Use of capillary electrophoresis and endogenous fluorescent substrate to monitor intracellular activation of protein kinase A

Here we demonstrate for the first time the use of an endogenous multiphosphorylatable substrate for monitoring the intracellular activation of kinase with capillary electrophoresis. First, we devised a novel PCR-based strategy for controlled generation of short multirepeat DNA sequences and applied this method to generate a green fluorescence protein (GFP)-tagged protein substrate containing eight phosphorylation sites for protein kinase A (PKA). The protein substrate was transiently expressed in C2C12 rat myoblast cells, and intracellular PKA was then activated by adding [8]-bromo-cyclic AMP to the cell culture medium. Phosphorylated product and nonphosphorylated substrate present in the crude cell extract were separated by capillary zone electrophoresis and detected with laser-induced fluorescence of the GFP tag. The identities of two electrophoretic peaks were confirmed by both phosphorylation of the substrate and dephosphorylation of the product in vitro. The proposed method was applied to monitoring the activation of PKA in single myoblast cells. It advantageously allowed us to avoid microinjection of the substrate, the procedure that is both hard to perform and excessively invasive when applied to small mammalian cells.     

Peptide biosensors for the electrochemical measurement of protein kinase activity

The kinase activities are elucidated using the novel redox-active cosubstrate adenosine 5'-[gamma-ferrocene] triphosphate (Fc-ATP), which enables the kinase-catalyzed transfer of a redox active gamma-phosphate-Fc to a hydroxyamino acid. In this report, a versatile electrochemical biosensor is developed for monitoring the activity and inhibition of a serine/threonine kinase, casein kinase 2 (CK2), and protein tyrosine kinases, Abl1-T315I and HER2, in buffered solutions and in cell lysates. The method is based on the labeling of a specific phosphorylation event with Fc, followed by electrochemical detection. The electrochemical response obtained from the "ferrocenylated" peptides enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. Kinetic information was extracted from the electrochemical measurements for the determination of K(m) and V(m) values, which were in agreement with those previously reported. Kinase reactions were also performed in the presence of well-defined inhibitors of CK2, 4,5,6,7-tetrabromo-2-azabenzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, and E-3-(2,3,4,5-tetrabromophenyl)acrylic acid as well as the nonspecific kinase inhibitors, staurosporine and N-benzoylstaurosporine. On the basis of the dependency of the Fc signal on inhibitor concentration, K(i) of the inhibitors was estimated, which were also in agreement with the literature values. The performance of the biosensor was optimized including the kinase reaction, incubation with Fc-ATP, and the small molecule inhibitors. Peptide modified electrochemical biosensors are promising candidates for cost-effective in vitro kinase activity and inhibitor screening assays.     

Microfluidic device for electric field-driven single-cell capture and activation

A microchip that performs directed capture and chemical activation of surface-modified single cells has been developed. The cell capture system is comprised of interdigitated gold electrodes microfabricated on a glass substrate within PDMS channels. The cell surface is labeled with thiol functional groups using endogenous RGD receptors, and adhesion to exposed gold pads on the electrodes is directed by applying a driving electric potential. Multiple cell types can thus be sequentially and selectively captured on desired electrodes. Single-cell capture efficiency is optimized by varying the duration of field application. Maximum single-cell capture is attained for the 10-min trial, with 63 +/- 9% (n = 30) of the electrode pad rows having a single cell. In activation studies, single M1WT3 CHO cells loaded with the calcium-sensitive dye fluo-4 AM were captured; exposure to the muscarinic agonist carbachol increased the fluorescence to 220 +/- 74% (n = 79) of the original intensity. These results demonstrate the ability to direct the adhesion of selected living single cells on electrodes in a microfluidic device and to analyze their response to chemical stimuli.     

Deformation measurement of individual cells in large populations using a single-cell microchamber array chip

We analyze the deformability of individual red blood cells (RBCs) using SiCMA technology. Our approach is adequate to quickly measure large numbers of individual cells in heterogeneous populations. Individual cells are trapped in a large-scale array of micro-wells, and dielectrophoretic (DEP) force is applied to deform the cells. The simple structures of micro-wells and DEP electrodes facilitate the analysis of thousands of RBCs in parallel. This unique method allows the correlation of red cell deformation with cell surface and cytosolic characteristics to define the distribution of individual cellular characteristics in heterogeneous populations.     

一种ICP-MS真空测量系统的研制

本文介绍了一种基于C8051F微控制器并应用于电感耦合等离子体质谱仪(ICP-MS)的真空测量系统。系统由真空计组、预处理电路、C8051F微控制器、串行接口和上位机组成,能同时测量接口、透镜和四级杆三个部分的真空状态,且具有较高的集成度,紧凑的体积和较低的功耗,经最后实验证实,系统能准确且稳定的测量各个部分的真空度,可为ICP-MS的正常工作提供相应的条件保障。     

Chromium silicide film on ceramic substrate for pressure measurement

In this paper we present a device, based on amorphous silicon technology, fabricated on ceramic substrate able to perform pressure measurement with both good linearity and sensitivity. The active material of the sensor is a thin film (below 5 nm) of chromium silicide formed at room temperature on an n-type amorphous silicon layer. Sensors with different shapes (square and rectangular) and positions on the ceramic membrane have been characterized. Sensitivity in the order of 400 μV/kPa has been achieved.     

Development of a laser-scattering-based probe for on-line measurement of surface roughness

The design and properties of an optical probe for on-line measurement of surface roughness are discussed. Based on light scattering, a probe that consists of a laser diode, a measuring lens, and a linear photodiode array was designed to detect surface roughness, in which the light scattered from a test surface at a relatively large scattering angle phi (=28 degrees) can be collected to enhance measuring range and repeatability. A coaxial design that incorporates a dual-laser probe and compressed air makes the proposed system insensitive to the position of the test surface and to surface conditions such as the presence of debris, vibration, and lubricants that result from machining. The results from measurements of several sets of specimens have demonstrated the feasibility of measuring surface roughness by using light scattering. On-line measurement on a diamond-turning lathe has shown that the proposed technique is stable and compact enough to be applicable to on-line measurement of surface roughness of an engineering surface.     

Aptameric peptide for one-step detection of protein kinase

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.     

手推式钢轨外形几何尺寸测量系统

研制了一种手推式钢轨外形几何尺寸测量系统。利用激光线光源投射于钢轨表面,钢轨外形轮廓的光截曲线成像在CCD上。经过灰度变换、边缘提取和中心线提取,获得截面处的钢轨外形曲线。将其标定后,与钢轨标准曲线进行比较,从而求得实测钢轨的磨损量。该系统具有体积小、重量轻、实时处理、操作方便等优点。     

Thickness measurement of a thin-film layer on an anisotropic substrate by phase-sensitive acoustic microscope

Complex V(z) curves for single thin-film layers on anisotropic substrates are studied both experimentally and theoretically, and the application of V(z) measurement to the determination of film thickness on anisotropic substrates is discussed. Complex V(z) curves for aluminum layers (with thicknesses between 0.5 and 2 mum) on a silicon wafer have been calculated. The inverse Fourier transform of the V(z) curves, which corresponds to the reflection coefficient, shows sharp changes at critical angles of pseudosurface waves, pseudo-Sezawa waves, and Rayleigh surface waves. These critical angles strongly depend on the thickness. Complex V(z) curves for these specimens have been measured using a phase-sensitive acoustic microscope with a point focus lens at 400 MHz. The critical angles of the surface waves obtained from the measured V(z) curves are in good agreement with those obtained from the calculated V(z) curves. On the basis of this result, it is shown that the V(z) measurement is applicable to the determination of film thickness on an anisotropic substrate.     

批量光电二极管弱光光敏特性测试仪

针对光照度小于101x的弱光使用环境,提出并设计了一种批量光电二极管弱光光敏特性测试仪。仪器包括可调弱光光源、照度均匀化装置、批量光电二极管固定板、多通道转换开关、微电流放大电路、环境照度监测通道、数据采集及微机接口电路和相关配套软件。仪器的核心为自行研制的光照度在0．01-101x范围内无级可调的弱光光源。实际应用中,在21x照度下,光电二极管弱光光电流一般为12．2-15．6nA,只有少数光电流低于10nA;因此,该仪器可用来进行光电二极管的定量筛选。     

Development of a silver nanoparticle-based method for the antioxidant capacity measurement of polyphenols

A sensitive colorimetric method for the detection of polyphenols (i.e., flavonoids, simple phenolic, and hydroxycinnamic acids) was proposed in this research based on the reduction of Ag(+) ions by polyphenols in the presence of citrate-stabilized silver seeds. The color of the stable suspension was controlled by varying the concentration of trisodium citrate, silver nitrate, and silver seeds. The reduction of Ag(+) to spherical silver nanoparticles (SNPs) by polyphenols in the presence of trisodium citrate and silver seeds produced a very intense surface plasmon resonance (SPR) absorption band of SNPs at 423 nm. The plasmon absorbance of SNPs allows the quantitative spectrophotometric detection of the polyphenols, and the developed method gave a linear response over a wide concentration range of standard polyphenolic compounds. In contrast to other reported NP-based antioxidant assays, it was established in this work that growth but not nucleation of SNPs gave a linear concentration-dependent response. The trolox equivalent antioxidant capacity (TEAC) values of various (hydrophilic and lipophilic) antioxidants using the developed method were comparable to those of the CUPRAC assay. Common food ingredients like oxalate, citrate, fruit acids, amino acids, and reducing sugars did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. The developed method was used to screen total antioxidant capacity (TAC) of some commercial fruit juices and herbal teas without preliminary treatment, and showed a promising potential for the preparation of antioxidant inventories of a wide range of food plants.     

Fluorescent peptide probes for high-throughput measurement of protein phosphatases

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.     

Measurements of the binding of a large protein using a substrate density-controlled DNA chip

The DNA chip that immobilizes DNA oligonucleotides on a solid plate surface is used for many diagnostic applications. For maximizing the detection sensitivity and accuracy, it is important to control the DNA density on a chip surface and establish a convenient method for optimizing the density. Here, the binding of DNA mismatch-binding protein MutS to the DNA substrate on the chip was investigated, which can be applied for high-throughput single-nucleotide polymorphism analysis in a genome. We prepared the DNA chips where the DNA substrate density was changed simply by using a mixed DNA solution. The binding of MutS was significantly influenced by the amount of DNA substrate on the chip as a consequence of steric crowding, and the moderate density that gave the distance between the DNA substrates greater than the size of the protein was appropriate to obtain accurate kinetic parameters. The substrate density-controlled DNA chip prepared using the mixed DNA solution has distinctive advantages for maximizing the detection capability and kinetic analysis of the binding of MutS and probably also other large proteins.     

Development of a comprehensiveness of Rasch measurement application scale

A large number of papers and technical reports are published every year describing researches where Rasch models are used. It has been observed, however, that not all the authors describe the application of the Rasch measurement with the same thoroughness. Some authors may leave behind important bits of information e.g. they may fail to investigate the person or item fit or may even fail to discuss the reliability of measurement. As a result, editorial guidelines have been published in order to suggest an informal minimum of thoroughness with which the authors may describe the application of Rasch measurement in their papers. This study presents stages for the development of a scale to investigate the comprehensiveness with which individual papers describe the application of Rasch models in practical settings. The scale is used to evaluate how comprehensively the papers published by the Journal of Applied Measurement present the application of Rasch models.     

Development of a variable launch attenuation and isolation measurement system for optical waveguides

In this paper we describe a system that measures the attenuation and isolation of optical waveguides and has the capability to fully explore these properties over a range of reproducible launch conditions. The system allows both the launch signal spot size and numerical aperture to be varied and can be correlated to the actual operating conditions of the board. Characterization of the optical system, including the magnification factor as well as the linearity, sensitivity, spatial uniformity of the charge-coupled device cameras, is shown. Initial results from a variety of waveguides, including planar, radii, and crossover designs, are discussed and an assessment of the key uncertainty contributions of the system is presented.     

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.     

Reusable amperometric biosensor for measuring protein tyrosine kinase activity

This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.     

Development of a TL detector for neutron measurement by CaSO4:Dy phosphors

Personal neutron dosimetry is quite a difficult area because a neutron is always accompanied with gamma radiation, which is required of a capability for mixed field dosimetry. CaSO4:Dy phosphor is known to have a very high sensitivity to gamma radiation, but the neutron capture cross section of the constituents of CaSO4:Dy are so small that the interactions between the thermal neutron and the phosphor are rare. One method to improve the neutron interaction is by introducing an impurity ion with a large thermal neutron captures cross section into the phosphor to act as a neutron target centre such as 6Li. In neutron-gamma mixed radiation fields, if two detectors for the 6Li-7Li compounds embedded CaSO4:Dy thermoluminescent (TL) pellets are used, a 6Li-compound embedded pellet can detect the neutron and gamma radiations together, and the other pellet can only detect the gamma radiation. Recently, the Korea Atomic Energy Research Institute (KAERI) has developed a new type of CaSO4:Dy TL materials embedded with phosphorous (KCT-300) to detect beta and gamma radiation with a very high sensitivity. This paper presents the development of CaSO4:Dy TL pellets embedded with 6Li compound for a thermal neutron measurement, and the detection method of the neutron and gamma dose in mixed fields with CaSO4:Dy TL pellets embedded with a 6Li compound (KCT-306) and CaSO4:Dy TL pellets embedded with a 7Li compound (KCT-307) is introduced. The net neutron sensitivity of CaSO4:Dy TL pellets embedded with 6Li compound developed in this study is about two times higher than that of the TLD-600 (Harshaw Chemical) dosemeter which is available commercially.