title: Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers.
abstract: We have designed a single-stranded DNAzyme-aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3' end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme-aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner.
authors.affiliation: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Hyoyoung
authors.initials: H
authors.lastname: Mun
authors.affiliation: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Eun-Jung
authors.initials: EJ
authors.lastname: Jo
authors.affiliation: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Taihua
authors.initials: T
authors.lastname: Li
authors.affiliation: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Hyou-Arm
authors.initials: HA
authors.lastname: Joung
authors.affiliation: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Dong-Gu
authors.initials: DG
authors.lastname: Hong
authors.affiliation: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Won-Bo
authors.initials: WB
authors.lastname: Shim
authors.affiliation: Department of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Cheulhee
authors.initials: C
authors.lastname: Jung
authors.affiliation: School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea; Advanced Photonics Research Institute, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea. Electronic address: mkim@gist.ac.kr.
authors.forename: Min-Gon
authors.initials: MG
authors.lastname: Kim
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation*; D021881:DNA, Catalytic / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer; D008163:Luminescent Measurements / Q000295:instrumentation*; D009793:Ochratoxins / Q000032:analysis* / Q000737:chemistry
chemical_list: D052157:Aptamers, Nucleotide; D021881:DNA, Catalytic; D009793:Ochratoxins; C025589:ochratoxin A
keywords: Aptamer; Aptasensor; Chemiluminescence resonance energy transfer; DNAzyme; Quenching