A label-free fluorescence assay for thrombin based on aptamer exonuclease protection and exonuclease III-assisted recycling amplification-responsive cascade zinc(ii)-protoporphyrin IX/G-quadruplex supramolecular fluorescent labels

A simple, label-free and sensitive fluorescence protein assay has been developed on the basis of aptamer exonuclease protection and exonuclease III (Exo III)-assisted recycling amplification-responsive cascade ZnPPIX/G-quadruplex supramolecular fluorescent labels. In the sensing system, a special aptamer probe containing the aptamer sequence at the 3'-terminus and the DNAzyme sequence at the 5'-terminus was applied, which has the capacity to recognize a protein target with high affinity and specificity. Exonuclease I (Exo I) can efficiently catalyze the degradation of free single stranded DNA probes in the 3' to 5' direction. In the presence of the target protein, the strong binding between the target protein and its aptamer can protect aptamer probes from degradation. Subsequently, the protected aptamer probes act as catalysators to trigger hybridization with the hairpin DNA probe that contains a partially "caged" G-quadruplex sequence. Upon interaction with the protected aptamer probes, the hairpin opens to yield the active G-quadruplex structure. In the presence of exonuclease III (Exo III), Exo III-assisted recycling amplification occurs generating numerous G-quadruplex supramolecular structures. The zinc(ii)-protoporphyrin IX (ZnPPIX) fluorophore binds to the G-quadruplexes and this results in the enhanced fluorescence of the fluorophore. The resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event. Thrombin is used as the model analyte in the current proof-of-concept. The developed method was demonstrated to have very high sensitivity for the detection of proteins with a limit of detection of 0.2 pM without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis.