title: Chemiluminescence resonance energy transfer imaging on magnetic particles for single-nucleotide polymorphism detection based on ligation chain reaction.
abstract: A novel ligation chain reaction (LCR) methodology for single-nucleotide polymorphism (SNP) detection was developed based on luminol-H2O2-horseradish peroxidase (HRP)-mimicking DNAzyme-fluorescein chemiluminescence resonance energy transfer (CRET) imaging on magnetic particles. For LCR, four unique target-complement probes (X and X(⁎), YG and Y(⁎)) for the amplification of K-ras (G12C) were designed by modifying G-quadruplex sequence at 3'-end of YG and fluorescein at 5'-end of Y(⁎). After the LCR, the resulting products of XYG/X(⁎)Y(⁎) with biotin-labeled X(⁎) were captured onto streptavidin-coated magnetic particles (SA-MPs) via specific biotin-SA interaction, which stimulated the CRET reaction from hemin/G-quadruplex-catalyzed luminol-H2O2 CL system to fluorescein. By collecting signals by a cooled low-light CCD, a CRET imaging method was proposed for visual detection and quantitative analysis of SNP. As low as 0.86fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000:1. This high sensitivity and specificity could be attributed to not only the exponential amplification and excellent discrimination of LCR but also the employment of SA-MPs. SA-MPs ensured the feasibility of the proposed strategy, which also simplified the operations through magnetic separation and separated the reaction and detection procedures to improve sensitivity. The proposed LCR-CRET imaging strategy extends the application of signal amplification techniques to SNP detection, providing a promising platform for effective and high-throughput genetic diagnosis.
authors.affiliation: Laboratory of Fiber Materials and Modern Textiles, the Growing Base for State Key Laboratory, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Qingdao University, Qingdao 266071, PR China. Electronic address: bisai11@126.com.
authors.forename: Sai
authors.initials: S
authors.lastname: Bi
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Laboratory of Fiber Materials and Modern Textiles, the Growing Base for State Key Laboratory, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Qingdao University, Qingdao 266071, PR China. Electronic address: wangzonghua@qdu.edu.cn.
authors.forename: Zonghua
authors.initials: Z
authors.lastname: Wang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D004247:DNA / Q000097:blood / Q000235:genetics*; D004735:Energy Transfer; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
chemical_list: D004247:DNA
keywords: Chemiluminescence resonance energy transfer; Ligation chain reaction; Magnetic particles; Single-nucleotide polymorphism