Aptamer-based exonuclease protection and enzymatic recycling cleavage amplification homogeneous assay for the highly sensitive detection of thrombin

Abstract

Critical challenges in homogeneous solution-based biomolecular detection are the separation and sensitivity compared to biomolecular detection in heterogeneous solutions. In this work, a novel, separation-free and sensitive homogeneous protein detection assay based on combining aptameric exonuclease protection with nicking enzyme assisted fluorescence signal amplification (NEFSA) is developed for highly sensitive protein detection. We applied a special oligonucleotide probe containing a protein aptamer sequence at the 3′-terminus, which has the capacity to recognize the protein target with high affinity and specificity. Specifically, the aptamer probe is protected from exonuclease-catalyzed digestion upon binding to the protein target. The protected aptamer probe hybridizes with the molecular beacon (MB) probe, a reporter signal oligo-DNA. Consequently, the NEFSA process is triggered in the presence of a nicking enzyme, resulting in the continuous enzyme cleavage of many MBs, providing a fluorescent cascadic amplification detection signal for the target. Thrombin was used as the model analyte in the current proof-of-concept experiments. This method permits the detection of human thrombin specifically with a detection limit as low as 1.0 pM without using washes or separations. Our method exhibits excellent sensitivity. In addition, this new method is simple and avoids the specific conformational design of an aptasensor probe for the elimination of washing and separation steps. The mechanism, moreover, may be generalized and used for other forms of protein analysis by changing the corresponding aptamer without changing the other conditions. So our new strategy may provide a homogeneous fluorescence detection platform for many proteins.