title: Colorimetric and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme.
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-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.
authors.affiliation: Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing, China 100084.
authors.forename: Longhua
authors.initials: L
authors.lastname: Tang
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Md Monsur
authors.initials: MM
authors.lastname: Ali
authors.forename: Dong Ku
authors.initials: DK
authors.lastname: Kang
authors.forename: Weian
authors.initials: W
authors.lastname: Zhao
authors.forename: Jinghong
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001360:Avidin; D000077214:Becaplermin; D052160:Benzothiazoles; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin; D002417:Cattle; D003124:Colorimetry; D017931:DNA Primers / Q000737:chemistry; D021881:DNA, Catalytic / Q000737:chemistry*; D004270:DNA, Circular / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide; D020574:Proto-Oncogene Proteins c-sis / Q000097:blood*; D011994:Recombinant Proteins / Q000097:blood; D012710:Serum Albumin, Bovine; D013451:Sulfonic Acids; D013844:Thiazoles
chemical_list: D052157:Aptamers, Nucleotide; D052160:Benzothiazoles; D017931:DNA Primers; D021881:DNA, Catalytic; D004270:DNA, Circular; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D013451:Sulfonic Acids; D013844:Thiazoles; D001360:Avidin; D000077214:Becaplermin; D012710:Serum Albumin, Bovine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D001710:Biotin; D006861:Hydrogen Peroxide