Photoactivated Nanoflares for mRNA Detection in Single Living Cells

Gold nanoparticles (AuNPs) have shown great promise as a universal platform for biosensing and are often functionalized with a densely packed DNA for intracellular detection. While DNA-AuNP conjugates, such as nanoflares, have been used for single and multiple mRNA molecules detection in living cells, the target recognition reaction is triggered once they enter into cells, making it impossible to control the initial reaction at the desired time. To solve this problem, we have designed photoactivated (PA) nanoflares for intracellular mRNA analysis with high spatiotemporal control. PA nanoflares consist of AuNP and photoresponsive DNA hairpin probes. Without UV irradiation, the DNA hairpin could be kept unawakened and show no reactivity to target the probe. Upon UV activation, the hairpin structures are destroyed and expose the sticky domains, which act as toeholds to mediate strand displacement reactions, making flares release from the gold surface and causing an increase of fluorescence. By tuning light irradiation, PA nanoflares for mRNA detection in living cells can be temporally controlled. With the benefit from two-photon laser illumination, PA nanoflares can detect mRNA in selective cells at a desired time point at the single-cell level. Compared to the traditional nanoflares, the novel PA nanoflares have increased the detection sensitivity and achieved intracellular biomarkers detection at the single-cell level with high spatiotemporal control.