Honghong Wang

Upconversion Nanophosphor: An Efficient Phosphopeptides-Recognizing Matrix and Luminescence Resonance Energy Transfer Donor for Robust Detection of Protein Kinase Activity

Protein kinases play important regulatory roles in intracellular signal transduction pathways. The aberrant activities of protein kinases are closely associated with the development of various diseases, which necessitates the development of practical and sensitive assays for monitoring protein kinase activities as well as for screening of potential kinase-targeted drugs. We demonstrate here a robust luminescence resonance energy transfer (LRET)-based protein kinase assay by using NaYF4:Yb,Er, one of the most efficient upconversion nanophosphors (UCNPs), as an autofluorescence-free LRET donor and a tetramethylrhodamine (TAMRA)-labeled substrate peptide as the acceptor. Fascinatingly, besides acting as the LRET donor, NaYF4:Yb,Er UCNPs also serve as the phosphopeptide-recognizing matrix because the intrinsic rare earth ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by protein kinases over the unphosphorylated ones. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read format without any requirement of surface modification, substrate immobilization, separation, or washing steps, showing great potential in protein kinases-related clinical diagnosis and drug discovery. To the best of our knowledge, this is the first report by use of rare earth-doped UCNPs as both the phospho-recognizing and signal reporting elements for protein kinase analysis.

Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single‐Cell Level

A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level.

Enzyme-free and multiplexed microRNA detection using microRNA-initiated DNA molecular motor

In this work, we have developed a sensitive, simple, and enzyme-free assay for detection of microRNAs (miRNAs) by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains. In the presence of miRNA target, it can hybridize with one of the stem-loop DNA to open the stem and to produce a miRNA/DNA hybrid and a single strand (ss) DNA, the ssDNA will in turn hybridize with another stem-loop DNA and finally form a double strand (ds) DNA to release the miRNA. One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence. The formation of dsDNA can produced specific fluorescence signal for miRNA detection. The released miRNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor, which results in great fluorescence amplification. With the efficient signal amplification, as low as 1 pmol/L miRNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained. Moreover, by designing different stem-loop DNAs specific to different miRNA targets and labeling them with different fluorophores, multiplexed miRNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum (SFS) technique.

Digital quantitative analysis of microRNA in single cell based on ligation-depended polymerase colony (Polony)

The ability to dissect cell-to-cell variations of microRNA (miRNA) expression with single-cell resolution has become a powerful tool to investigate the regulatory function of miRNAs in biological processes and the pathogenesis of miRNA-related diseases. Herein, we have developed a novel scheme for digital detection of miRNA in single cell by using the ligation-depended DNA polymerase colony (polony). Firstly, two simply designed target-specific DNA probes were ligated by using individual miRNA as the template. Then the ligated DNA probe acted as polony template that was amplified by PCR process in the thin polyacrylamide hydrogel. Due to the covalent attachment of a PCR primer on polyacrylamide matrix and the retarding effect of the polyacrylamide hydrogel matrix itself, as the polony reaction proceeds, the PCR products diffused radially near individual template molecule to form a bacteria colony-like spots of DNA molecules. The spots can be counted after staining the polyacrylamide gel with SYBR Green I and imaging with a microarray scanner. Our polony-based method is sensitive enough to detect 60 copies of miRNA molecules. Meanwhile, the new strategy has the capability of distinguishing singe-base difference. Due to its high sensitivity and specificity, the proposed method has been successfully applied to analysis of the expression profiling of miRNA in single cell.

Sensitive detection of tumor cells based on aptamer recognition and isothermal exponential amplification

The noninvasive detection of tumor cells is significantly important for early diagnosis of cancers and monitoring of their progress. Herein, we have developed a novel aptamer-based isothermal exponential amplification reaction (EXPAR) for sensitive detection of tumor cells. In this new assay, biotinylated sgc8c DNA aptamers are immobilized on streptavidin-coated magnetic beads. After incubation with target CCRF-CEM cells and magnetic isolation, the tumor cells are detected by translating the structure-switching of an aptamer upon tumor cell binding into an input of DNA trigger for EXPAR. Since more and more specific aptamers towards different tumor cells would be identified in the future, our strategy can be easily extended to the detection of different tumor cells just by simply altering the corresponding aptamers. Therefore, this EXPAR-based strategy may serve as a generic platform for the accurate detection of tumor cells, which has great potential for the early diagnosis of cancers.

Ultrasensitive detection of site-specific DNA methylation by loop-mediated isothermal amplification

A novel loop-mediated isothermal amplification (LAMP)-based methylation assay for simple, robust and cost-effective detection of site-specific DNA methylation has been developed. DNA targets are first treated with methylation-sensitive restriction endonuclease (HpaII), where the DNA targets will be cleaved at specific unmethylated-cytosine residues while leaving the methylated DNA intact. Subsequently, the methylated DNA targets can serve as templates to perform LAMP for the detection of DNA methylation with real-time fluorescence measurements by using a common fluorescent dye (SYBR Green I). Taking advantage of the simplicity and high specificity of HpaII digestion and the isothermal nature and high sensitivity of LAMP, the proposed assay can greatly simplify the detection of DNA methylation and achieve ultrahigh sensitivity and specificity. With this assay, as low as 10 aM methylated DNA can be detected and 0.1% methylated DNA can be determined in the presence of a large excess of unmethylated DNA.