Hybridization-activated spherical DNAzyme for cascading two-photon fluorescence emission: Applied for intracellular miRNA measurement by two-photon microscopy

Two-photon excitation (TPE) has excellent properties, such as lower tissue self-absorption and autofluorescence, reduced photodamage and photobleaching, and higher spatial resolution. Meanwhile, DNAzyme has significant signal amplification ability, and the gold nanoparticle (AuNP)-based spherical nucleic acid (SNA) has the properties of resistance to enzymatic degradation, enhanced nucleic acid binding and excellent cellular uptake. Taking advantage of their properties, in this work, we constructed a novel SNA-based TPE fluorescent DNAzyme probe for the selective and sensitive detection of target miRNA in living cells. Briefly, AuNPs were first functionalized with the substrate sequences hybridized to two split fragments of the 8-17 DNAzyme-contained sequence. Then, numerous two-photon absorption (TPA)-dye molecules of Ethyl-4-[3,6-Bis (1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] (EBMVC) were inserted into the substrate/split-DNAzyme duplexes on the surface of the AuNP to form the TPE SNA nanoprobe in the "off" fluorescence emission state. When the target miRNA is bound, the conformation of the split DNAzymes will change resulting in the activation of their catalytic ability, thus leading to cleavage of the substrate sequences and the release of the TPA-dye molecules-inserted DNAzyme motif from the surface of the AuNP. Concurrently, the released target miRNA would autonomously hybridize with another split DNAzyme motif and activate further cleavage reactions. This would induce the gradual dissociation of the TPA-dye molecules-inserted DNAzyme motifs from the surface of the AuNP through the repeated target miRNA hybridization-activated cleavage reaction and thereby trigger the cascade TPE fluorescence emission, ultimately achieving the detection of the miRNA through amplification of the fluorescence signals. The assay was successfully validated with clean buffer conditions as well as with cells.