Signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A.

Abstract:

In this work, a new signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A (OTA) is reported. OTA aptamer (DNA1) and OTA aptamer complementary (DNA2) were immobilized onto a magnetic bead (MB). In the presence of OTA, DNA2 was dissociated and released from the MB. The released DNA2 then hybridized with DNA3, which was linked at the 5' terminus of the amplification template and can extend along the template in the presence of Phi 29 DNA polymerase. The formed double-stranded DNA was cleaved by nicking endonuclease Nb.BbvCI and produced a short single-stranded DNA. The cleaved DNA strand generated a new site by Phi 29 DNA polymerase and the process of extension and cleavage was cyclical. Thus, a amount of the short single-stranded DNA were produced. Using DNA and ABEI labeled carboxylic silica nanoparticles chemiluminescence (CL) probe, the short single-stranded DNA could be sensitively detected. The CL intensity (ΔI) versus the concentration of OTA was linear in the range from 1.0×10(-12) to 5.0×10(-8)gmL(-1). The detection limit was 3.0×10(-13)gmL(-1), and the RSD was 3.4% at 1.0×10(-10)gmL(-1) (n=7). The developed method has been applied to detect OTA in naturally contaminated wheat samples. Due to its simplicity, sensitivity and no need of specific recognition of aptamer for cleavage, this CL bioassay offers a promising approach for the detection of OTA and other biomolecules.

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