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

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• 作者：Jun Zhou ; Wenqiang Lai ; Junyang Zhuang ; Juan Tang ; Dianping Tang
• 刊名：ACS Applied Materials & Interfaces
• 出版年：2013
• 出版时间：April 10, 2013
• 年：2013
• 卷：5
• 期：7
• 页码：2773-2781
• 全文大小：484K
• 年卷期：v.5,no.7(April 10, 2013)
• ISSN：1944-8252

文摘

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 (S₀) and detection antibody (mAb₂) with a large ratio (mAb₂鈥揂uNP鈥揝₀), and then two auxiliary DNA strands S₁ and S₂ were designed for in situ propagation of DNAzyme concatamers with the hemin/G-quadruplex format. The quadruple signal amplification was implemented by using the avidin鈥揵iotin 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 mAb₂鈥揂uNP鈥揝₀. The carried S₀ initiator strands could progress a chain reaction of hybridization events between alternating S₁/S₂ DNA strands to form a nicked double-helix. Upon addition of hemin, the hemin-binding aptamers could be bound to form the hemin/G-quadruplex-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^鈥? to 20 ng mL^鈥? toward CEA standards with a low detection limit of 0.5 fg mL^鈥?. 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.

Keywords:

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