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Proximity Ligation Assay‐induced Structure‐switching Hairpin DNA toward Development of Electrochemical Immunosensor
Author(s) -
Lu Minghua,
Tang Dianping
Publication year - 2016
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/elan.201501173
Subject(s) - dna , chemistry , oligonucleotide , proximity ligation assay , ferrocene , chromatography , microbiology and biotechnology , biochemistry , electrochemistry , biology , electrode , receptor
A novel electrochemical immunosensor was designed for sensitive detection of mycotoxins (aflatoxin B 1 , AFB 1 , 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‐AFB 1 antibody‐conjugated DNA 1 (mAb‐DNA 1 ), AFB 1 ‐BSA‐labeled DNA 2 (AFB 1 ‐DNA 2 ) 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‐DNA 1 and AFB 1 ‐DNA 2 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 AFB 1 introduction, the competitive‐type immunoassay was executed between the analyte and AFB 1 ‐DNA 2 for mAb‐DNA 1 . Under the optimal conditions, the electrochemical signal was indirectly proportional to target AFB 1 concentration, and allowed the detection of target AFB 1 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 AFB 1 ELISA kit.