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At least 919 records · Page 51Linked to original sources
PubMed2004

Separation of long DNA molecules by quartz nanopillar chips under a direct current electric field.

We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1-38 kbp) and large DNA fragments (lambda DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of lambda DNA and T4 DNA was successfully separated by a 380-microm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 microm long) was 1000-3000 (0.7 x 10(6)-2.1 x 10(6) plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas lambda DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.

DNA↗
PubMed2003

3D protein microarrays: performing multiplex immunoassays on a single chip.

The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.

Antibodies↗
PubMed2004

Cell-free protein expression and functional assay in nanowell chip format.

The expression and characterization of large protein libraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology.

Cell-Free System↗
PubMed2004

Fully automated chip-based mass spectrometry for complex carbohydrate system analysis.

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.

Automation↗
PubMed2004

Electrokinetically controlled DNA hybridization microfluidic chip enabling rapid target analysis.

Biosensors and more specifically biochips exploit the interactions between a target analyte and an immobilized biological recognition element to produce a measurable signal. Systems based on surface nucleic acid hybridization, such as microarrays, are particularly attractive due to the high degree of selectivity in the binding interactions. One of the drawbacks of this reaction is the relatively long time required for complete hybridization to occur, which is often the result of diffusion-limited reaction kinetics. In this work, an electrokinetically controlled DNA hybridization microfluidic chip will be introduced. The electrokinetic delivery technique provides the ability to dispense controlled samples of nanoliter volumes directly to the hybridization array (thereby increasing the reaction rate) and rapidly remove nonspecific adsorption, enabling the hybridization, washing, and scanning procedures to be conducted simultaneously. The result is that all processes from sample dispensing to hybridization detection can be completed in as little as 5 min. The chip also demonstrates an efficient hybridization scheme in which the probe saturation level is reached very rapidly as the targets are transported over the immobilized probe site enabling quantitative analysis of the sample concentration. Detection levels as low as 50 pM have been recorded using an epifluorescence microscope.

Adsorption↗
PubMed2004

Stable microstructured network for protein patterning on a plastic microfluidic channel: strategy and characterization of on-chip enzyme microreactors.

Chemical modification of a poly(methyl methacrylate) (PMMA) microchannel surface has been explored to functionalize microfluidic chip systems. A craft copolymer was designed and synthesized to introduce the silane functional groups onto the plastic surface first. Furthermore, it has been found that, through a silicon-oxygen-silicon bridge that formed by tethering to these functional groups, a stable patterning network of gel matrix could be achieved. Thus, anchorage of proteins could be realized onto the hydrophobic PMMA microchannels with bioactivity preserved as far as possible. The protein homogeneous patterning in a microfluidic channel has been demonstrated by performing microchip capillary electrophoresis with laser-induced fluorescence detection and confocal fluorescence microscopy. To investigate the bioactivity of enzymes entrapped within stable silica gel-derived microchannels, the suggested scheme was employed to the construction of immobilized enzyme microreactor-on-a-chip. The proteolytic activity of immobilized trypsin has been demonstrated with the digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL/min, which affords the short residence time less than 5 s. The digestion products were characterized using MALDI-TOF MS with sequence coverage of 75 and 31% observed, respectively. This research exhibited a simple but effective strategy of plastic microchip surface modification for protein immobilization in biological and proteomic research.

Amino Acid Sequence↗
PubMed2005

Chip-based microelectrodes for detection of single-nucleotide mismatch.

Microelectrode arrays having eight 10-microm-diameter gold microelectrodes arranged on a gold-covered Si chip were designed and characterized. The chips prove useful for the detection of single-nucleotide mismatches in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy.

Base Pair Mismatch↗
PubMed2005

On-chip microfluidic transport and mixing using electrowetting and incorporation of sensing functions.

An integrated system was developed that performs microfluidic transport, mixing, and sensing on a single chip. The operation principle for the microfluidic transport was based on electrowetting. A solution to be transported was confined in a space between a row of gold working electrodes and a protruding poly(dimethylsiloxane) (PDMS) structure. When a negative potential was applied to one of the gold working electrodes, it became hydrophilic, and the solution was transported through the flow channel. The solution could be transported in any desired direction in a network of flow channels by switching on necessary electrodes one by one. Furthermore, two solutions transported through two flow channels could be mixed using a mixing electrode based on the same principle. To demonstrate the applicability of a lab-on-a-chip, an air gap ammonia electrode was integrated by taking advantage of the open structure of the flow channel. Gaseous ammonia that was produced after pH adjustment and diffused through an air gap caused a pH change in the electrolyte layer, which was measured with an iridium oxide pH indicator electrode. The 90% response time was less than 1 min for the millimolar order of ammonia. The calibration curve was linear down to 10 microM. The ammonia-sensing system was also applied to construct biosensing systems for urea and creatinine. A linear relationship was observed between the potential and the logarithm of the concentration of the analytes down to 50 microM for both urea and creatinine. The developed microfluidic system can be a basic building block for future systems.

Journal Article↗
PubMed2005

Controlled protein precipitation in combination with chip-based nanospray infusion mass spectrometry. An approach for metabolomics profiling of plasma.

Liquid chromatography-mass spectrometry (LC-MS) is a common method for profiling biological samples in metabolomics. However, LC-MS data of metabolomic studies are often affected by high noise levels, retention time shifts, and high variability in signal intensities. With a new chip-based nanoelectrospray source it becomes possible to directly infuse complex biological samples such as plasma without any chromatographic separation beforehand. In combination with highly diluted samples and long data acquisition times, the parallel analysis of hundreds of compounds is now possible. In a proof-of-concept study, 10 human plasma samples from females and males were analyzed with the intention to separate the two groups by their different metabolomes. The reproducibility was so high that statistical analysis of the data could be performed without prior normalization. Two groups of female and male samples were separated by a supervised machine learning algorithm, principal component analysis, and hierarchical clustering. Peaks contributing to the group separation were characterized by accurate mass measurement and MS-MS fragmentation and by spiking experiments. The feasibility of direct sample infusion using the new chip-based nanoelectrospray device opens a new dimension for the rapid parallel analysis of complex biological mixtures.

Adult↗
PubMed2005

Microfluidic chip for low-flow push-pull perfusion sampling in vivo with on-line analysis of amino acids.

Multilayer soft lithography was used to prepare a poly(dimethylsiloxane) microfluidic chip that allows for in vivo sampling of amino acid neurotransmitters by low-flow push-pull perfusion. The chip incorporates a pneumatically actuated peristaltic pump to deliver artificial cerebrospinal fluid to a push-pull perfusion probe, pull sample from the probe, perform on-line derivatization with o-phthaldialdehyde, and push derivatized amino acids into the flow-gated injector of a high-speed capillary electrophoresis-laser-induced fluorescence instrument. Peristalsis was achieved by sequential actuation of six, 200 microm wide by 15 microm high control valves that drove fluid through three fluidic channels of equal dimensions. Electropherograms with 100,000 theoretical plates were acquired at approximately 20-s intervals. Relative standard deviations of peak heights were 4% in vitro, and detection limits for the excitatory amino acids were approximately 60 nM. For in vivo measurements, push-pull probes were implanted in the striatum of anesthetized rats and amino acid concentrations were monitored while sampling at 50 nL/min. o-Phosphorylethanolamine, glutamate, aspartate, taurine, glutamine, serine, and glycine were all detected with stable peak heights observed for over 4 h with relative standard deviations of 10% in vivo. Basal concentrations of glutamate were 1.9 +/- 0.6 microM (n = 4) in good agreement with similar methods. Monitoring of dynamic changes of neurotransmitters resulting from 10-min applications of 70 mM K(+) through the push channel of the pump was demonstrated. The combined system allows temporal resolution for multianalyte monitoring of approximately 45 s with spatial resolution 65-fold better than conventional microdialysis probe with 4-mm length. The system demonstrates the feasibility of sampling from a complex microenvironment with transfer to a microfluidic device for on-line analysis.

Amino Acids↗
PubMed2006

Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics.

A polymeric microfluidic device for solid-phase extraction (SPE)-based isolation of nucleic acids is demonstrated. The plastic chip can function as a disposable sample preparation system for different biological and diagnostic applications. The chip was fabricated in a cyclic polyolefin by hot-embossing with a master mold. The solid phase consisted of a porous monolithic polymer column impregnated with silica particles. The extraction was achieved due to the binding of nucleic acids to the silica particles in the monolith. The solid phase was formed within the channels of the device by in situ photoinitiated polymerization of a mixture of methacrylate and dimethacrylate monomers, UV-sensitive free-radical initiator, and porogenic solvents. The channel surfaces were pretreated via photografting to covalently attach the monolith to the channel walls. The solid phase prepared by this method allowed for successful extraction and elution of nucleic acids in the polymeric microchip.

Combinatorial Chemistry Techniques↗
PubMed2006

Construction of a biomimetic surface on microfluidic chips for biofouling resistance.

A biomimetic surface has been formed on the poly(methyl methacrylate) (PMMA) microfluidic chips for biofouling resistance on the basis of a simple modification. Accordingly, an amphiphilic phospholipid copolymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate (PMB) was developed to introduce the phosphorylcholine functional groups onto the PMMA surface via the anchoring of hydrophobic n-butyl methacrylate units. The 2-methacryloyloxyethyl phosphorylcholine segments could form hydrophilic domains, considered to be located on the surface, to provide a biocompatible surface. X-ray photoelectron spectroscopy and Fourier transform infrared spectra confirmed the success of surface functionalization. The PMB-modified microchips containing phosphorylcholine moieties exhibited more stable electroosmotic mobility compared with the untreated one. In addition to being characterized for minimized nonspecific adhesion of serum proteins and plasma platelets, the PMB-functionalized microchannels have been exemplified by electrophoresis of proteins. This one-step procedure offers an effective approach for a biomimetic surface design on microfluidic chips, which is promising in high-throughput and complex biological analysis.

Biomimetics↗
PubMed2006

Multiplex measurement of seven tumor markers using an electrochemical protein chip.

An electrochemical immunosensor for performing multianalyte measurements of tumor markers is described. The sensor consisted of an array of immunosensing electrodes fabricated on a glass substrate. Each electrode contained a different immobilized antigen and was capable of measuring a specific tumor marker using electrochemical enzyme-based competitive immunoassay. Using this arrangement, multiple analytes could be measured simultaneously by performing the technical operations for a single assay. The biosensor was used to measure the concentrations of seven important tumor markers: AFP, ferritin, CEA, hCG-beta, CA 15-3, CA 125, and CA 19-9. The sensor had excellent precision and accuracy and was comparable in performance to single-analyte ELISAs (1.9-8.1% interassay CV; <2 ng/mL (or units/mL) detection limit for most analytes). Multianalyte assays provide significant advantages over single-analyte tests in terms of cost per test, labor, test throughput, and convenience. We anticipate that chip-based sensors, as described herein, will be suitable for the mass production of economical, miniaturized lab-on-a-chip devices that will have applications in a wide range of clinical, environmental, and biodefense applications.

Antigens, Neoplasm↗
PubMed2006

Detection and discrimination capabilities of a multitransducer single-chip gas sensor system.

The performance of a single-chip, three-transducer, complementary metal oxide semiconductor gas sensor microsystem has been thoroughly evaluated. The monolithic gas sensor system includes three polymer-coated transducers, a mass-sensitive cantilever, a thermoelectric calorimetric sensor, and an interdigitated capacitive sensor that are integrated along with all electronic circuits needed to operate these sensors. The system additionally includes a temperature sensor and a serial interface unit so that it can be directly connected to, for example, a microcontroller. Several multitransducer chips have been coated with various partially selective polymers and then have been exposed to different volatile organic compounds. The sensitivities of the three different polymer-coated transducers to defined sets of gaseous analytes have been determined. The obtained sensitivity values have then been normalized with regard to the partition coefficients of the respective analyte/polymer combination to reveal the transducer-specific effects. The results of this investigation show that the three different transducers respond to fundamentally different molecular properties, such as the analyte molecular mass (mass-sensitive), its dielectric coefficient (capacitive), and its sorption heat (calorimetric) so that correlations between the determined sensitivity values and the different molecular properties of the absorbed analytes could be established. The information as provided by the system, hence, represents a body of orthogonal data that can serve as input to appropriate signal processing and pattern recognition techniques to address issues such as the quantification of analytes in mixtures.

Journal Article↗
PubMed1997

High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.

Capillary array electrophoresis (CAE) chips have been designed and fabricated with the capacity to rapidly (< 160 s) analyze 12 different samples in parallel. Detection of all lanes with 0.3 s temporal resolution was achieved using a laser-excited confocal-fluorescence scanner. The operation and capabilities of these CAE microdevices were first determined by performing electrophoretic separations of pBR322 MspI DNA samples. Genotyping of HLA-H, a candidate gene for the diagnosis of hereditary hemochromatosis, was then performed to demonstrate the rapid analysis of biologically relevant samples. Two-color multiplex fluorescence detection of HLA-H genotypes was accomplished by prelabeling the standard pBR322 MspI DNA ladder with a red emitting bis-intercalation dye (butyl TOTIN) and on-column labeling of the HLA-H DNA with thiazole orange. This work establishes the feasibility of using CAE chips for high speed, high-throughput genotyping.

Benzothiazoles↗
PubMed1997

Surface plasmon resonance biomolecular interaction analysis mass spectrometry. 1. Chip-based analysis.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in concert with surface plasmon resonance-based biomolecular interaction analysis (SPR-BIA) is reported. A chip-based biosensor unit was used to simultaneously monitor biomolecular interactions taking place on four different regions of the sensor chip (flow cells). Species retained during SPR-BIA were then identified by performing MALDI-TOF directly from within the area of the flow cells. Analyses were performed on an antibody/antigen/antibody system with detection limits in the low-femtomole range. The combined assay demonstrates the use of SPR-BIA to evaluate the relative stability of sequential solution-phase interactions, as well as, upon MALDI-TOF analysis, the ability to unambiguously confirm the presence of species retained during the interaction analysis.

Animals↗
PubMed1999

Disposable optical sensor chip for medical diagnostics: new ways in bioanalysis.

An optical sensor system is described which is particularly well suited for medical point-of-care diagnostics. The system allows for all kinds of immunochemical assay formats and consists of a disposable sensor chip and an optical readout device. The chip is built up from a ground and cover plate with in- and outlet and, between, of an adhesive film with a capillary aperture of 50 microns. The ground plate serves as a solid phase for the immobilization of biocomponents. In the readout device, an evanescent field is generated at the surface of the ground plate by total internal reflection of a laser beam. This field is used for the excitation of fluorophor markers. The generated fluorescence light is detected by a simple optical setup using a photomultiplier tube. Because of the evanescent field excitation, washing or separation steps can be avoided. With this system the pregnancy hormone chorionic gonadotropin (hCG) could be determined in human serum with a detection limit of 1 ng/mL. Recovery values were 86, 106, and 102% for 5, 50, and 100 ng/mL hCG, respectively. The SD in repeated measurements (n = 10) was 5.6%. Furthermore, the feasibility of the system in competitive-type immunoassays was demonstrated for serum theophylline. A linear calibration curve of signal vs theophylline between 1 and 50 mg/L was obtained. Recovery values varied between 118% (10 mg/L) and 81.0% (20 mg/L).

Biosensing Techniques↗
PubMed2000

Electroosmosis- and pressure-driven chromatography in chips using continuous beds.

The application range of microchips can be extended to any mode of chromatography by filling the narrow channels with continuous polymer beds, exemplified by electrochromatography and ion-exchange chromatography. "Wall effects" are eliminated by anchoring the bed to the wall of the channel, an arrangement which has the additional advantage that no frits to support the bed are required. The design of the equipment is based on a quartz chip with all auxiliary pieces (for example, electrode vessels and fluid transfer fittings) placed in a rack, which permits a flexibility of great importance for automation. The same resolution and van Deemter plots were obtained in experiments performed in fused-silica capillaries and in chips for both low-molecular-weight (alkyl phenones, antidepressants) and high-molecular-weight substances (proteins). A sample of uracil, phenol, and benzyl alcohol was separated by electrochromatography in less than 20 s.

Chromatography, High Pressure Liquid↗