Target-mediated rolling circle amplification/transcription coupling with double signal amplification of exonuclease III-assisth most of them are low-ed CRISPR/Cas12a-Cas13a for simultaneously ultrasensitive detection of aflatoxin B1 and ochratoxin A

Abstract

Aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) are among the more toxic natural carcinogens that threaten human and animal health. Herein, we reported a target-mediated rolling circle amplification/transcription fluorescent aptasensor for the simultaneous ultrasensitive detection of AFB₁ and OTA by hydrothermal synthesis of double emitting CQDs (CQDs) based on dual signal amplification of Exonuclease III (Exo Ⅲ) and CRISPR-Cas12a/Cas13a. CQDs was combined with probes for the signal marker. In the presence of AFB₁, the aptamer (Apt¹) specifically bonded to the target resulting in cDNA¹ detachment, Apt¹ was digested by the addition of Exo Ⅲ and AFB₁ was released to interact with Apt¹-cDNA¹ again, releasing more cDNA¹ to activate the trans-cleavage activity of the Cas12a protein and to digest the fluorescent probe (Probe¹) from cobalt hydroxide nanosheet (CoOOH) surface detachment, leading to fluorescence recovery to achieve AFB₁ detection. The system of crRNA²/cDNA²/Probe²/Cas13a was used for OTA detection with the similar principles and processes. This strategy utilized both enzymatic activities of Exo III without interfering with each other. In addition, the simultaneous cutting of DNA probe and RNA probe was realized by utilizing the difference in the cutting targets of Cas12a and Cas13a proteins. The double sensitization signal amplification could further reduce the detection limit to achieve the ultrasensitive detection. The linear range for AFB₁ determination was 0.01–50 ng mL⁻¹ with a detection limit of 3.1 pg mL⁻¹. The OTA was 0.01–50 ng mL⁻¹ with a detection limit of 3.5 pg mL⁻¹. Finally, this developed method was employed to detect three kinds of real grain flour samples to demonstrate the reliability.