Application of microchip phosphate-affinity electrophoresis to measurement of protease activity in complex samples.

We previously demonstrated microchip phosphate-affinity electrophoresis (μPAE) for activity measurement of kinases and phosphatases. In the current work, we extend the μPAE application to a protease: caspase-3 (CASP3). We designed a substrate peptide modified with both a fluorescent tag for label and a phosphate tag for separation.

Rapid microRNA detection using power-free microfluidic chip: coaxial stacking effect enhances the sandwich hybridization.

We present a new method for rapid microRNA detection with a small volume of sample using the power-free microfluidic device driven by degassed PDMS. Target microRNA was detected by sandwich hybridization taking advantage of the coaxial stacking effect. This method allows us to detect miR-21 in 20 min with a 0.

Activity measurement of protein kinase and protein phosphatase by microchip phosphate-affinity electrophoresis.

We previously proposed microchip-based phosphate-affinity electrophoresis (μPAE) and demonstrated its application to activity measurement of a tyrosine kinase, c-Src. In this study, we extended the μPAE application to a serine/threonine kinase, protein kinase A (PKA), and to a tyrosine phosphatase, leukocyte antigen-related protein tyrosine phosphatase (LAR PTPase). For standard peptide samples, we obtained linear calibration plots, and the limits of detection were 1.

SNP genotyping of unpurified PCR products by sandwich-type affinity electrophoresis on a microchip with programmed autonomous solution filling.

We demonstrate rapid single-nucleotide polymorphism (SNP) genotyping on a poly(dimethylsiloxane)-glass microchip. Sandwich-type affinity electrophoresis was employed to achieve sufficient specificity for reliable genotyping of unpurified PCR products. We tested three SNPs in different genes: CYP2D6 of artificial templates, and ALDH3A1 and CYP1A1 of human genomic samples.

Phosphate-affinity electrophoresis on a microchip for determination of protein kinase activity.

We describe microchip-based phosphate-affinity electrophoresis (microPAE) for separation of peptides aimed at determination of kinase activity. The microPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form.

Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor.

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe.

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C.

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T.

A MutS protein-immobilized au electrode for detecting single-base mismatch of DNA.

A novel electrochemical biosensor was developed to detect gene mutation by using a DNA-mismatch binding protein: MutS from Escherichia coli. The MutS protein was immobilized onto an Au-electrode surface via complex formation between a histidine tag of the MutS protein and a thiol-modified nitrilotriacetic acid chemically adsorbed on the Au-electrode surface. When a target double-stranded DNA having a single-base mismatch was captured by the MutS protein on the electrode, some electrostatic repulsion arose between polyanionic DNA strands and anionic redox couple ions.

Development of a fluorescence peptide chip for the detection of caspase activity.

Proteases play a key role in cell functions, and it is very important to monitor their activities for drug screening and diagnosis of diseases. In the present study, a new class of fluorescence probe, into which a fluorophore and a quencher have been introduced, was developed and applied to the on-chip detection of caspase-3 activity. This probe is non fluorescent in the absence of caspase-3.

Gene mutation assay using a MutS protein-modified electrode.

A novel electrochemical biosensor for gene mutation detection was developed using a DNA mismatch recognizing protein MutS from E. coli. The MutS protein was immobilized onto an Au electrode by coordination of His-tag at its C-terminus to vacant sites of Ni(II)-nitrilotriacetato complex attached to the surface of electrode.