Silver ion-stabilized DNA triplexes for completely enzyme-free and sensitive fluorescence detection of transcription factors via catalytic hairpin assembly amplification

Abstract

Transcription factors play important roles in gene regulation and have been identified as promising biomarkers for disease diagnosis. On the basis of a new Ag⁺-stabilized DNA triplex probe and catalytic hairpin assembly (CHA) signal amplification, we have established a completely enzyme-free and sensitive method for simple fluorescence detection of NF-κB p50 (nuclear factor-kappa B), a transcription factor. We found that the employment of Ag⁺ to stabilize the DNA triplex structure could effectively reduce the background noise. The association of the target NF-κB p50 with the recognition hairpin in the Ag⁺-stabilized DNA triplex leads to the release of a single stranded DNA, which is used as the trigger to initiate subsequent CHA between a fluorescently quenched signal hairpin and the recognition hairpin in the triplex DNA structure, thereby resulting in drastically amplified fluorescence recovery for sensitive detection of NF-κB p50. This assay method shows a dynamic concentration range of 5 to 150 pM and a detection limit of 1.5 pM for the detection of NF-κB p50. Besides, the presence of the target molecules can also be selectively discriminated from other non-specific proteins. Moreover, the presence of low concentrations of NF-κB p50 in human serum samples could be monitored with this approach. With the successful demonstration for NF-κB p50, such a method can be potentially extended to detecting other transcription factors in a convenient and sensitive manner without using any enzymes for signal amplification.