Yucong Wang

Dual-Readout Fluorescent Assay of Protein Kinase Activity by Use of TiO2-Coated Magnetic Microspheres

A simple, highly sensitive, and dual-readout fluorescent assay is developed for the detection of protein kinase activity based on the specific recognition utility of TiO2-coated Fe3O4/SiO2 magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When the fluorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they can bind specifically to the TiO2 layer of TMSPs by means of phosphate groups, resulting in fluorophore enrichment on the TMSP surfaces. The accumulated fluorophores on the TMSPs are proportional to the kinase activity, and the fluorescence signal readout could be run through either direct fluorescent imaging of the TMSPs or measurement of the fluorescence intensity by simply detaching the fluorescent phosphopeptides into the solution. The TMSPs exhibit extremely high selectivity for capturing phosphorylated peptides over the nonphosphorylated ones, resulting in an ultrahigh fluorescence signal-to-background ratio of 42, which is the highest fluorescence change thus far in fluorescent assays for detection of protein kinase activities. Therefore, the proposed fluorescent assay presents high sensitivity, low detection limit of 0.1 milliunit/μL, and wide dynamic range from 0.5 milliunit/μL to 0.5 unit/μL with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fluorescent assay can simultaneously quantify multiple kinase activities with their specific peptides labeled with different dyes. This new strategy is also successfully applied to monitoring drug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fluorescent assay is very promising in high-throughput screening of kinase inhibitors and in highly sensitive detection of kinase activity, and thus it is a valuable tool for development of targeted therapy, clinical diagnosis, and studies of fundamental life science.

A cytometric bead assay for sensitive DNA detection based on enzyme-free signal amplification of hybridization chain reaction

A versatile flow cytometric bead assay (CBA) is developed for sensitive DNA detection by integrating the advantages of hybridization chain reaction (HCR) for enzyme-free signal amplification, flow cytometry for robust and rapid signal readout as well as magnetic beads (MBs) for facile separation. In this HCR-CBA, a biotinylated hairpin DNA (Bio-H1) is firstly immobilized on streptavidin-functionalized MBs. Upon the addition of target DNA, each target would hybridize with one Bio-H1 to open its hairpin structure and subsequently initiate a cascade of hybridization events between two species of fluorescent DNA hairpin probes (H1*/H2*) to form a nicked double helical DNA structure, resulting in amplified accumulation of numerous fluorophores on the MBs. Finally, the fluorescent MBs are directly analyzed by flow cytometry. This technique enables quantitative analysis of the HCR products anchored on the MBs as a function of target DNA concentration, and analysis of each sample can be completed within few minutes. Therefore, the HCR-CBA approach provides a practical DNA assay with greatly improved sensitivity. The detection limit of a model DNA target is 0.5 pM (3σ), which is about 3 orders of magnitude lower compared with traditional hybridization methods without HCR. Furthermore, the signal of complementary target can be clearly distinguished from that of single-base mismatched sequences, indicating the high specificity of the HCR-CBA. Moreover, this strategy is also successfully applied to the DNA analysis in complex biological samples, showing great potential in gene analysis and disease diagnosis in clinical samples.

Highly sensitive detection of protein kinase activity using upconversion luminescent nanoparticles

An upconversion nanophosphor (UCNP)-based highly sensitive assay for the detection of protein kinase activity is developed with the assistance of Zr4+-functionalized magnetic beads.

Label-free detection of histone based on cationic conjugated polymer-mediated fluorescence resonance energy transfer

A simple and homogeneous histone assay is developed based on histone-induced DNA compressing coupled with cationic conjugated polymer (CCP)-mediated fluorescence resonance energy transfer (FRET). In this strategy, the CCP serves as the FRET donor and SYBR Green I (SG), which can strongly fluoresce not at its free state but after intercalated into the double stranded calf thymus DNA (dsDNA), serves as the acceptor of FRET. In the absence of histone, the dsDNA-SG and CCP combine with each other through electrostatic interaction and the strong FRET from CCP to SG occurs due to the overlapping between the fluorescent emitting spectrum of the CCP and the absorption spectrum of SG. Upon the introduction of histone, the formed compact complex of histone/dsDNA will lead to the compression of dsDNA structure and prevent SG binding to dsDNA and fluorescing, which gives rise to a significant decrease of FRET efficiency between CCP and SG. Thus, the quantitative analysis of histone is realized by monitoring the change of FRET ratio, namely, the intensity ratio of the two emission bands of CCP and SG. Due to the light harvesting and fluorescence amplification properties of CCP, high sensitivity is achieved with a low detection limit of 0.74ng/mL histone. This strategy provides a simple, homogeneous and sensitive strategy for histone analysis in the study of histone-related biological processes.

A new two-mode fluorescence signal amplification strategy for protease activity assay based on graphene oxide

A graphene oxide (GO)-based two-mode fluorescence signal amplification assay of protease activity has been established. GO can adsorb the FITC-labeled substrate peptide and quench the fluorescence of FITC. In the presence of the target protein, carboxypeptidase Y (CPY), the FITC-labeled substrate peptide is hydrolyzed by CPY, leading to the turn-on of fluorescence. The fluorescence intensity increases significantly after the hydrolysis. More interestingly, it is even much higher than the fluorescence intensity of the added FITC-labeled substrate peptides. It is deduced that the extraordinary growing of fluorescence intensity is attributed to the hydrolysis also. The strong quenching efficiency of GO significantly improved the signal-to-noise ratio (SNR) of the proposed method for protease analysis. By combining the GO-based fluorescence turn-on with the fluorescence signal amplification induced by hydrolysis, the proposed method obtained higher sensitivity and specificity for CPY activity detection. The detection limit for CPY activity assay is estimated to be 1.0 × 10−5 U μL−1. The other proteins, proteases and a complicated matrix cannot disturb the assay of CPY activity.