Ultrasensitive electrochemiluminescent aptasensor for ochratoxin A detection with the loop-mediated isothermal amplification.
In this study, we for the first time presented an efficient, accurate, rapid, simple and ultrasensitive detection system for small molecule ochratoxin A (OTA) by using the integration of loop-mediated isothermal amplification (LAMP) technique and subsequently direct readout of LAMP amplicons with a signal-on electrochemiluminescent (ECL) system. Firstly, the dsDNA composed by OTA aptamer and its capture DNA were immobilized on the electrode. After the target recognition, the OTA aptamer bond with target OTA and subsequently left off the electrode, which effectively decreased the immobilization amount of OTA aptamer on electrode. Then, the remaining OTA aptamers on the electrode served as inner primer to initiate the LAMP reaction. Interestingly, the LAMP amplification was detected by monitoring the intercalation of DNA-binding Ru(phen)3(2+) ECL indictors into newly formed amplicons with a set of integrated electrodes. The ECL indictor Ru(phen)3(2+) binding to amplicons caused the reduction of the ECL intensity due to the slow diffusion of Ru(phen)3(2+)-amplicons complex to the electrode surface. Therefore, the presence of more OTA was expected to lead to the release of more OTA aptamer, which meant less OTA aptamer remained on electrode for producing LAMP amplicons, resulting in less Ru(phen)3(2+) interlaced into the formed amplicons within a fixed Ru(phen)3(2+) amount with an obviously increased ECL signal input. As a result, a detection limit as low as 10 fM for OTA was achieved. The aptasensor also has good reproducibility and stability.
Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China; College of Resources and Environments, Southwest University, Chongqing 400715, PR China.
Yali
Y
Yuan
College of Resources and Environments, Southwest University, Chongqing 400715, PR China.
Shiqiang
S
Wei
College of Resources and Environments, Southwest University, Chongqing 400715, PR China.
Guangpeng
G
Liu
Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Shunbi
S
Xie
Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. Electronic address: yqchai@swu.edu.cn.
Yaqin
Y
Chai
Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. Electronic address: yuanruo@swu.edu.cn.
Ruo
R
Yuan
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D055664:Electrochemical Techniques; D004566:Electrodes; D061925:Immobilized Nucleic Acids / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements; D021141:Nucleic Acid Amplification Techniques; D009793:Ochratoxins / Q000032:analysis*; D009942:Organometallic Compounds / Q000737:chemistry; D010618:Phenanthrolines / Q000737:chemistry; D013917:Thrombin / Q000032:analysis; D014920:Wine / Q000032:analysis
D052157:Aptamers, Nucleotide; D061925:Immobilized Nucleic Acids; D009793:Ochratoxins; D009942:Organometallic Compounds; D010618:Phenanthrolines; C025589:ochratoxin A; C034282:tris-(1,10-phenanthroline)ruthenium; D004247:DNA; D013917:Thrombin
Aptasensor; Electrochemiluminescent; Loop-mediated isothermal amplification; Signal-on; Small molecule