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At least 955 records · Page 53Linked to original sources
PubMed2004

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments.

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses. Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK), phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms. In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid). Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions. In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.

1-Phosphatidylinositol 4-Kinase↗
PubMed1998

Applications of DNA chips for genomic analysis.

A major frontier in medical genetics is the definition of the molecular basis of multifactorial diseases. This is especially relevant in the field of clinical psychiatry where the majority of common disorders display complex inheritance patterns, and are further influenced by environmental interactions. New technologies are needed to help address the pressing needs for discovering and deciphering the nature of such disease-associated genes. One such technology which has emerged within the past 3 years involves hybridization-based nucleic acid array (DNA chip) analysis. This technology has the potential to have a lasting impact on diverse genomic-based applications such as large-scale gene mapping studies, mutational analysis, and global expression level monitoring of all human genes. In this review we will describe the fundamental principles behind nucleic acid array-based assays, while focusing on their applications towards genome-wide DNA and RNA analysis. The current capabilities and limitations of these technologies will be discussed, with a focus on areas where future development will be needed for DNA chip-based assays to achieve their full potential.

Chromosome Mapping↗
PubMed2000

A chip-scale universal detector for electrophoresis based on backscattering interferometry.

An on-chip detector based on backscatter interferometry has been developed to perform sub-nanoliter volume refractive index measurements. The detection system consists of a simple, folded optical train based on the interaction of a laser beam and an etched channel in a silica (glass) plate. This etched channel is composed of two radii joined by a flat portion which define a curved surface in the shape of a half cylinder in a silica (glass) plate. The backscattered light from the channel takes on the form of a high contrast interference pattern that contains information related to the bulk properties of the fluid located within the probe volume. Positional changes of the interference pattern extrema (fringes) allow for the determination of refractive index changes at the 10(-6) level in a detection volume of 188 x 10(-12) L. Under capillary electrophoresis (CE) conditions, the injected mass detection limits for small molecules with little native absorption ranges from 530 fmol (0.18 ng) for sucrose to 720 fmol (0.43 nanograms) for raffinose. Fluorescein was also used to evaluate the technique for universal CE and under further optimized conditions can be quantified at the 150 microM level. Separation performance for the solutes tested ranged from about 2300 to 15,500 plates or 61,000 to 400,000 N m-1. The results presented here indicate there is potential for using the simple optical train of backscattering interferometry for on-chip universal solute analysis.

Electrophoresis, Capillary↗
PubMed2001

Quantitative 3-dimensional profiling of channel networks within transparent lab-on-a-chip microreactors using a digital imaging method.

We have developed a method for the quantitative 3-dimensional profiling of micron sized channel networks within optically transparent "lab-on-a-chip" microreactor devices. The method involves capturing digitised microscope images of the channel network filled with an optically absorbing dye. The microscope is operated in transmission mode using light filtered through a narrow bandpass filter with a maximum transmission wavelength matching the wavelength of the absorbance maximum of the dye solution. Digitised images of a chip filled with solvent and dye solution are analysed pixel by pixel to yield a spatially resolved array of absorbance values. This array is then converted to optical path length values using the Beer-Lambert law, thereby providing the 3D profile of the channel network. The method is capable of measuring channel depths from 10 to 500 microm (and probably even smaller depths) with an accuracy of a few percent. Lateral spatial resolution of less than 1 microm is achievable. It has been established that distortion of the measured profiles resulting from a mismatch in refractive index between the dye solution and the glass of the microreactors is insignificant. The method has been successfully used here to investigate the effects of thermal bonding and etch time on channel profiles. The technique provides a convenient, accurate and non-destructive method required to determine channel profiles; information which is essential to enable optimisation of the operating characteristics of microreactor devices for particular applications.

Journal Article↗
PubMed2002

A chip-based capillary electrophoresis-contactless conductivity microsystem for fast measurements of low-explosive ionic components.

A miniaturized analytical system for separating and detecting inorganic explosive residues, based on the coupling of a micromachined capillary electrophoresis (CE) chip with a contactless conductivity detector is described. The low electroosmotic flow (EOF) of the poly(methylmethacrylate) (PMMA) chip material facilitates the rapid switching between analyses of cations and anions using the same microchannel and run buffer (and without an EOF modifier), and hence offers rapid (< 1 min) measurement of seven explosive-related cations and anions. Experimental parameters relevant to the separation and detection processes have been optimized. Addition of a 18-crown-6 ether modifier has been used for separating the peaks of co-migrating potassium and ammonium ions. The ionic-explosive microchip system combines the distinct advantages of contactless conductivity detection with the attractive features of plastic CE microchips. The new microsystem offers great promise for monitoring explosive-related ions at the sample source, with significant advantages of speed/warning, efficiency, cost, or sample size.

Electrophoresis, Capillary↗
PubMed2004

Microfluidic chip for high efficiency DNA extraction.

A high efficiency DNA extraction microchip was designed to extract DNA from lysed cells using immobilized beads and the solution flowing back and forth. This chip was able to increase the extraction efficiency by 2-fold when there was no serum. When serum existed in the solution, the extraction efficiency of immobilized beads was 88-fold higher than that of free beads. The extraction efficiency of the microchip was tested under different conditions and numbers of E. coli cells. When the number of E. coli cells was between 10(6) and 10(8) in 25 microl of whole blood, the extraction efficiency using immobilized beads was only slightly higher than that using free beads (10(0) to 10(1) fold). When the number of E. coli cells was in the range 10(4) to 10(6) in 25 microl of whole blood, the extraction efficiency of immobilized beads was greater than that of the free beads (10(1) to 10(2) fold). When the number of E. coli cells was lower, in the range 10(3) to 10(4) in 25 microl of whole blood, the extraction efficiency of immobilized beads was much higher than that of the free beads (10(2) to 10(3) fold). This study indicated that DNA could be efficiently extracted even when the number of bacterial cells was smaller (10(5) to 10(3)). This microfluidic extraction chip could find potential applications in rare sample genomic study.

Blood↗
PubMed2004

A new on-chip ESI nozzle for coupling of MS with microfluidic devices.

This paper presents a new on-chip electrospray ionisation (ESI) nozzle, which can be used as an interface for coupling microfluidic devices with mass spectrometric (MS) detection. The nozzle was micromilled in a polymer foil (polymethylmethacrylate (PMMA) 750 microm thick), normally used as a cover for microfluidic chips. The performance of this device was examined in the ESI-MS analysis of the tetrapeptide MRFA (methionine-argenine-phenylalanine-alanine). The spray quality is basically dependent on the inner diameter of the nozzle, beside the part of the organic modifier in the solution to be sprayed. Three different inner nozzle diameters (30, 50, 100 microm) and two different apex angles were investigated. Stable electrospray conditions can be generated with a relative standard deviation less than 10% of the total ion current, and down to a concentration of 0.01 micromol L(-1). The production of this ESI interface is relatively simple for the purpose of a low-cost batch fabrication of miniaturized analytical instruments.

Journal Article↗
PubMed2004

Perfusion and chemical monitoring of living cells on a microfluidic chip.

A microfluidic device that incorporates continuous perfusion and an on-line electrophoresis immunoassay was developed, characterized, and applied to monitoring insulin secretion from single islets of Langerhans. In the device, a cell chamber was perfused with cell culture media or a balanced salt solution at 0.6 to 1.5 microL min(-1). The flow was driven by gas pressure applied off-chip. Perfusate was continuously sampled at 2 nL min(-1) by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and monoclonal anti-insulin antibody and allowed to react for 60 s as the mixture traveled down a 4 cm long reaction channel. The cell chamber and reaction channel were maintained at 37 degrees C. The reaction mixture was injected onto a 1.5 cm separation channel as rapidly as every 6 s, and the free FITC-insulin and the FITC-insulin-antibody complex were separated under an electric field of 500 to 600 V cm(-1). The immunoassay had a detection limit of 0.8 nM and a relative standard deviation of 6% during 2 h of continuous operation with standard solutions. Individual islets were monitored for up to 1 h while perfusing with different concentrations of glucose. The immunoassay allowed quantitative monitoring of classical biphasic and oscillatory insulin secretion with 6 s sampling frequency following step changes in glucose from 3 to 11 mM. The 2.5 cm x 7.6 cm microfluidic system allowed for monitoring islets in a highly automated fashion. The technique should be amenable to studies involving other tissues or cells that release chemicals.

Animals↗
PubMed2004

A single cell electroporation chip.

Increasing the cell membrane's permeability can be accomplished via single cell electroporation. Polar substances that cannot otherwise permeate the plasma membrane (such as dyes, drugs, DNA, proteins, peptides, and amino acids) can thus be introduced into the cell. We developed a polymeric chip that can selectively immobilize and locally electroporate single cells. This easy-to-use chip focuses the electric field, eliminating the need to manipulate electrodes or glass pipettes. Moreover, this device allows parallel single cell electroporation. We demonstrate the effectiveness of our device design by electroporating HeLa cells using low applied voltages (< 1 V). We found the average transmembrane potential required for electroporation of HeLa cells to be 0.51 +/- 0.13 V. Membrane permeation is assessed electrically by measuring characteristic 'jumps' in current that correspond to drops in cell resistance, and microscopically by recording either the escape of cytoplasmic dye Calcein AM or the entrance of Trypan blue stain.

Cell Membrane Permeability↗
PubMed2004

On-chip generated mercury microelectrode for heavy metal ion detection.

In this paper, the on-chip generated mercury microelectrode for heavy metal ion detection has been presented. A mercury droplet (approximately 150 microm in diameter) is on-chip generated to form a microelectrode through the mercury microfluidics control. The mercury microelectrode is used to electrochemically detect the heavy metal ions. The sample solutions with different concentration of heavy metal ions (Pb(2+) and Cd(2+)) have been successfully detected on the mercury droplet microelectrode using the square wave stripping voltammetry.

Journal Article↗
PubMed2004

Dry powder injection on chip.

To match the current speed of drug development there is a serious requirement for the pharmaceutical industry to upgrade powder handling processes. Miniaturization is a key concept especially in the early stages of drug development when only small amounts of the drug compound are available. This paper shows for the first time the application of miniaturization through chip technology for dry powder handling. Two methods to separate and transport minute amounts of dry powder particles on chip are described. Both techniques could be employed in small scale mixing devices, which depend on reliable particle feeding systems to substitute balances and the time consuming weighing process.

Biosensing Techniques↗
PubMed2004

On-chip cell lysis by local hydroxide generation.

We present a novel method for on-chip cell lysis based on local hydroxide electro-generation. Hydroxide ions porate the cell membrane, leading to cell lysis. After lysis occurs, hydrogen ions, also generated on chip, react with excess hydroxide, creating a neutral pH lysate and eliminating the need for a wash step. Three different cell types are shown to be effectively lysed by this method: red blood cells, HeLa (human tumor line) and Chinese Hamster Ovary (CHO) cell lines. The release of cytoplasmic molecules from HeLa and CHO cells is demonstrated by monitoring the escape of a membrane impermeant dye from the cytoplasm. In the vicinity of the cathode, the hydroxide concentration is predicted by finite element simulations and shown to fit the lysis rates at different distances from the generating cathode. For flow-through experiments, a second device integrating a mechanical filter with hydroxide generation is fabricated and tested. The purpose of the filter is to trap whole cells and only allow lysate to pass through. The flow rate dependence of hydroxide concentration at the lysis filter is modeled and lysis efficiency is experimentally determined to be proportional to the hydroxide concentration for flow rates from 15 to 30 microl min(-1).

Animals↗
PubMed2005

Highly sensitive signal detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip: confocal surface-enhanced Raman spectroscopic study.

Rapid and highly sensitive detection of duplex dye-labelled DNA sequences in a PDMS microfluidic channel was investigated using confocal surface enhanced Raman spectroscopy (SERS). This method does not need either an immobilization procedure or a PCR amplification procedure, which are essential for a DNA microarray chip. Furthermore, Raman peaks of each dye-labelled DNA can be easily resolved since they are much narrower than the corresponding broad fluorescence bands. To find the potential applicability of confocal SERS for sensitive bio-detection in a microfluidic channel, the mixture of two different dye-labelled (TAMRA and Cy3) sex determining Y genes, SRY and SPGY1, was adsorbed on silver colloids in the alligator teeth-shaped PDMS microfluidic channel and its SERS signals were measured under flowing conditions. Its major SERS peaks were observable down to the concentration of 10(-11) M. In the present study, we explore the feasibility of confocal SERS for the highly sensitive detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip.

Colloids↗
PubMed2005

Microfluidic chip-based liquid-liquid extraction and preconcentration using a subnanoliter-droplet trapping technique.

A robust and simple approach for microfluidic liquid-liquid (L-L) extraction at the subnanoliter-scale was developed for on-chip sample pretreatment. Organic solvent droplets of a few hundred pL were trapped within micro recesses fabricated in the channel walls of a microfabricated glass chip. L-L extraction was performed by delivering aqueous samples through the channel, with the sample stream continuously flowing adjacent to the droplets. The analytes in aqueous streams were enriched within the droplet with high preconcentration factors owing to both phase transfer and dissolution of organic solvent into the bypassing aqueous sample. An aqueous solution of butyl rhodamine B (BRB) and 1-hexanol were used, respectively, as sample and extractant to demonstrate the performance of the system. The fluorescence intensity of the dye extracted into the droplet was monitored in situ by LIF. The system proved to be an efficient means for achieving high enrichment factors of over 1000, with sample consumption of a few microL. Quantitative measurement of the extracted analyte was achieved with a linear response in the range 1 x 10(-9)-8 x 10(-7) M BRB. The precision of the measured fluorescence values for a 10(-7) M BRB standard with a 12.5 min preconcentration period was 6.6% RSD (n = 5).

Journal Article↗
PubMed2005

Isolation of plasma from whole blood using planar microfilters for lab-on-a-chip applications.

Researchers are actively developing devices for the microanalysis of complex fluids, such as blood. These devices have the potential to revolutionize biological analysis in a manner parallel to the computer chip by providing very high throughput screening of complex samples and massively parallel bioanalytical capabilities. A necessary step performed in clinical chemistry is the isolation of plasma from whole blood, and effective sample preparation techniques are needed for the development of miniaturized clinical diagnostic devices. This study demonstrates the use of passive, operating entirely on capillary action, transverse-flow microfilter devices for the microfluidic isolation of plasma from whole blood. Using these planar microfilters, blood can be controllably fractionated with minimal cell lysis. A characterization of the device performance reveals that plasma filter flux is dependent upon the wall shear rate of blood in the filtration channel, and this result is consistent with macroscale blood filtration using microporous membranes. Also, an innovative microfluidic layout is demonstrated that extends device operation time via capillary action from seconds to minutes. Efficiency of these microfilters is approximately three times higher than the separation efficiencies predicted for microporous membranes under similar conditions. As such, the application of the microscale blood filtration designs used in this study may have broad implications in the design of lab-on-a-chip devices, as well as the field of separation science.

Cell Separation↗
PubMed2005

Use of self assembled magnetic beads for on-chip protein digestion.

The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.

Dimethylpolysiloxanes↗
PubMed2005

Lab-on-a-chip with integrated optical transducers.

Taking the next step from individual functional components to higher integrated devices, we present a feasibility study of a lab-on-a-chip system with five different components monolithically integrated on one substrate. These five components represent three main domains of microchip technology: optics, fluidics and electronics. In particular, this device includes an on-chip optically pumped liquid dye laser, waveguides and fluidic channels with passive diffusive mixers, all defined in one layer of SU-8 polymer, as well as embedded photodiodes in the silicon substrate. The dye laser emits light at 576 nm, which is directly coupled into five waveguides that bring the light to five different locations along a fluidic channel for absorbance measurements. The transmitted portion of the light is collected at the other side of this cuvette, again by waveguides, and finally detected by the photodiodes. Electrical read-out is accomplished by integrated metal connectors. To our knowledge, this is the first time that integration of all these components has been demonstrated.

Equipment Design↗
PubMed2006

Monolithically integrated dye-doped PDMS long-pass filters for disposable on-chip fluorescence detection.

We report the fabrication of high quality monolithically integrated optical long-pass filters, for use in disposable diagnostic microchips. The filters were prepared by incorporating dye molecules directly into the microfluidic chip substrate, thereby providing a fully integrated solution that removes the usual need for discrete optical filters. In brief, lysochrome dyes were added to a poly(dimethylsiloxane) (PDMS) monomer prior to moulding of the microchip from a structured SU-8 master. Optimum results were obtained using 1 mm layers of PDMS doped with 1200 microg mL(-1) Sudan II, which resulted in less than 0.01% transmittance below 500 nm (OD 4), >80% above 570 nm, and negligible autofluorescence. These spectral characteristics compare favourably with commercially available Schott-glass long-pass filters, indicating that high quality optical filters can be straightforwardly integrated into the form of PDMS microfluidic chips. The filters were found to be robust in use, showing only slight degradation after extended illumination and negligible dye leaching after prolonged exposure to aqueous solutions. The provision of low cost high quality integrated filters represents a key step towards the development of high-sensitivity disposable microfluidic devices for point-of-care diagnostics.

Azo Compounds↗