Colorimetric and Ultrasensitive Bioassay Based on a Dual- Amplification System Using Aptamer and DNAzyme Longhua Tang, † Yang Liu, † Md Monsur Ali, ‡ Dong Ku Kang, ‡ Weian Zhao,* ,‡ and Jinghong Li* ,† † Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing, China 100084 ‡ The Sue and Bill Gross Stem Cell Research Center, the Chao Family Comprehensive Cancer Center and Department of Pharmaceutical Sciences, University of California, Irvine, California 92697, United States * S Supporting Information ABSTRACT: 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′-azino-bis(3-ethylbenzothiazo- line-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. R apid and sensitive assays for the quantitative detection of biomarkers (i.e., proteins, peptides, lipids, and small molecules) are of tremendous importance in clinical settings for disease diagnosis, proteomics, and metabolomics. 1-4 In particular, many biomarkers that are used to diagnose the early stage of a disease or pathological condition, including cancer, are often present at a very low concentration, which demands ultrasensitive assays. 3,4 In addition, there is an urgent need for simple and rapid point-of-care diagnostics that can be used by nontrained personnel in the developing countries. 5-7 Tradi- tional bioassays, which often use antibodies as biorecognition molecules, include enzyme-linked immunosorbent assay (ELISA), 8 immunomagnetic assay, 9 electrochemical assay, 10 and surface plasma resonance. 11 Although these are well- established and have been widely used, they are complex and time-consuming, often lack sensitivity, and sometimes rely on sophisticated and expensive instruments to process the signal. Functional nucleic acid (FNA), 12 including DNA aptamers (single-stranded DNA molecules that can specifically bind to a non-nucleic acid target) 13 and DNA enzymes (DNAzymes) (DNA molecules that are capable of catalyzing chemical reactions) 14 represent an emerging class of biomolecules that finds great advantages in bioassay development. Aptamers and DNAzymes for a given target can be isolated using an in vitro method called systematic evolution of ligands by exponential enrichment (SELEX) or in vitro selection. 15,16 Once the sequence is identified, it can be synthesized in a large scale by a DNA synthesizer with almost no batch-to-batch variation and various functional molecules such as biotin (surface anchor) and organic dyes (signaling molecules) can be incorporated at desirable positions without compromising their functions. 17-19 In addition to their high affinity, specificity, and chemical stability at polymerization (e.g., polymerase chain reaction (PCR) and at room temperature, FNA-based assays can be integrated with a variety of established molecular biology tools such as DNA rolling circle amplification (RCA)) and ligation. 20-28 Indeed, the simplicity and versatility have made FNA an emerging candidate in the past decade for bioassay platform development including fluorescent, electrochemical, colorimetric, and others. 12 In particular, the colorimetric FNA assays can be readily applied onto a solid substrate such as cellulose membrane, microfluidic chip and paper which holds great potential in point-of-care applications. 5-7,12 Received: December 10, 2011 Accepted: April 25, 2012 Published: April 25, 2012 Article pubs.acs.org/ac © 2012 American Chemical Society 4711 dx.doi.org/10.1021/ac203274k | Anal. Chem. 2012, 84, 4711-4717