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At least 1,729 records · Page 96Linked to original sources
PubMed2005

Liquid-phase binding assay of alpha-fetoprotein using DNA-coupled antibody and capillary chip electrophoresis.

An immunoassay using DNA-coupled antibody for bound/free separation in a liquid-phase binding assay format is described. Anti-alpha-fetoprotein monoclonal antibody was conjugated with DNA, mixed with alpha-fetoprotein (AFP), and incubated, and then 1 muL of the mixture was applied to capillary electrophoresis on a microchip. The DNA molecule of the antibody-DNA conjugate and the DNA-conjugated immune complex peak were detectable fluorophotometrically using intercalator dye within 90 s, whereas the Alexa-labeled antibody was detected as a broad and slower migrating peak. The electrophoretic mobility of the immune complex could be optimized for resolution and sharpness by changing the length of the DNA coupled to the antibody. The detection limit of AFP was approximately 300 pM in a sample. This immunoassay method utilizing a liquid-phase binding assay format is simple and convenient for antigen measurements on microchips.

Antibodies↗
PubMed2005

Giant magnetoresistive sensors and superparamagnetic nanoparticles: a chip-scale detection strategy for immunosorbent assays.

Thin structures of alternating magnetic and nonmagnetic layers with a total thickness of a few hundred nanometers exhibit a phenomenon known as giant magnetoresistance. The resistance of microfabricated giant magnetoresistors (GMRs) is dependent on the strength of an external magnetic field. This paper examines magnetic labeling methodologies and surface derivatization approaches based on protein-protein binding that are aimed at forming a general set of protocols to move GMR concepts into the bioanalytical arena. As such, GMRs have been used to observe and quantify the immunological interaction between surface-bound mouse IgG and alpha-mouse IgG coated on superparamagnetic particles. Results show the response of a GMR network connected together as a set of two sense GMRs and two reference GMRs in a Wheatstone bridge as a means to compensate for temperature effects. The response can be readily correlated to the amount of the magnetically labeled alpha-mouse IgG that is captured by an immobilized layer of mouse IgG, the presence of which is confirmed with X-ray photoelectron spectroscopy and atomic force microscopy. These results, along with a detailed description of the experimental testing platform, are described in terms of sensitivity, detection limits, and potential for multiplexing.

Animals↗
PubMed2005

Microfluidic electrophoresis chip coupled to microdialysis for in vivo monitoring of amino acid neurotransmitters.

Microfluidic electrophoresis devices were coupled on-line to microdialysis for in vivo monitoring of primary amine neurotransmitters in rat brain. The devices contained a sample introduction channel for dialysate, a precolumn reactor for derivatization with o-phthaldialdehyde, a flow-gated injector, and a separation channel. Detection was performed using confocal laser-induced fluorescence. In vitro testing revealed that the initial device design had detection limits for amino acids of approximately 200 nM, relative standard deviation of peak heights of 2%, and separations within 95 s with up to 30,200 theoretical plates when applying an electric field of 370 V/cm. A second device design that allowed electric fields of 1320 V/cm to be applied while preserving the reaction time allowed separations within 20 s with up to 156,000 theoretical plates. Flow splitting into the electrokinetic network from hydrodynamic flow in the sample introduction channel was made negligible for sampling flow rates from 0.3 to 1.2 microL/min by placing a 360-microm-diameter fluidic access hole that had flow resistance (0.15-7.2) x 10(8)-fold lower than that of the electrokinetic network at the junction of the sample introduction channel and the electrokinetic network. Using serial injections, the device allowed the dialysate stream to be analyzed at 130-s intervals. In vivo monitoring was demonstrated by using the microdialysis/microfluidic device to record glutamate concentrations in the striatum of an anesthetized rat during infusion of the glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid. These results prove the feasibility of using a microfabricated fluidic system coupled to sampling probes for chemical monitoring of complex media such as mammalian brain.

Amino Acids↗
PubMed2006

On-chip amperometric measurement of quantal catecholamine release using transparent indium tin oxide electrodes.

Carbon-fiber amperometry has been extensively used to monitor the time course of catecholamine release from cells as individual secretory granules discharge their contents during the process of quantal exocytosis, but microfabricated devices offer the promise of higher throughput. Here we report development of a microchip device that uses transparent indium tin oxide (ITO) electrodes to measure quantal exocytosis from cells in microfluidic channels. ITO films on a glass substrate were patterned as 20-mum-wide stripes using photolithography and wet etching and then coated with polylysine to facilitate cell adherence. Microfluidic channels (100 mum wide by 100 mum deep) were formed by molding poly(dimethylsiloxane) (PDMS) on photoresist and then reversibly sealing the PDMS slab to the ITO-glass substrate. Bovine adrenal chromaffin cells were loaded into the microfluidic channel and adhered to the ITO electrodes. Cells were stimulated to secrete by perfusing a depolarizing "high-K" solution while monitoring oxidation of catecholamines on the ITO electrode beneath the cell using amperometry. Amperometric spikes with charges ranging from 0.1 to 1.5 pC were recorded with a signal-to-noise ratio comparable to that of carbon-fiber electrodes. Further development of this approach will enable high-throughput measurement of quantal catecholamine release simultaneously with optical cell measurements such as fluorescence.

Adrenal Glands↗
PubMed2006

Monitoring and kinetics study of a single particle on a simple microfluidic chip.

A single-channel poly(dimethylsiloxane) microchip was developed for the desorption process monitoring and kinetics studies of a single particle. The microchannel consisting of a narrow section following a relatively wide part enabled particle introduction, transfer, and location. A microinfusion pump was employed to delivery eluting solution at a precise rate. Once the particle contacted with the eluting solution, the solute transferred from particle into eluting solution and would be detected by laser-induced fluorescence or a chemiluminescence detector. Desorption process of a single particle was sensitively monitored. Depending on the desorption curves obtained, kinetics studies were carried out. The sediment desorption process analyses of single resin particles and single active carbon particles were performed.

Carbon↗
PubMed2006

On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system.

This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable surface plasmon resonance (SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction with acetylcholine esterase-labeled antibody (conjugate), and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction with the trapped conjugate was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We achieved a detectable concentration range of 5 pg/mL-100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This success resulted from the use of a T-shaped microfluidic device structure, which prevents the sample solution from flowing over the gold film used for SPR detection. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device. We employed the procedure to detect serum BNP by using spiked samples in human serum and achieved satisfactory recovery for heat-treated samples to denature the esterase in the serum before the immunoreaction.

Biomarkers↗
PubMed1997

Miniaturized ion-selective chip electrode for sensor application.

The performance of miniaturized potentiometric cells, with multilayer, planar ion-selective sensors in aqueous electrolyte solutions, human serum, urine, and whole blood, is presented. The basic steps of the fabrication with silicon technology are summarized. The effect of the contact surface between the internal reference system and the ion-sensitive membrane on the analytical characteristics of potassium- and calcium-sensitive sensors is studied. Silicone rubber-, high molecular weight PVC-, carboxylated PVC and aliphatic polyurethane (Tecoflex)-based solvent polymeric membranes were dispensed into anisotropically etched wells on silicon wafers, and the resulted planar sensors were tested in terms of their ion sensitivity (slopes of the cell voltage-pK or pCa calibration curves), long-term stability, and reproducibility. For the assay of potassium in whole blood, the miniaturized potentiometric cell was built in a flow-through manifold. To achieve the required precision, the flow conditions were optimized and the sensors calibrated periodically. The results prove the feasibility of the new sensor design and satisfy the particularly difficult requirements for the analysis of biological samples.

Biosensing Techniques↗
PubMed2000

Trapping of bead-based reagents within microfluidic systems: on-chip solid-phase extraction and electrochromatography

A 330-pL chromatographic bed was fabricated on a glass substrate as part of an electroosmotically pumped microfluidic system. Two weirs within a sample channel formed a cavity in which octadecylsilane (ODS) coated silica beads (1.5-4 microns diameter) were trapped. ODS beads were mobilized into and out of the cavity using electroosmotic flow through a bead-introduction channel which accessed the cavity. This procedure allowed the beads in the cavity to be repeatedly exchanged. A 1 nM solution of a nonpolar analyte (BODIPY 493/503) in buffer was loaded onto the beads for different lengths of time using an electroosmotic flow of 1.2 nL/s. The material retained on the ODS phase was then eluted by electroosmotic flow of acetonitrile with a concentration enhancement of up to 500 times. Capillary electrochromatography was conducted using a similar device. BODIPY and fluorescein were loaded onto a 200-micron-long chromatographic bed and then separated in an isocratic CEC run with an acetonitrile/buffer mobile phase. Complete separation was achieved in less than 20 s with a 2-micron plate height.

Journal Article↗
PubMedDate not supplied

Chip-based polyketide biosynthesis and functionalization.

We demonstrate construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microfluidic platform. THNS immobilized to Ni-NTA agarose beads is prepacked into a microfluidic channel, while SBP is covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result is a tandem, two-step biochip that enables the synthesis of novel polyketide derivatives. The first microchannel, consisting of THNS, results in the conversion of malonyl-CoA to flaviolin in yields up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and malonyl-CoA (extender substrate) feed rates, high yields of the pyrone products could be achieved, which is further structurally diversified from self- and cross-coupling in the SBP microchannel. The ability to rapidly evaluate the effects of reaction conditions and synthetic multienzyme pathways on a microfludic platform provides a new paradigm for performing metabolic pathway engineering, namely, the reconstruction of pathways for use in new compound discovery.

Catalysis↗
PubMed2003

On-chip integration of enzyme and immunoassays: simultaneous measurements of insulin and glucose.

A microfluidic device coupling immunological and enzymatic assays within a single microchannel has been developed for simultaneous measurements of insulin and glucose. Such a dual-mode (enzyme/immuno) protocol involves precolumn reactions of insulin and glucose with the enzyme-labeled anti-human insulin and glucose-dehydrogenase/NAD+, respectively, followed by the electrophoretic separation of the free antibody, antibody-antigen complex, and the NADH product of the enzymatic reaction. The separation is followed by a postcolumn reaction of the alkaline-phosphatase tracer with the p-NPP substrate and a downstream amperometric detection of the p-nitrophenol and NADH products. Despite the huge concentration difference [millimolar (glucose) and nanomolar (insulin)] and the use of different assay principles, the new biochip responds independently to the corresponding target analytes, with linear dynamic ranges over their clinically relevant ranges. Complete assays, carried out within less than 4 min, lead to good precision (RSD 0.36%) for the insulin/glucose ratio. The resulting biochip allows simultaneous testing for insulin and glucose to be performed more rapidly, easily, and economically, and hence it holds great promise for improved management of diabetes.

Antibodies↗
PubMed2003

Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets.

Protein crystallization is a major bottleneck in determining tertiary protein structures from genomic sequence data. This paper describes a microfluidic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization droplet. The droplets are formed by mixing protein, precipitant, and additive stock solutions in variable ratios in a flow of water-immiscible fluids inside microchannels. Each droplet represents a discrete trial testing different conditions. The system has been validated by crystallization of several water-soluble proteins.

Crystallization↗
PubMed2005

A polydiacetylene-based fluorescent sensor chip.

Self-assembled diacetylene vesicles were spotted and immobilized on aldehyde-modified glass substrates using conventional microarray technology. Irradiation of the immobilized diacetylenes allowed generation of nonfluorescent "blue-phase" polydiacetylene (PDA) arrays. Specific interaction of the PDA vesicle arrays with carbohydrates or poly(acrylic acid) solutions afforded fluorescent profiles.

Acetylene↗