A new class of intracellular nanoprobe, termed fluorescence resonance energy transfer (FRET) nanoflares, was developed to sense mRNA in living cells. It consists of a gold nanoparticle (AuNP), recognition sequences, and flares. Briefly, the AuNP functionalized with recognition sequences hybridized to flares, which are designed as hairpin structures and fluorescently labeled donors and acceptors at two ends, respectively. In the absence of targets, the flares are captured by binding with the recognition sequences, separating of the donor and acceptor, and inducing low FRET efficiency. However, in the presence of targets, the flares are gradually displaced from the recognition sequences by the targets, subsequently forming hairpin structures that bring the donor and acceptor into close proximity and result in high FRET efficiency. Compared to the conventional single-dye nanoflares, the upgraded FRET nanoflares can avoid false positive signals by chemical interferences (such as nuclease and GSH) and thermodynamic fluctuations. Moreover, the signal generation in FRET nanoflares can be easily made with ratiometric measurement, minimizing the effect of system fluctuations.
A new class of intracellular nanoprobe, termed AuNP loaded split-DNAzyme probe, was developed to sense miRNA in living cells. Briefly, it consists of an AuNP and substrates hybridized with two half of split DNAzymes. In the absence of target miRNA, the split DNAzymes form an inactive DNAzyme motif with their substrate through partial paring at the end of each strand, and the fluorescence is quenched. Inside the cells, the target miRNA binds with both of the two half of split DNAzymes, forming the active secondary structure in the catalytic cores, which can cleave the substrates, resulting in the rupture of the substrate and recovery of the fluorescence. Meanwhile, the target is released and binds to another inactive DNAzyme motif to drive another cycle of activation. During the cyclic process, a very small number of target miRNAs can initiate the cleavage of many fluorophore-labeled substrate strands from AuNP surface, providing an amplified fluorescent signal of the target miRNA and, thus, offering high detection sensitivity.
The peroxidase‐like activity of nanozymes is promising for chemodynamic therapy by catalyzing H2O2 into .OH. However, for most nanozymes, this activity is optimal just in acidic solutions, while the pH of most physiological systems is beyond 7.0 (even >8.0 in chronic wounds) with inadequate H2O2. We herein communicate an activatable nanozyme with targeting capability to simultaneously break the local pH and H2O2 limitations under physiological conditions. As a proof of concept, aptamer‐functionalized nanozymes, glucose oxidase, and hyaluronic acid constitute an activatable nanocapsule “APGH”, which can be activated by bacteria‐secreted hyaluronidase in infected wounds. Nanozymes bind onto bacteria through aptamer recognition, and glucose oxidation tunes the local pH down and supplements H2O2 for the in‐situ generation of .OH on bacteria surfaces. The activity switching and enhanced antibacterial effect of the nanocapsule were verified in vitro and in diabetic wounds. This strategy for directly regulating local microenvironment is generally accessible for nanozymes, and significant for facilitating biological applications of nanozymes.
A new class of intracellular nanoprobe, termed AuNP-based hairpin-locked-DNAzyme probe, was developed to sense miRNA in living cells. Briefly, it consists of an AuNP and hairpin-locked-DNAzyme strands. In the absence of target miRNA, the hairpin-locked-DNAzyme strand forms a hairpin structure by intramolecular hybridization, which could inhibit the catalytic activity of DNAzyme strand and the fluorescence is quenched by the AuNP. However, in the presence of target, the target-probe hybridization can open the hairpin and form the active secondary structure in the catalytic cores to yield an "active" DNAzyme, which then cleaves the self-strand with the assist of Mg. The cleaved two shorter DNA fragments are separated with the target. As a result, the fluorophores are released from the AuNP and the fluorescence is enhanced. Meanwhile, the target is also released and binds to another hairpin-locked-DNAzyme strand to drive another cycle of activation. In such a way, the target-recycling amplification leads to significant signal enhancement and thus offers high detection sensitivity.
To date, a few of DNAzyme-based sensors have been successfully developed in living cells; however, the intracellular aptazyme sensor has remained underdeveloped. Here, the first aptazyme sensor for amplified molecular probing in living cells is developed. A gold nanoparticle (AuNP) is modified with substrate strands hybridized to aptazyme strands. Only the target molecule can activate the aptazyme and then cleave and release the fluorophore-labeled substrate strands from the AuNP, resulting in fluorescence enhancement. The process is repeated so that each copy of target can cleave multiplex fluorophore-labeled substrate strands, amplifying the fluorescence signal. Results show that the detection limit is about 200 nM, which is 2 or 3 orders of magnitude lower than that of the reported aptamer-based adenosine triphosphate (ATP) sensors used in living cells. Furthermore, it is demonstrated that the aptazyme sensor can readily enter living cells and realize intracellular target detection.
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