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Results for “Microchip Analytical Procedures”

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At least 109 records · Page 6Linked to original sources

PubMed2005

On-chip native gel electrophoresis-based immunoassays for tetanus antibody and toxin.

By integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device, we have developed a microanalytical platform for performing electrophoresis-based immunoassays. The microfluidic immunoassays are performed by gel electrophoretic separation and quantitation of bound and unbound antibody or antigen. To retain biological activity of proteins and maintain intact immune complexes, nondenaturing polyacrylamide gel electrophoresis conditions were investigated. Both direct (noncompetitive) and competitive immunoassay formats are demonstrated in microchips. A direct immunoassay was developed for detection of tetanus antibodies in buffer as well as diluted serum samples. After an off-chip incubation step, the immunoassay was completed in less than 3 min and the sigmoidal dose-response curve spanned an antibody concentration range from 0.17 to 260 nM. The minimum detectable antibody concentration was 0.68 nM. A competitive immunoassay was also developed for tetanus toxin C-fragment by allowing unlabeled and fluorescently labeled tetanus toxin C-fragment compete to bind to a limited fixed concentration of tetanus antibody. The immunoassay technique described in this work shows promise as a component of an integrated microfluidic device amenable to automation and relevant to development of clinical diagnostic devices.

Antibodies↗

PubMed2005

Surface modification method of microchannels for gas-liquid two-phase flow in microchips.

A capillarity restricted modification method for microchannel surfaces was developed for gas--liquid microchemical operations in microchips. In this method, a microstructure combining shallow and deep microchannels and the principle of capillarity were utilized for chemical modification of a restricted area of a microchannel. A hydrophobic--hydrophilic patterning in microchannels was prepared as an example for guiding gas and liquid flows along the respective microchannels. Validity of the patterning was confirmed by measuring aqueous flow leak pressure from the hydrophilic microchannel to the hydrophobic one. The leak pressure of 7.7-1.1 kPa agreed well with that predicted theoretically from the Young-Laplace equation for the microchannel depth of 8.6-39 microm. In an experiment to demonstrate usefulness and effectiveness of the method, an air bubble was first introduced into the hydrophilic microchannel and purged from the hydrophobic-hydrophilic patterned microchannels. Next, the patterning structure was applied to remove dissolved oxygen by contacting the aqueous flow with a nitrogen flow. The concentration of dissolved oxygen decreased with contact time, and its time course agreed well with numerical simulation. These demonstrations showed that the proposed patterning method can be used in general microfluidic gas-liquid operations.

Biomechanical Phenomena↗

PubMed2004

Electrochemical detection method for nonelectroactive and electroactive analytes in microchip electrophoresis.

In this work, we establish an indirect amperometric detection method via mounting a single carbon fiber disk working electrode in the end part of a microchannel. This in-channel configuration for microchip capillary electrophoresis brings about that the potential of the working electrode in the case of electrochemical reduction reaction is coupled by the separation electric field, while the potential of the working electrode in the case of electrochemical oxidation reaction is not coupled by the separation electric field. Such a special performance provides a convenient and sensitive approach for indirectly detecting nonelectroactive analytes that relies on amperometric response of dissolved oxygen in solution and directly detecting electroactive analytes based on their own amperometric response on the carbon fiber electrode. This method has shown its essential importance in the analysis of inorganic cations, biomolecules, and electroosmotic flow rates. Based on preliminary results, a detection limit of 1.0 microM for K(+) and Na(+) have been achieved.

Carbon↗

PubMed2004

A three-dimensional flow control concept for single-cell experiments on a microchip. 1. Cell selection, cell retention, cell culture, cell balancing, and cell scanning.

An ideal microchip for single-cell experiments should be able to allow us to culture cells, to select any desired single cell from a group, to retain the cell for convenient cellular signal detection, and to deliver any buffer or reagent directly to the cell at any time during continual detection and observation. Most importantly, any negative impact on the live cell should be minimized. To accomplish all these functions, we developed a three-dimensional liquid flow control concept and employed special liquid flow fields to manipulate and retain a single yeast cell freely in the chip. A zero-speed point was controlled to retain the cell for three-dimensional cell balancing and cell scanning. A dispersive flow delivered reagents at a high speed to very near the cell and provided them to the cell at a low speed. No force stronger than its gravitational force was exerted on the cell, which could be balanced on different positions on an arc-sloping wall, thus minimizing any negative impact on the cell due to strong liquid flows. Specifically, we demonstrate on-chip single-cell culture, cell wall removal, and reagent delivery. Subsequently, single-cell fluorescence detection was performed, and noise filtering and background correction were applied for data processing.

Cell Culture Techniques↗

PubMed2004

Deformation-induced release of ATP from erythrocytes in a poly(dimethylsiloxane)-based microchip with channels that mimic resistance vessels.

The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 x 38 micron (2.22 +/- 0.50 microM ATP). Microscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 +/- 0.15 microM) vs a microchip (2.59 +/- 0.32 microM) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation.

Adenosine Triphosphate↗

PubMed2004

Microchip capillary electrophoresis with electrochemical detection of thiol-containing degradation products of V-type nerve agents.

A microchip protocol for the capillary electrophoresis separation and electrochemical detection of thiol-containing degradation products of V-type nerve agents is described. The microchip assay relies on the derivatization reaction of 2-(dimethylamino)ethanethiol (DMAET), 2-(diethylamino)ethanethiol (DEAET), and 2-mercaptoethanol (ME) with o-phthaldialdehyde in the presence of the amino acid valine along with amperometric monitoring of the isoindole derivatives. Both off-chip and on-chip derivatization reactions have led to highly sensitive and rapid detection of the thiol degradation products. Various parameters influencing the derivatization, separation, and detection processes were examined and optimized. These include the amino acid co-reagent, reagent-mixing ratio, reaction time, injection time, separation voltage, and detection potential. The chip microsystem offers a rapid (<4 min) simultaneous detection of micromolar concentrations of DMAET, DEAET, and ME. Linear calibration plots were observed for the V-type nerve agent thiol degradation products, along with good stability and reproducibility (RSD < 8.0%). Detection limits of 5 and 8 microM were obtained for the off-chip reaction of DMAET and DEAET, respectively, following a 2-s injection. The suitability for assays of environmental matrixes was demonstrated for the determination of DMAET and DEAET in untreated tap and river water samples. The favorable analytical performance makes the new microfluidic device attractive for addressing the needs of various security scenarios.

Biodegradation, Environmental↗

PubMed2004

Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing.

A new method for on-chip sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins is reported. Miniaturization of SDS-PAGE has attracted significant attention because it offers rapid analysis times, excellent resolution, high throughput, and the potential for integration and automation, as compared to conventional counterparts. The presented on-chip SDS-PAGE technique employed photolithographically patterned, cross-linked gels fabricated in situ in <20 min. The effects of sieving gel composition on the migration properties of fluorescently labeled protein standards (ranging in molecular weight from 14.2 to 66 kDa) were quantified, as was the ability of the gels to function as a sieving matrix for biologically relevant species. Ferguson analysis was employed to calculate retardation coefficients and free solution mobilities. In conjunction with fluorescence imaging, the on-chip SDS-PAGE separation mechanism was evaluated in terms of separation performance indexes, as well as limiting behaviors (i.e., free solution mobility, exclusion characteristics). The photolithographically fabricated gels employed for on-chip SDS-PAGE allowed rapid (<30 s) separations of proteins in short separation lengths (4 mm) with efficiencies as high as 4.41 x 10(5) plates/m. The on-chip SDS-PAGE separations were approximately 100 times faster than conventional slab gel SDS-PAGE (60 min) and occurred in a fraction of the separation length required by slab gels. The migration behavior of protein standards correlated well with molecular weight and allowed molecular weight determination for interleukin-2, fibroblast growth factor, insulin-like growth factor, and tetanus toxin C-fragment.

Cross-Linking Reagents↗

PubMed2004

Electrophoretic concentration of proteins at laser-patterned nanoporous membranes in microchips.

Laser-patterning of nanoporous membranes at the junction of a cross channel in a microchip is used to integrate protein concentration with an electrokinetic injection scheme. Upon application of voltage, linear electrophoretic concentration of charged proteins is achieved at the membrane surface because buffer ions can easily pass through the membrane while proteins larger than the molecular weight cutoff of the membrane (>5700) are retained. Simple buffer systems can be used, and the concentration results constitute outward evidence that the uniformity of buffer ion concentration is maintained throughout the process. Local and spatially averaged concentration are increased by 4 and 2 orders of magnitude, respectively, upon injection with moderate voltages (70-150 V) and concentration times (100 s). The degree of concentration is limited only by the solubility limit of the proteins. The porous polymer membrane can be used repeatedly as long as care is taken to avoid protein precipitation.

Electrophoresis↗

PubMed2005

Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow.

We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using alpha-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.

Antibodies, Monoclonal↗

PubMed2005

Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule.

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.

Microchip Analytical Procedures↗

PubMed2005

Microchip immobilized enzyme reactors for hydrolysis of methyl cellulose.

Microchip immobilized enzyme reactors (microIMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two microIMERs containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the microIMER could be confirmed from the average molar masses and molar mass distributions measured by size exclusion chromatography (SEC) with online multiangle light scattering and refractive index detection. Methylated cellooligosaccharides with degrees of polymerization (DP) between 1 and 6 formed during hydrolysis were analyzed by direct infusion electrospray ionization ion-trap mass spectrometry (ESI-ITMS). Mass spectra of microIMER- and batch-hydrolyzed samples were compared and no significant differences were found, indicating that microIMER hydrolysis was as efficient as conventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved by connecting the microIMER to high-performance liquid chromatography and ESI-ITMS. This online separation reduced the relative intensities of interfering signals and increased the signal-to-noise ratios in MS. The microIMER hydrolysates were also subjected to SEC interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With this technique, oligomers with DP 3-30 could be detected. The hydrolysis by the microIMER was performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for at least 24 h. The microIMER also allowed hydrolysis after 10 days of continuous use. The method presented in this work offers new approaches for the analysis of derivatized cellulose and provides the possibility of convenient online, fast, and more versatile analysis compared with the traditional batch method.

Bacillus↗

PubMed2005

Aptamer-derived nucleic acid oligos: applications to develop nucleic acid chips to analyze proteins and small ligands.

The specificity and affinity of aptamers for their cognate ligands are comparable to those of antibodies for antigens. To use aptamers effectively in high-throughput assays in a microarray format, to analyze various analytes, we developed a strategy in which the aptamer was split into two nonfunctional units and allowed to reassemble into the functional aptamer by the cognate ligand. We have named this method "analyte-dependent oligonucleotide modulation assay" (ADONMA). As proof-of-principle, we used oligonucleotides derived from the aptamer RNA against HIV-1 Tat and demonstrated, with both titer plates and plastic slide chips, that specifically in the presence of Tat or its peptide, the two oligos reconstituted the core binding regions of Tat. Thus, these results suggest that ADNOMA has the potential for use in nucleic acid microarrays for detecting various ligands.

Aptamers, Nucleotide↗

PubMed2005

Temperature alternation by an on-chip microheater to reveal enzymatic activity of beta-galactosidase at high temperatures.

A method to measure enzymatic activity at high temperatures by rapid temperature alternation of a microreactor with a microheater is proposed. On-chip microreactor and microheater were integrated on a glass plate by MEMS technology; this microheater can control the temperature of the microreactor with a response speed of 34.2 and 31.5 K/s for temperature rise and fall, respectively, with an accuracy of 3 degrees C. The enzyme, beta-galactosidase, was revealed to survive short exposure (4-s pulses) to temperatures above that which would "normally" denature them. Its activity at 60 degrees C was revealed to be approximately 4 times greater than that at room temperature. This method not only gives new kinetic information in biochemistry but also enables application in highly sensitive biosensors.

Hot Temperature↗

PubMed2005

Recognition of dengue virus protein using epitope-mediated molecularly imprinted film.

Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required.

Animals↗

PubMed2005

Modification of micronozzle surfaces using fluorinated polymeric nanofilms for enhanced dispensing of polar and nonpolar fluids.

In this work, we report on the surface modification of a micronozzle surface to enhance fluid dispensing in the nanoliter range. Unmodified dispensing chips usually suffer from lateral wetting of the nozzle surfaces by low surface tension liquids resulting in poor control of the volume of the dispensed fluid. Covalent attachment of a fluorinated acrylate polymer to the outer surface of the micronozzle using a novel UV irradiation process helps to overcome capillary and adhesive forces and results in an enhancement of the control on fluid dispensing in the nanoliter range. The modified nozzles have been tested with a variety of fluids having a wide range of surface tensions. The surface modification allows precise control of the dispensing of nanoliter droplets with a high degree of reproducibility.

Fluorine Compounds↗

PubMed2005

Intrinsic viscosity of polymers and biopolymers measured by microchip.

Intrinsic viscosity provides insight to molecular structure and interactions in solution. A new microchip method is described for fast and accurate measurements of viscosity and intrinsic viscosity of polymer and biopolymer solutions. Polymer samples are diluted with solvent in the microfluidic chip by imposing pressure gradients across the channel network. The concentration and flow dilutions of the polymer sample are calculated from the fluorescent signals recorded over a range of dilutions. The viscosities at various polymer dilutions are evaluated using mass and momentum balances in the pressure-driven microchannel flow. The technique is particularly important to many chemical, biological, and medical applications where sample is available in very small quantities. The intrinsic viscosity experiments were performed for three classes of polymer solutions: (a) poly(ethylene glycol), polymers with linear hydrocarbon chains; (b) bovine serum albumin, biopolymer chains with hydrophobic and hydrophilic amino acids, and (c) DNA fragments, biological macromolecules with double-stranded polymeric chains. The measured values of intrinsic viscosity agree remarkably well with the available data obtained using different methods. The data exhibit power law behavior for molecular weight as described by the Mark-Houwink-Sakurada equation. Experiments were performed to understand the effect of solvent quality and salt concentration on molecular conformations and the intrinsic viscosity of the polymers. This method offers a new way to study the conformational changes in proteins and DNA solutions in various buffer conditions such as pH, ionic strength, and surfactants. The effects of shear rate in the microchannel and mixing time on the accuracy and limitation of the measurement method are discussed.

Animals↗

PubMed2005

Functional membrane-implanted lab-on-a-chip for analysis of percent HDL cholesterol.

A functional lab-on-a-chip has been developed for simultaneous quantitative analyses of high-density lipoprotein (HDL) cholesterol (HDL-C) and total cholesterol (total-C) in a submicroliter plasma sample. The analytical device was fabricated by placing commercial membranes, traditionally used for rapid diagnostics, within microfluidic channels engraved on the surface of a plastic chip. The concentration of HDL-C was measured using enzymatic reactions to produce a colorimetric signal after separation of the single plasma lipoprotein from a mixture. Two small pieces of different membrane pads were used to provide each group of reagents, for HDL separation and enzyme reactions, deposited within their tiny pores in a dry state. To maintain a connection toward the capillary action of the medium, the pads were arranged in a sequence within the fluidic channel that controlled the inlet and outlet of the flow. Upon the addition of a sample, the fluid was delivered through the pads of the chip and a color signal was subsequently generated in proportion to the concentration of HDL-C. The level of total-C was concurrently determined by following identical processes, except absent HDL separation. The two signals were simultaneously determined by employing optical detectors based on transmittance of a light. Such total analyses were completed within 2 min, and the sample sizes were able to be reduced to 0.4 microL for HDL-C and 0.1 microL for total-C, enough to cover the clinically required dynamic ranges.

Cholesterol↗

PubMed2006

Rapid melting curve analysis on monolayered beads for high-throughput genotyping of single-nucleotide polymorphisms.

This report describes a rapid solid-phase melting curve analysis method for single-nucleotide polymorphism (SNP) genotyping. The melting curve analysis is based on dynamic allele-specific hybridization (DASH). The DNA duplexes are conjugated on beads that are immobilized on the surface of a microheater chip with integrated heaters and temperature sensors. SNP on PCR products were scored, illustrating the sensitivity and robustness of the system. The method is based on random bead immobilization by microcontact printing. Single-bead detection and multiplexing were performed with a heating rate more than 20 times faster than conventional DASH. Analyses that took more than 15 min could be performed in less that 1 min, enabling ultrarapid SNP analysis. In addition, an array version of the chip was implemented enabling the preparation of an array of bead arrays for high-throughput and rapid SNP genotyping.

Alleles↗