Charge-coupled device operated in a time-delayed integration mode as an approach to high-throughput flow-based single molecule analysis

Single molecule detection (SMD) readouts are particularly attractive for assays geared toward high-throughput processing, because they can potentially reduce assay time by eliminating various processing steps. Unfortunately, most flow-based SMD experiments have generated low throughputs due primarily to the fact that they are configured in single assay formats. The use of a charge-coupled device (CCD) with flow-based SMD can image multiple single molecule assays simultaneously to realize high-throughput processing capabilities. We present, for the first time, the ability to simultaneously track and detect single molecules in multiple microfluidic channels by employing a CCD camera operated in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement. As an example of the technology, we have configured a CCD to operate in a TDI mode to detect single double-stranded DNA molecules (lambda and pBR322) labeled with an intercalating dye (TOTO-3) in a series of microfluidic channels poised on a poly(methyl methacrylate), PMMA, chip. A laser beam was launched into the side of the chip, which irradiated a series of fluidic channels (eight) with the resulting fluorescence imaged onto a CCD. Using this system, we were able to identify single DNA molecules based on the fluorescence burst intensity arising from differences in the extent of dye labeling associated with the DNA molecule length. The CCD/TDI approach allowed increasing sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s (-1) per channel and 2208 molecules s (-1) for an eight channel microfluidic system was demonstrated. Operated in its full capacity, this multichannel format was projected to yield a sample throughput of 1.7 x 10 (7) molecules s (-1), which represents a 170-fold improvement over previously reported single molecule sampling rates.     

Development of multichannel devices with an array of electrospray tips for high-throughput mass spectrometry

The basic principles of multichannel devices with an array of electrospray tips for high-throughput infusion electrospray ionization mass spectrometry (ESI-MS) have been developed. The prototype plastic devices were fabricated by casting from a solvent-resistant resin. The sample wells on the device were arranged in the format of the standard 96-microtiter well plate, with each sample well connected to an independent electrospray exit port via a microchannel with imbedded electrode. A second plastic plate with distribution microchannels was employed as a cover plate and pressure distributor. Nitrogen gas was used to pressurize individual wells for transport of sample into the electrospray exit port. The design of independent microchannels and electrospray exit ports allowed very high throughput and duty cycle, as well as elimination of any potential sample carryover. The device was placed on a computer-controlled translation stage for precise positioning of the electrospray exit ports in front of the mass spectrometer sampling orifice. High-throughput ESI-MS was demonstrated by analyzing 96 peptide samples in 480 s, corresponding to a potential throughput of 720 samples/h. As a model application, the device was used for the MS determination of inhibition constants of several inhibitors of HIV-1 protease.     

Microfluidic devices for the high-throughput chemical analysis of cells

A microfluidic device is reported that integrated cell handling, rapid cell lysis, and electrophoretic separation and detection of fluorescent cytosolic dyes. The device function was demonstrated using Jurkat cells that were loaded with the fluorogenic dyes - carboxyfluorescein diacetate, Oregon green carboxylic acid diacetate, or Calcein AM. The loaded cells were hydrodynamically transported from the cell-containing reservoir to a region on the microfluidic device where they were focused and then rapidly lysed using an electric field. Complete lysis was accomplished in <33 ms. The hydrolyzed, fluorescent dyes in the cell lysate were automatically injected into a separation channel on the device and detected 3 mm downstream of the injection point. The total separation time was approximately 2.2 s with absolute migration time reproducibilities of <1% and efficiencies ranging from 2300 to 4000 theoretical plates. Results from 139 cells are reported. A small fraction of these cells, approximately 9%, were found to enzymatically hydrolyze the loaded dyes in a manner significantly different from the majority of the cells. Cell analysis rates of 7-12 cells/min were demonstrated and are >100 times faster than those reported using standard bench-scale capillary electrophoresis.     

Parallel electrophoretic analysis of segmented samples on chip for high-throughput determination of enzyme activities

Capillary electrophoresis (CE) on microfabricated structures has achieved impressive sample throughput by combining fast separation speed and parallel operations. One obstacle to further increasing throughput has been lack of methods for loading and injecting individual samples at a rate that matches analysis speed. To address this issue, we have developed a microfluidic device in which samples stored as nanoliter volume plugs segmented by a fluorocarbon oil are introduced sequentially to an array of three electrophoresis channels. A microfluidic interface consisting of patterned surface chemistry and geometric restriction was used to extract samples from each segmented flow channel and transfer to the respective electrophoresis channel for separation. Fluorescence detection was achieved by imaging the chip using a fluorescence microscope equipped with a charge-coupled device. Characterization of the system shows that injection volume is controlled by sample plug volume, flow rate during introduction, and voltage applied to the electrophoresis channel. The system was tested for a GTPase assay. Peak area ratios of enzyme product and internal standard had 6% relative standard deviations. Cross-contamination between peaks was 7%. Throughput of 120 samples in 10 min was achieved. Further development of the system may allow application to high-throughput applications such as drug screening.     

Multilayer polymer microchip capillary array electrophoresis devices with integrated on-chip labeling for high-throughput protein analysis

It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis, drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to eight samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to eight samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/mL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid, and high-throughput analysis.     

Sensing array for coherence analysis of modulated aquatic chemical plumes

Here we show that an array of sensors can provide information about the spatial and temporal distribution of chemicals in liquid turbulent plumes. Planar laser induced fluorescence (PLIF) and amperometric sensor arrays were used to record signals from modulated chemical plumes released into a recirculating flume. Coherence analysis was applied to extract the frequency components contained in the sensor response. Effects due to release distance, modulation frequency, and array orientation were investigated. This study has demonstrated that frequency encoded information can be extracted from a turbulent chemical plume using an array of amperometric sensors with optimized three-dimensional geometry and tuning.     

Metric tensor approach to surveillance analysis of phased array radar

The author shows that the metric tensor gab is sufficient to accurately derive some of the most important system surveillance characteristics of static and rotating phased array radars. The author presents a novel and generalised method using metric spaces on Riemann manifolds as a viable approach for investigating phased array radar system design characteristics that is mathematically tractable and avoids complex iterative and non-analytic procedures.     

A semi-quantitative approach to risk analysis, as an alternative to QRAs

In quantitative risk analysis (QRA) risk is quantified using probabilities and expected values, for example expressed by PLL values, FAR values, IR values and F–N curves. The calculations are tedious and include a strong element of arbitrariness. The value added by the quantification can certainly be questioned. In this paper, we argue that such analyses often are better replaced by semi-quantitative analyses, highlighting assessments of hazards and barriers, risk influencing factors (RIFs) and safety improvement measures. The assessments will be based on supporting information produced by risk analysts, including hard data and analyses of failure causes and mechanisms, barrier performance, scenario development, etc. The approach acknowledges that risk cannot be adequately described and evaluated simply by reference to summarising probabilities and expected values. There is a need for seeing beyond the standard probabilistic risk results of a QRA. Key aspects to include are related to uncertainties in phenomena and processes, and manageability factors. Such aspects are often ignored in standard QRAs.     

Experimental analysis of charge redistribution due to chemical bonding by high-resolution transmission electron microscopy

The electronic charge density distribution or the electrostatic atomic potential of a solid or molecule contains information not only on the atomic structure, but also on the electronic properties, such as the nature of the chemical bonds or the degree of ionization of atoms. However, the redistribution of charge due to chemical bonding is small compared with the total charge density, and therefore difficult to measure. Here, we demonstrate an experimental analysis of charge redistribution due to chemical bonding by means of high-resolution transmission electron microscopy (HRTEM). We analyse charge transfer on the single-atom level for nitrogen-substitution point defects in graphene, and confirm the ionicity of single-layer hexagonal boron nitride. Our combination of HRTEM experiments and first-principles electronic structure calculations opens a new way to investigate electronic configurations of point defects, other non-periodic arrangements or nanoscale objects that cannot be studied by an electron or X-ray diffraction analysis.     

Remarks on the use of multilayer perceptrons for the analysis of chemical sensor array data

Multilayer perceptrons (MLPs) are a standard tool for establishing relationships between data in many real world problems, in the absence of a parametric model. In the last decade, they have often been used for analyzing data produced by arrays of chemical sensors [electronic noses (e-noses)]. Still, the central issue of controlling the complexity of an MLP for optimal generalization is frequently overlooked by chemical sensors practitioners causing incorrect or suboptimal results (over or underfitting). In this paper, we will: 1) present different ways of controlling the complexity of an MLP (model order selection, early stopping, and regularization); 2) shortly review the literature on complexity control, inside and outside the e-nose community; and 3) give examples of effective complexity control for two e-noses datasets of different size and learning difficulty. It will be shown that, if early stopping or regularization are adopted, overfitting is avoided whatever the number of hidden units (and, hence, network weights). Another issue tackled in this paper is the influence on the generalization error of the number of principal components over which data are projected (before being fed into the MLP). Simulations show that (test set) performance depends strongly on the number of principal components and that even components with less than 1% of the global variance enhance classification.     

An internal state variable approach to constitutive theories for granular materials with snow as an example

In this paper, a microphysical constitutive theory is developed for a class of rate dependent granular materials under finite deformation. The theory is based on non-equilibrium thermodynamics with internal state variables. The state variables may be thought of as representing the current pattern of microstructural arrangemenp and hence characterize the plastic state of the material. A significant feature of this theory is that the state variables are identified at the granular level, as opposed to the crystalline level. This allows one to develop a microdynamical theory in terms of experimentally observable quantities and is a unique feature of granular materials.The theory is used to describe the mechanical properties of snow under high rate multiaxial deformation. Snow is a highly nonlinear, rate dependent material which exhibits significant microstructural alternations under finite strain. These alternations are tracked mathematically by temporal evolution equations governing the internal state variables. The change in the state variables is directly related to the plastic strain of the material.     

How to make an autonomous robot as a partner with humans: design approach versus emergent approach

In this paper, we discuss what factors are important to realize an autonomous robot as a partner with humans. We believe that it is important to interact with people without boring them, using verbal and non-verbal communication channels. We have already developed autonomous robots such as AIBO and QRIO, whose behaviours are manually programmed and designed. We realized, however, that this design approach has limitations; therefore we propose a new approach, intelligence dynamics, where interacting in a real-world environment using embodiment is considered very important. There are pioneering works related to this approach from brain science, cognitive science, robotics and artificial intelligence. We assert that it is important to study the emergence of entire sets of autonomous behaviours and present our approach towards this goal.     

Phospholipid complex as an approach for bioavailability enhancement of echinacoside

Context: Echinacoside (ECH) has been shown to possess a multitude of pharmacological activities, however, oral administered ECH failed to fulfill its therapeutic potential due to poor absorption and low bioavailability. Thus, there is a pressing need to develop a new oral dosage form to enhance its intestinal absorption and improve bioavailability.

Objective: The aim of this study was to formulate ECH into phospholipid complex (phytosome, PHY) to enhance intestinal absorption and oral bioavailability of ECH in vivo.

Methods: The PHY was prepared by a solvent evaporation method and was characterized by differential scanning calorimetry (DSC) and infrared spectroscopy (IR), and then the physicochemical properties, intestinal absorption and bioavailability of the PHY were investigated.

Results: Compared with the physical mixture (MIX) or ECH alone, the n-octanol/water partition coefficient (P) determination results showed that the lipophilicity of ECH was significantly enhanced by formation of PHY. Accordingly, the intestinal absorption rate (K_a) was improved to 2.82-fold and the effective permeability coefficient (P_eff) increased to 3.39-fold. The concentrations of ECH in rat plasma at different times after oral administration of PHY were determined by HPLC. Pharmacokinetic parameters of the PHY in rats were T_max?=?1.500?h, C_max?=?3.170?mg/mL, AUC_0–∞?=?9.375?mg/L?h and AUC_0–24?=?7.712?mg/L?h, respectively.

Conclusions: Compared with ECH alone or the MIX group, the relative bioavailability of ECH was increased significantly after formulation into PHY (p?相似文献   

Optical retrodirective tracking system approach using an array of phase conjugators for communication and power transmission

A new concept for a retrodirective tracking system applicable for communication and power transmission is proposed. In the proposed concept, the power transmitter utilizes a receiver's pilot signal to obtain information about its direction by conjugating the signal's phase inside a nonlinear medium. Power is therefore transmitted back to the receiver by the phase-conjugated signal beam. The power can be concentrated by an array of phase conjugators, which provides a large aperture so that the intensity can be increased on the receiver's photovoltaic panels compared to a single element. Controlling the phase and the direction of the readout beams in the four-wave-mixing process provides control over the interference pattern, its position, and its size. A numerical analysis is given for the phase and spot size control, and measurements with two Co-doped Sr(x)Ba(1-x)Nb(2)O(6) (Co:SBN) crystals confirm the occurrence of interference that is achieved for the case of two beams.     

Column precipitation chromatography: an approach to quantitative analysis of eigencolloids

A new column precipitation chromatography (CPC) technique, capable of quantitatively measuring technetium eigencolloids in aqueous solutions, is presented. The CPC technique is based on the destabilization and precipitation of eigencolloids by polycations in a confined matrix. Tc(IV) colloids can be quantitatively determined from their precipitation onto the CPC column (separation step) and their subsequent elution upon oxidation to pertechnetate by peroxide (elution step). A clean-bed particle removal model was used to explain the experimental results.     

Enzymatic tissue digestion as an alternative sample preparation approach for quantitative analysis using liquid chromatography-tandem mass spectrometry

Compound extraction from biological tissue often presents a challenge for the bioanalytical chemist. Labor-intensive homogenization or sonication of whole or powdered tissue is performed before compounds can be extracted and analyzed. Enzymatic digestion is commonly used for tissue dissociation and cell harvesting and offers the advantages of unattended sample preparation, potential automation, and low cost. The feasibility of enzymatic digestion as an alternate tissue preparation technique was evaluated for bioanalysis of drugs in conjunction with LC/MS/MS. Two different enzymes (collagenase and proteinase K) that are known to degrade connective tissues to allow tissue dissolution were chosen for evaluation, employing well-known antidepressants desipramine and fluoxetine as test compounds in dog and rat brain tissue. Comparison between enzymatic digestion and conventional homogenization tissue preparation was performed, including investigation of matrix ionization suppression of both methods using a postcolumn infusion system. Results showed that enzymatic digestion has extraction efficiency comparable to homogenization. Matrix ionization suppression was not observed for either the test compounds evaluated or the sample extraction method. Test compound levels of incurred tissue samples prepared by enzymatic digestion were in good agreement with the values obtained by the conventional homogenization tissue preparation, indicating that enzymatic digestion is an appropriate tissue sample preparation method.     

Hair analysis as an indicator of exposure to uranium

Medical examinations performed on four monks of a monastery in the northern Greece revealed heavy metal contamination. Hair analysis, performed by a toxicological laboratory abroad, indicated, among other, the presence of uranium. The uranium concentrations determined in a laboratory of "Elemental Hair Analysis' indicated a uranium level that was about five times the maximum value of the reference range, which has been adopted by the measuring laboratory. After these diagnostic findings, on request of 10 monks, uranium determination in hair and urine samples was performed by means of alpha spectrometry in GAEC's laboratory. The measured uranium concentrations in hair varied from 0.15 to 2.10 mBq g(-1), which correspond to 12.1 and 170 ng g(-1), respectively. The uranium concentrations in urine were between 41 and 174 ng d(-1). For comparison purposes, urine and non-dyed hair samples from the staff of the laboratory were analysed. Because one of the major sources of uranium intake is through drinking water, water samples were also analysed. The mean value of the uranium concentration in the two drinking water samples collected from the residence area was found to be 2.35 μg l(-1).     

Interdigitated array electrode as an alternative to the rotated ring-disk electrode for determination of the reaction products of dioxygen reduction

Electrochemical reduction of dioxygen in aqueous media can proceed to water, hydrogen peroxide, or a mixture of the two. The production of hydrogen peroxide, classically established with the rotated ring-disk electrode, can also be quantitatively assessed at interdigitated array (IDA) electrodes, where dioxygen is reduced at the set of microband generator electrodes and any H(2)O(2) produced is detected by its oxidation (back to O(2)) at the interdigitated set of microband collector electrodes. The sensitivity of the IDA for H(2)O(2) detection is higher owing to its more complete collection, and to the ensuing regeneration of O(2), which leads to an amplification of the generator currents. The production of H(2)O(2) is thus reflected both in the ratio of collector and generator electrode currents [the collection efficiency, coll(τ)] and in the ratio of the generator current with the collector potential on to that with it off [amplification factor, ampl(τ)]. The necessary theory for interpretation of the fraction ε of H(2)O(2) produced per dioxygen reduced is presented, based on conformal mapping techniques. Explicit equations are derived for ε at long times that are independent of the IDA dimensions and that can be used with any two-product electrochemical reaction analogous to the dioxygen reduction. Experimental data are presented for dioxygen reduction in acidic and basic media to illustrate application of the theory.     

Angular acceptance analysis of an infrared focal plane array with a built-in stationary Fourier transform spectrometer

Stationary Fourier transform spectrometry is an interesting concept for building reliable field or embedded spectroradiometers, especially for the mid- and far- IR. Here, a very compact configuration of a cryogenic stationary Fourier transform IR (FTIR) spectrometer is investigated, where the interferometer is directly integrated in the focal plane array (FPA). We present a theoretical analysis to explain and describe the fringe formation inside the FTIR-FPA structure when illuminated by an extended source positioned at a finite distance from the detection plane. The results are then exploited to propose a simple front lens design compatible with a handheld package.