Le Ying

FRET nanoflares for intracellular mRNA detection: avoiding false positive signals and minimizing effects of system fluctuations

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.

Ratiometric Fluorescent Sensing of pH Values in Living Cells by Dual-Fluorophore-Labeled i-Motif Nanoprobes

We designed a new ratiometric fluorescent nanoprobe for sensing pH values in living cells. Briefly, the nanoprobe consists of a gold nanoparticle (AuNP), short single-stranded oligonucleotides, and dual-fluorophore-labeled i-motif sequences. The short oligonucleotides are designed to bind with the i-motif sequences and immobilized on the AuNP surface via Au-S bond. At neutral pH, the dual fluorophores are separated, resulting in very low fluorescence resonance energy transfer (FRET) efficiency. At acidic pH, the i-motif strands fold into a quadruplex structure and leave the AuNP, bringing the dual fluorophores into close proximity, resulting in high FRET efficiency, which could be used as a signal for pH sensing. The nanoprobe possesses abilities of cellular transfection, enzymatic protection, fast response and quantitative pH detection. The in vitro and intracellular applications of the nanoprobe were demonstrated, which showed excellent response in the physiological pH range. Furthermore, our experimental results suggested that the nanoprobe showed excellent spatial and temporal resolution in living cells. We think that the ratiometric sensing strategy could potentially be applied to create a variety of new multicolor sensors for intracellular detection.

Aptazyme–Gold Nanoparticle Sensor for Amplified Molecular Probing in Living Cells

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.