Zhengping Li

An enzyme-free signal amplification strategy for sensitive detection of microRNA via catalyzed hairpin assembly

In this work, a simple fluorescence turn-on assay is developed for a sensitive detection of microRNA (miRNA) through an enzyme-free signal amplification strategy. This design is based on the miRNA-catalyzed hairpin assembly (CHA). Two metastable hairpin DNA probes, one of which is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence, are rationally designed to preclude unexpected hybridization events between themselves. Therefore, in the absence of miRNAs, the two types of hairpins could stably coexist in the solution with low background fluorescence because of the stringent design. When target miRNAs are added, they work as catalysts to trigger the self-assembly pathway of the two probes and initiate the cycling of CHA circuits, which results in greatly enhanced fluorescence signal. With the help of an efficient signal amplification of CHA and the low-background design of hairpin probes, at concentrations as low as 1 pM miRNA can be detected using this simple and low-cost protocol. High specificity and a wide dynamic range from 1 pM to 2 nM are also obtained. Therefore, this method may have great potential for miRNA-related biological studies.

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.

A general and versatile fluorescence turn-on assay for detecting the activity of protein tyrosine kinases based on phosphorylation-inhibited tyrosyl oxidation

A simple, homogeneous and generic method for detecting protein tyrosine (Tyr) kinase activity is developed based on a tyrosinase-assisted fluorescence turn-on strategy. The tyrosinase-mediated oxidation of the Tyr residue in a fluorescently-labeled peptide may lead to efficient fluorescence quenching, while the tyrosine kinase-catalyzed phosphorylation of the peptide can prevent the Tyr oxidation and thus maintain strong fluorescence.

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.

An electrospun micro/nanofibrous mesh based nontoxic sensor for optical detection of high humidity

Optical transition of a polyethylene oxide electrospun micro/nanofibrous mesh from opaque to transparent has been studied by UV-Vis spectroscopy and scanning electron microscopy. This transition occurs only above approximately 88% relative humidity and could be used for humidity monitoring by the naked eye for food quality control in which low toxicity and irreversibility are highly required.

A Clamp-Based One-Step Droplet Digital Reverse Transcription PCR (ddRT-PCR) for Precise Quantitation of Messenger RNA Mutation in Single Cells

Precise detection of the low copy numbers of messenger RNA (mRNA) mutation in single cells is of great significance but still remains challenging. Herein, by integrating the outstanding features of a rationally designed peptide nucleic acid (PNA) clamp for highly selective discrimination of single-nucleotide variation, and droplet digital PCR for ultrasensitive and precise quantification, we have developed a robust one-step droplet digital reverse transcription PCR (ddRT-PCR) method which enables precise mRNA mutation detection in single cells with ultrahigh specificity to clearly discern as low as 0.01% mutated mRNA in a high background of wild-type mRNA. Because of its outstanding single-molecule level sensitivity and ultrahigh specificity, this ddRT-PCR method holds great promise for studying cellular heterogeneity at the single cell level, as well as for the precise quantification of mutant mRNAs in complex plasma or serum for liquid biopsy.

Single Microbead-Anchored Fluorescent Immunoassay (SMFIA): A Facile and Versatile Platform Allowing Simultaneous Detection of Multiple Antigens

A new concept of single microbead (MB)-anchored fluorescent immunoassay (SMFIA) is proposed with greatly improved sensitivity. In the SMFIA, a single MB is manipulated as the reaction carrier so that the target-tethered fluorescent immunocomplexes will be highly concentrated on one MB. By monitoring the enriched fluorescence signal on the single MB through imaging, highly sensitive target quantification can be realized just by employing the most common sandwich immunoreactions without requirement of further signal amplification routes. The high sensitivity of the SMFIA can fully meet the demand of current medical diagnosis. Furthermore, we have further advanced a fluorescence-encoding mechanism for the proposed SMFIA which allows the simultaneous detection of multiple antigens in a single reaction. Sharing the distinct advantages of simple operation, high sensitivity and multiplexed detection capability, the SMFIA provides a general platform for the detection of various biomarkers.