4.8 Article

Nanogold-Functionalized DNAzyme Concatamers with Redox-Active Intercalators for Quadruple Signal Amplification of Electrochemical Immunoassay

期刊

ACS APPLIED MATERIALS & INTERFACES
卷 5, 期 7, 页码 2773-2781

出版社

AMER CHEMICAL SOC
DOI: 10.1021/am400652g

关键词

electrochemical immunoassay; gold nanoparticles; DNAzyme concatamers; quadruple single amplification

资金

  1. 973 National Basic Research Program of China [2010CB732403]
  2. Research Fund for the National Science Foundation of Fujian Province [2011J06003]
  3. Doctoral Program of Higher Education of China [20103514120003]
  4. National Natural Science Foundation of China [21075019, 41176079]
  5. Program for Changjiang Scholars and Innovative Research Team in University [IRT1116]

向作者/读者索取更多资源

A novel and in situ amplified immunoassay strategy with quadruple signal amplification was designed for highly efficient electrochemical detection of low-abundance proteins (carcinoembryonic antigen, CEA, as a model) by using nanogold-functionalized DNAzyme concatamers with redox-active intercalators. To construct such an in situ amplification system, streptavidin-labeled gold nanoparticles (AuNP-SA) were initially used for the labelling of initiator strands (So) and detection antibody (mAb(2)) with a large ratio (mAb(2)-AuNP-S-0), and then two auxiliary DNA strands S-1 and S-2 were designed for in situ propagation of DNAzyme concatamers with the hemin/G-quadruplex format. The quadruple signal amplification was implemented by using the avidinbiotin chemistry, nanogold labels, DNA concatamers, and DNAzymes. In the presence of target CEA, the sandwiched immunocomplex was formed between the immobilized primary antibodies on the electrode and the conjugated detection antibodies on the mAb(2)-AuNP-S-0. The carried So initiator strands could progress a chain reaction of hybridization events between alternating S-1/S-2 DNA strands to form a nicked double-helix. Upon addition of hemin, the hemin-binding aptamers could be bound to form the hemin/Gquadruplex-based DNAzymes. The formed double-helix DNA polymers could cause the intercalation of numerous electroactive methylene blue molecules. During the electrochemical measurement, the formed DNAzymes could catalyze the reduction of H2O2 in the solution to amplify the electrochemical signal of the intercalated methylene blue. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of 1.0 fg mL(-1) to 20 ng mL(-1) toward CEA standards with a low detection limit of 0.5 fg Intra-assay and inter-assay coefficients of variation (CV) were less than 8.5% and 11.5%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 14 clinical serum specimens between the developed immunoassay and commercialized electrochemiluminescent (ECL) method for detection of CEA.

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