Recently, we have successfully developed a two-photon excitation fluorescence resonance energy transfer (TPE-FRET)-based homogeneous immunoassay using two-photon excitable small organic molecule as the energy donor. In the present work, the newly emerging TPE-FRET technique was extended to the determination of oligonucleotide. A new TPE molecule with favorable two-photon action cross section was synthesized [2-(2,5-bis(4-(dimethylamino)styryl)-1H-pyrrol-1-yl)acetic acid, abbreviated as TP-COOH], with the tagged reactive carboxyl group allowing facile conjugation with streptavidin (SA). Employing the TP-COOH molecule as energy donor and black hole quencher 1 (BHQ-1) as acceptor, a TPE-FRET-based homogeneous competitive hybridization model was constructed via a biotin−streptavidin bridge. Through the hybridization between a biotinylated single-stranded DNA (ssDNA) and a BHQ-1-linked ssDNA, and the subsequent capture of the as-formed hybrid by TP-COOH labeled SA, the donor fluorescence was quenched due to the FRET between TP-COOH and BHQ-1. Upon the competition between a target ssDNA and the quencher-linked ssDNA toward the biotinylated oligonucleotide, the donor fluorescence was recovered in a target-dependent manner. Good linearity was obtained with the target oligonucleotide ranging from 0.08 to 1.52 μM. The method was applied to spiked serum and urine samples with satisfying recoveries obtained. The results of this work verified the applicability of TPE-FRET technique in hybridization assay and confirmed the advantages of TPE-FRET in complicated matrix.

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a “plug-and-play” fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil–DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil–DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.