Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification

• Published in: Biosensors & bioelectronics Vol. 55; pp. 324 - 329
• Main Authors: Xie, Shunbi, Chai, Yaqin, Yuan, Yali, Bai, Lijuan, Yuan, Ruo
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 15.05.2014; Elsevier
• Subjects: Aptamer; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - genetics; Biological and medical sciences; Biosensing Techniques - instrumentation; Biotechnology; Conductometry - instrumentation; Electrochemical; Equipment Design; Equipment Failure Analysis; Fundamental and applied biological sciences. Psychology; Loop-mediated isothermal amplification; Nucleic Acid Amplification Techniques - instrumentation; Ochratoxin A; Ochratoxins - analysis; Ochratoxins - chemistry; Reproducibility of Results; Sensitivity and Specificity; Temperature; Loop-mediated isothermal amplification; Aptamer; Ochratoxin A; Electrochemical; Amplification; Toxin; Ochratoxin; Electrochemistry; Method; Detection
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2013.11.009

Loop-mediated isothermal amplification (LAMP) is an outstanding DNA amplification procedure, in which the reaction can accumulate 109 copies from less than 10 copies of input template within an hour. While the amplification reaction is extremely powerful, the quantitative detection of LAMP products is still analytically difficult. Besides, the type of targets that LAMP can detect is also less, which to some extent limited the application of LAMP. In this study, we are reporting for the first time an efficient and accurate detection system which employs the integration of LAMP, aptamer and the electrochemical method for the sensitive detection of Ochratoxin A (OTA). Aptamers were designed as the forward outer primer to trigger the LAMP reaction, and then the LAMP amplification products were combined with a redox active molecule methylene blue (MB) and analyzed by an electrode using differential pulse voltammograms (DPV). As the reaction progresses, the MB intercalated into double-stranded regions of LAMP amplicons reduces the free MB concentration. Hence, the peak current of reaction mixture decreased with the amplification because of the slow diffusion of MB-amplified DNA complex to the electrode surface. The peak height of the current was related to the input amount of the aptamers, providing a ready means to detection the concentration of OTA. With such design, the proposed assay showed a good linear relationship within the range of 0.001–50nM with a detection limit of 0.3pM (defined as S/N=3) for OTA. •We employed the integration of LAMP, aptamer and the electrochemical method for the sensitive detection of Ochratoxin A (OTA) for the first time.•This assay was demonstrated that the LAMP reaction can be applied to ultrasensitive detection of OTA.•The prepared aptasensor exhibited low detection limit and wide linear range to OTA.