Colorimetric and Ultrasensitive Bioassay Based on a Dual-Amplification System Using Aptamer and DNAzyme

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• 作者：Longhua Tang ; Yang Liu ; Md Monsur Ali ; Dong Ku Kang ; Weian Zhao ; Jinghong Li
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：June 5, 2012
• 年：2012
• 卷：84
• 期：11
• 页码：4711-4717
• 全文大小：399K
• 年卷期：v.84,no.11(June 5, 2012)
• ISSN：1520-6882

文摘

Rapid detection of ultralow amount of biomarkers in a biologically complex mixture remains a major challenge. Herein, we report a novel aptamer-based protein detection assay that integrates two signal amplification processes, namely, polymerase-mediated rolling-circle amplification (RCA) and DNA enzyme-catalyzed colorimetric reaction. The target biomarker is captured in a sandwich assay by primary aptamer-functionalized microbeads (MBs) and a secondary aptamer that is connected to a RCA primer/circular template complex. RCA reaction, which amplifies the single biomarker binding events by a factor of hundreds to thousands (the first amplification) produces a long DNA molecule containing multiple DNAzyme units. The peroxidase-like DNAzyme catalyzes the oxidation of 2,2鈥?azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (the second amplification), which generates a blue-green colorimetric signal. This new biosensing platform permits the ultrasensitive, label-free, colorimetric detection of biomarker in real time. Using platelet-derived growth factor B-chain (PDGF-BB) as a model system, we demonstrated that our assay can detect a protein marker specifically in a serum-containing medium, at a concentration as low as 0.2 pg/mL in 2 h, which rivals traditional assays such as ELISA. We anticipate this simple methodology for biomarker detection can find utility in point-of-care applications.