title: Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.
abstract: To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, PR China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, PR China.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Du
authors.affiliation: Department of Chemistry, Tianjin University, Tianjin 300072, PR China.
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, PR China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, PR China.
authors.forename: De-Ming
authors.initials: DM
authors.lastname: Kong
mesh_terms: D052160:Benzothiazoles; D015374:Biosensing Techniques / Q000295:instrumentation*; D003199:Computer Systems; D015342:DNA Probes / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D054856:G-Quadruplexes; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013844:Thiazoles / Q000032:analysis / Q000737:chemistry*
chemical_list: D052160:Benzothiazoles; D015342:DNA Probes; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T
keywords: G-quadruplex; Real-time; Sensor; Strand displacement amplification; Thioflavin T (ThT)