Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays

In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.     

Synthesis and evaluation of superparamagnetic silica particles for extraction of glycopeptides in the microtiter plate format

Solid-phase extraction of glycopeptides (SPEG) coupled with quantitative proteomic analysis using mass spectrometry has shown great potential for investigating glycoproteins in an effort to discover new diagnostic biomarkers or therapeutic targets. As a solid-phase approach, SPEG can be performed with a microtiter plate to provide a high-throughput platform for large-scale screening of clinical samples. Here we describe the synthesis of superparamagnetic silica particles with hydrazide groups on the surface and further evaluate their use as the solid support for SPEG. We produced nonspherical silica particles containing superparamagnetic iron oxide cores using a modified St?ber method and then derivatized their surface with hydrazide-terminated silane. Such composite particles displayed a strong response to the external magnetic field, and this feature enabled us to capture and release the particles easily for automated, high-throughput sample preparation of glycopeptides. When measured with standard glycoproteins, the adsorption capacity of these particles was >36 mg of glycoproteins per g of nanoparticles. The nanoparticles were used in a microtiter plate format for glycopeptide capture using a liquid handler. The captured glycopeptides were then analyzed by LC-MS and LC-MS/MS to determine the specificity and reproducibility of glycopeptide isolation. Our results demonstrate the potential of these superparamagnetic colloidal particles for high-throughput analysis of glycoproteins.     

Immunoassay with a microtiter plate incorporated multichannel electrochemical detection system

To extend the application of a multichannel electrochemical detector (MED) for immunoassay, a MED system consisting of 8 sets of Pt electrodes in an arrangement fitted with the dimensions of a row of microtiter wells in a microtiter plate and a microcomputer-assisted 16-channel potentiostat was constructed. With this developed MED system, electroactive enzymatic products produced in eight microtiter wells can be analyzed simultaneously with a developed amperometric procedure. To demonstrate the applicability of the MED system for immunoassay, an immunosystem containing rabbit-IgG and alkaline phosphatase-conjugated goat anti rabbit-IgG was studied. From the dynamic range of 10-1000 ng/mL (0.064-6.4 pM) and the detection limit of 1.0 ng/mL (6.4 pM) obtained, the developed MED shows a tremendous improvement in sensitivity, detection limit, and efficiency compared with that obtained from conventional electrochemical immunoassay.     

Automated high-throughput infusion ESI-MS with direct coupling to a microtiter plate

This paper describes the design and application of instrumentation for automated high-throughput infusion ESI-mass spectrometry. The approach, based on a subatmospheric ESI interface, allows sample introduction from a commercially available microtiter plate without the need for a separate fluid delivery system. The microtiter plate was placed vertically on a three-dimensional translation stage in front of the sampling ESI interface. A single, 7-cm, 20-microm-i.d. fused-silica capillary (total volume, 70 nL), with a tapered tip, served as a combination of sample delivery and spraying capillary. The tapered tip of the capillary was enclosed in a subatmospheric chamber attached in front of the orifice of the mass spectrometer. The sample aspiration rate (flow rate) was regulated by computer-controlled pneumatic valves, which allowed fast switching of the pressure in the subatmospheric ESI chamber. A flow-through wash device was positioned between the microtiter plate and the ESI interface. This design allowed alternate filling of the capillary with (a) sample from the wells and (b) wash solution from the wash device. Sample turnaround times of 10 s/sample, with a 120-nL sample consumption/analysis, and a duty cycle (percentage of total analysis time spent acquiring data) of 40% were achieved. The infusion system was demonstrated in the analysis of preparative HPLC fractions from a small molecule combinatorial library.     

Cross-reactive metal ion sensor array in a micro titer plate format

A cross-reactive array in a micro titer plate (MTP) format is described that is based on a versatile and highly flexible scheme. It makes use of rather unspecific metal ions probes having almost identical fluorescence spectra, thus enabling (a) interrogation at identical analytical wavelengths, and (b) imaging of the probes contained in the wells of the MTP using a CCD camera and an array of blue-light-emitting diodes as a light source. The unselective response of the indicators in the presence of mixtures of five divalent cations generates a characteristic pattern that was analyzed by chemometric tools. The fluorescence intensity of the indicators was transferred into a time-dependent parameter applying a scheme called dual lifetime referencing. In this method, the fluorescence decay profile of the indicator is referenced against the phosphorescence of an inert reference dye added to the system. The intrinsically referenced measurements also were performed using blue LEDs as light sources and a CCD camera without intensifiers as the detector. The best performance was observed if each well was excited by a single LED. The assembly allows the detection of dye concentrations in the nanomoles-per-liter range without amplification and the acquisition of 96 wells simultaneously. The pictures obtained form the basis for evaluation by pattern recognition algorithms. Support vector machines are capable of predicting the presence of significant concentrations of metal ions with high accuracy.     

Development of a new passive integral dosemeter for gamma ray monitoring using an imaging plate

A new passive integral dosemeter for gamma ray monitoring is being developed using an imaging plate (IP). In the application of IPs to dosimetry, a fading effect causes serious problems. The fading is considered to be caused by the recombination or holes and charges trapped shallowly in the F centres, which have several activation energies. Appropriate annealing procedures allow elimination of trapped charges with low activation energies, and quantitative estimation of the radiation dose is possible. The optimum condition for minimising the effect of fading on dose estimation was obtained by post-irradiation annealing of BAS-MS (BAS-MS2025, fabricated by Fuji Film Co. Ltd) IPs at 80 degrees C for 24 h. This was confirmed by calculation. Under this condition, the detection limit was calculated to be 3.15 microSv when 10% error is allowed. A 1-month integral dose was measured by the IP. The results were compared with those obtained using a commercially available fluoro-glass dosemeter.     

Microfluidic devices constructed by a marker pen on a silica gel plate for multiplex assays

In this report, we describe a simple-to-fabricate microfluidic device constructed on a silica gel plate by a waterproof marker pen. We call it pen-based assay on silica, which was successfully applied to simultaneously analyze multiple targets qualitatively and quantitatively based on colorimetric assays for protein, glucose, and pH value.     

A method for connecting solution-phase enzyme activity assays with immobilized format analysis by mass spectrometry

This paper reports an enzyme activity assay that combines the assets of both homogeneous and solid-phase formats. In this method, enzyme reactions are carried out in solution using substrates that are tagged with an immobilization reagent that allows the substrates to be selectively immobilized to self-assembled monolayers (SAMs), for direct analysis by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). As a model enzyme reaction, this work examined the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to an arginine side chain of a peptide substrate by the enzyme protein arginine methyltransferase 1 (RMT1). A cysteine-terminated peptide substrate was methylated by RMT1 in solution and then applied to a maleimide-presenting SAM to give selective immobilization of the peptide. Time-dependent analysis of methylation using MALDI-TOFMS clearly showed that both the presence and relative amount of the two reaction products-the mono- and dimethylated peptides-can be conveniently evaluated. This assay strategy is rapid, takes advantage of solution-phase assay conditions, avoids the use of labels and complicated purification steps, and is applicable to multianalyte analyses.     

Raman spectroscopic method for identification of clinically relevant microorganisms growing on solid culture medium

Routine clinical microbiological identification of pathogenic microorganisms is largely based on nutritional and biochemical tests. In the case of severely ill patients, the unavoidable time delay associated with such identification procedures can be fatal. We present a novel identification method based on confocal Raman microspectroscopy. With this approach it is possible to obtain Raman spectra directly from microbial microcolonies on the solid culture medium, which have developed after only 6 h of culturing for the most commonly encountered organisms. Due to the limited thickness of microcolonies, some of the underlying culture medium is sampled together with the bacteria. Spectra measured at different depths in a microcolony contain different amounts of the medium signal. A mathematical routine, involving vector algebra, is described for the nonsubjective correction of spectra for variable signal contributions of the medium. To illustrate the possibilities of our approach for the identification of microorganisms, Raman spectra were collected from 6-h microcolonies of five bacterial strains on solid culture medium. The classification results show that confocal Raman microspectroscopy has great potential as a powerful new tool in clinical diagnostic microbiology.     

Analysis of a biphase-based servo format for hard-disk drives

Biphase modulation in an embedded-servo format for hard-disk drives is investigated. It is shown that for biphase, at the low linear densities typical of servo information, near-maximum-likelihood performance can be attained by a simple bit detector consisting of a full-response linear equalizer and a binary slicer. Compared to the commonly used method of dibit coding, a signal-to-noise ratio gain of some 4 dB is achieved. The same equalizer may be used as the basis for near-maximum-likelihood position error signal amplitude estimation and timing recovery. Simulations of a practical servo demodulator based on a fifth-order analog filter show that at typical linear densities, this ideal performance is closely approached. The equalizer has a band-pass character and yields excellent suppression of the effects of thermal asperities and magneto-resistive head asymmetry     

Evaluation of a new quadrilateral thin plate bending element

A review of 4-node, 12 degrees-of-freedom quadrilateral elements for thin plates is presented. A new element called DKQ is discussed. The formulation is based on a generalization of the efficient and reliable triangular element DKT presented in References 1 and 2 and on the rectangular element QC presented in Reference 3. These elements are derived using the so-called discrete Krichhoff technique. A detailed numerical evaluation of the behaviour of the DKQ element for the computation of displacements and stresses for thin plate bending problems is presented and discussed. The DKQ element appears to be a simple and reliable 12 degrees-of-freedom thin plate bending element.     

Anti-plane stress state of a plate with a V-notch for a new class of elastic solids

The main purpose of this study is to investigate the efficacy and usefulness of a class of recently proposed models that could be reasonable candidates for describing the response of brittle elastic materials. The class of models that are considered allows for a non-linear relationship between the linearized elastic strain and the Cauchy stress, and this allows one to describe situations wherein the stress increases while the strain yet remains small. Thus one would be in a position to model the response of brittle elastic bodies in the neighborhood of the tips of cracks and notches. In this paper we study the behavior of such models in a plate with a V-notch subject to a state of anti-plane stress. This geometrical simplification enables us to characterize the governing equation for the problem by means of the Airy stress function, though the constitutive relation is a non-linear relation between the linearized strain and the stress. We study the problem numerically by appealing to the finite element method. We find that the numerical solutions are stable. We are able to provide some information regarding the nature of the solution near the tip of the V-notch. In particular we find stress concentration in the vicinity of the singularity.     

A numerical study of a plate with a hole for a new class of elastic bodies

It has been shown recently that the class of elastic bodies is much larger than the classical Cauchy and Green elastic bodies, if by an elastic body one means a body incapable of dissipation (converting working into heat). In this paper, we study the boundary value problem of a hole in a finite nonlinear elastic plate that belongs to a subset of this class of the generalization of elastic bodies, subject to a uniaxial state of traction at the boundary (see Fig. 1). We consider several different specific models, including one that exhibits limiting strain. As the plate is finite, we have to solve the problem numerically, and we use the finite element method to solve the problem. In marked contrast to the results for the classical linearized elastic body, we find that the strains grow far slower than the stress.     

Development of a high-throughput format for solid-phase extraction of enantiomers using an immunosorbent in 384-well plates

An antibody-based solid-phase extraction method for filtered 384-well plates was developed for a medical drug candidate having two enantiomeric forms in order to demonstrate the potential of the use of recombinant antibody fragments as specific and efficient immunosorbents. An immobilization method using a six-histidine tag of the antibody fragment and mild oxidation was applied in order to immobilize antibody fragments in an oriented and kinetically stable way that ensured high capacity of the antibody support. Phosphate buffer or plasma spiked with enantiomers were used as samples. Selective solid-phase extraction was followed by liquid chromatography-mass spectrometry analysis. Average recoveries for buffer and plasma samples ranged from 79 to 122% and 80 to 108%, respectively. Good linearity was observed in the concentration range of 30-3000 ng/mL of the enantiomer.     

ISO新文档格式WARC助网络存储海量数据

按目前的网络模式,今天的网页也许明天就消失了。ISO新发布的国际标准ISO28500：2009《信息与文档——WARC格式》,将对解决上述问题有所帮助——若网页被变更或删除后,其中发布的信息不管多重要以及数据量多大,都不会丢失。     

Two-dimensional paper network format that enables simple multistep assays for use in low-resource settings in the context of malaria antigen detection

The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.     

A new special element for stress concentration analysis of a plate with elliptical holes

Special hole elements are presented for analyzing the stress behavior of an isotropic elastic solidcontaining an elliptical hole. The special hole elements are constructed using the special fundamental solutions for an infinite domain containing a single elliptical hole, which are derived based on complex conformal mapping and Cauchy integrals. During the construction of the special elements, the interior displacement and stress fields are assumed to be the combination of fundamental solutions at a number of source points, and the frame displacement field defined over the element boundary is independently approximated with conventional shape functions. The hybrid finite element model is formulated based on a hybrid functional that provides a link between the two assumed independent fields. Because the fundamental solutions used exactly satisfy both the traction-free boundary conditions of the elliptical hole under consideration and the governing equations of the problems of interest, all integrals can be converted into integrals along the element boundary and there is no need to model the elliptical hole boundary. Thus, the mesh effort near the elliptical hole is significantly reduced. Finally, the numerical model is verified through three examples, and the numerical results obtained for the prediction of stress concentration factors caused by elliptical holes are extremely accurate.