影响因子：
3
作者：
M. Tertiș;O. Hosu;L. Fritea;C. Farcǎu;Andreea Cernat;R. Sandulescu;C. Cristea
通讯作者：
M. Tertiș;O. Hosu;L. Fritea;C. Farcǎu;Andreea Cernat;R. Sandulescu;C. Cristea