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At least 1,225 records · Page 68Linked to original sources
PubMed2003

Multianalyte immunoassay with self-assembled addressable microparticle array on a chip.

This paper describes the random fluidic self-assembly of metallic particles into addressable two-dimensional microarrays and the use of these arrays as a platform for constructing a biochip useful for bioassays. The basic units in the assembly were the microfabricated particles carrying a straightforward visible code and the corresponding array template patterned on a glass substrate. The particles consisted of a hydrophobic and magnetic Ni-polytetrafluoroethylene (PTFE) composite layer on one face, and on the other face a gold layer that was modified for biomolecular attachment. An array template was photoresist-patterned with spatially discrete microwells in which an electrodeposited Ni-PTFE hydrophobic composite layer and a hydrophobic photo-adhesive coating were deposited. The particles, after biomaterial attachment and binding processes in bulk, were self-assembled randomly onto the lubricated bonding sites on the chip substrate, driven by a combination of magnetic, hydrophobic, and capillary interactions. The encoding symbol carried by the particles was used as the signature for the identification of each target/assay attached to the particle surface. We demonstrate here the utility of microfabricated-encoded particle arrays for conducting multianalyte immunoassays in a parallel fashion with the use of imaging detection.

Biological Assay↗
PubMed2003

Methods for on-chip protein analysis.

The unambiguous identification of peptides/proteins is crucial for the definition of the proteome. Using ProteinChip Array technology also known as surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS), we developed experimental protocols and probed test conditions required for the protein identification on ProteinChip surfaces. We were able to directly digest peptides/proteins on-chip surfaces by specific proteases, such as trypsin, and to obtain the peptide mass fingerprint of the sample under investigation by its direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via the ProteinChip interface, thus allowing the unambiguous identification of the protein(s) within the sample. In addition, we were able to identify the C-terminal sequence of peptides by their digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF MS analysis of the resulting peptide mass ladders employing the instrument's protein ladder sequence software. Moreover, the removal of up to nine amino acid residues from the C-terminal end of a peptide extends the functional range of Qq-TOF MS/MS sequence determination to over 3000 m/z. The utility of these procedures for the proteome exploration are discussed.

Amino Acid Sequence↗
PubMed1999

Ergonomics evaluation of a manually operated cassava chipping machine.

A manually operated machine for chipping cassava was evaluated. Six farmers took part in the study, with physiological, postural, and subjective measurements being taken. Using the machine resulted in drudgery and postural discomfort. Following an iterative design process and using appropriate anthropometric measurements, an improved, adjustable prototype was developed. This was tested with the six farmers and six novice users. It was found to reduce discomfort and physiological strain, allowed a faster work-rate (with novice users) and was preferred by all users. The study demonstrated how ergonomics can play an important role in reducing drudgery and improving user satisfaction in technology development and transfer in developing countries.

Adult↗
PubMed2003

On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells.

We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes. This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers. After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles. When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h. The results indicate that the cell's growth and division are directly related to their nutrient condition. The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing.

Adaptation, Physiological↗
PubMed2003

Brief episode of ischemia activates protective genetic program in rat heart: a gene chip study.

OBJECTIVE: Brief episodes of ischemia of 20 min or less have the potential to protect the heart. Such episodes are associated primarily with reversible ischemic injury yet they induce changes in gene expression. The purpose of the study was to determine whether activation of protective genes takes place within 4 h following a brief episode of ischemia that would mimic angina pectoris. METHODS: Three groups of rats were studied. In the control (Ctrl) group, hearts were immediately excised following anesthesia; in the sham-operated (SO) group, opened-chest rats received 4 h and 20 min of no intervention; and in the group subjected to ischemia (SI) hearts received 20 min of proximal coronary occlusion followed by 4 h of reperfusion. Hearts from the SI group were divided into nonischemic (NI) and ischemic (Isc) areas. Changes in gene expression pattern were analyzed by using Affymetrix Gene Chips. RESULTS: Ischemia led to strong upregulation of mRNA transcripts for heat shock proteins 70, 27, 105, 86 and 40 kDa, vascular endothelial growth factor, brain-derived neurotrophic factor, plasminogen activator inhibitor-1, activating transcription factor 3, B-cell translocation gene 2, and growth arrest and DNA damage inducible 45 alpha protein compared to the NI tissue. The majority of mRNAs whose levels increased following brief ischemia were of a protective nature. CONCLUSION: Genetic reprogramming emerging during or following brief episodes of ischemia that simulate angina, can be characterized as protective in nature. Developing new therapeutic strategies aimed to promote this protective response represents a legitimate target for future research.

Animals↗
PubMed1999

Identification of the genes responsive to etoposide-induced apoptosis: application of DNA chip technology.

DNA chip technology was used in an attempt to identify target genes responsible for apoptosis induced by etoposide, a p53 activating topoisomerase II inhibitor used clinically as an antitumor agent. 62 Individual mRNAs whose mass changed significantly were identified after screening oligonucleotide arrays capable of detecting 6591 unique human mRNA species. 12 (Nine induced and three repressed) of the etoposide-responsive genes were further studied by Northern analysis and an agreement rate of 92%, was reached. Among the 12 genes studied, two (WAF1/p21 and PCNA) are known p53 regulatory genes, two (glutathione peroxidase and S100A2 calcium-binding protein) appear to be the novel p53 target genes and the others appear to be p53-independent. Based upon these findings, the signalling pathways that possibly mediate etoposide-induced apoptosis are proposed.

Apoptosis↗
PubMed2001

Channel-specific coatings on microfabricated chips.

This paper reports channel-specific immobilization of fluorescein-5-isothiocyanate (FITC)-labeled bovine serum albumin and beta-galactosidase on microchips with a central channel and two crossing channels; referred to as a double cross channel configuration. Solvent wells at the termini of all channels were used to store reagents. Coatings were applied in multiple steps using electroosmotically driven flow to deliver reagents to specific channels in the chip. The first step in all coating reactions was derivatization of the capillary walls with an organosilane having a reactive pendant functional group. As the silylating reagent was transported from the reagent storage well to a specific waste well, capillary walls in the route of transport were silylated. Flow was maintained throughout a reaction. The route of transport, and thus the specificity of channel coating, were controlled by the well to which negative potential was applied. Flow in a multichannel network takes the shortest route between the electrodes delivering the motive potential. The second reagent in the reaction was delivered from a different well and took a different path through the channel network, as did other reagents. Only the channel being coated was in the flow path of all the reagents used in the coating process. The zone of immobilization in the case of FITC-labeled albumin was determined with confocal fluorescence microscopy. Enzyme activity of immobilized beta-galactosidase (beta-Gal) was monitored by following the hydrolysis of fluorescein mono-beta-D-galactopyranoside to fluorescein with laser-induced fluorescence.

Bioreactors↗
PubMed2003

Miniaturised isotachophoretic analysis of inorganic arsenic speciation using a planar polymer chip with integrated conductivity detection.

A new method allowing the analysis of inorganic arsenic species using isotachophoresis has been developed. This method has been shown to be suitable for use on both miniaturised planar polymer separation devices and capillary scale devices. A poly(methyl methacrylate) chip with integrated conductivity electrodes has been successfully used for the rapid analysis of inorganic arsenic species in under 600 s. Limits of detection of 1.8 mg l(-1) and 4.8 mg l(-1) for arsenic(V) and arsenic(II), respectively, have been achieved with the miniaturised device. The device has also been used to perform the simultaneous separation of arsenic(III), arsenic(V), antimony(III), molybdenum(VI) and tellurium(IV).

Electric Conductivity↗
PubMed2003

On-chip protein sample desalting and preparation for direct coupling with electrospray ionization mass spectrometry.

A membrane-based desalting step integrated in a MS microchip is presented: drugs, peptides and proteins are adsorbed on a hydrophobic poly(vinylidene difluoride) membrane, which allows the washing out of salts. The integration with microfluidics permits a controlled elution of analytes from the membrane and their direct mass spectrometric analysis by electrospray ionisation MS. The desalting process is demonstrated with picomole amounts of propanolol, insulin and cytochrome c. Moreover, this stop-and-go desalting process is tolerant to high concentrations of urea, and to the presence of reductants such as dithiothreitol. This particular feature allowed the chemical tagging of cysteines in beta-lactoglobulin A with iodoacetamide. Finally, the integration of chemical tagging, on-chip desalting and MS microchip paves the way for the development of high-throughput analytical procedure for structural proteomics.

Adsorption↗
PubMed1997

Conventional capillary electrophoresis in comparison with short-capillary capillary electrophoresis and microfabricated glass chip capillary electrophoresis for the analysis of fluorescein isothiocyanate anti-human immunoglobulin G.

Fast, efficient analysis of fluorescein isothiocyanate anti-human IgG was achieved in a short-capillary and in a microfabricated glass chip. The capillary was 6 cm long from injection end to detector with electric field strength of 0.268 kV/cm. Analyses were performed within 1 min. A glass microchip device was fabricated using standard photolithographic procedures and chemical wet etching. The channels were sealed using a direct bonding technique. For an effective length of 2.8 cm with electric field strength of 0.526 kV/cm, electrophoretic analysis was achieved in less than 16 s. Conventional capillary electrophoresis gave highly reproducible results and good detection limits. Analysis time was 2.5 min for a capillary with 47 cm effective length and electric field strength of 0.383 kV/cm.

Electrophoresis, Capillary↗
PubMed2003

Visualization and numerical modelling of microfluidic on-chip injection processes.

Sample injection processes accomplished using a microfluidic-cross chip are investigated experimentally and numerically. Fluorescent dye is employed to differentiate the sample solution from the pure buffer. Different sample geometries are achieved using different applied electric fields and dyes with different electrophoretic mobilities. Of particular interest here are concentration-dense samples with large axial extent (extending beyond the intersection). The ability to load and subsequently dispense these large axial extent samples is predicted numerically and verified experimentally by direct visualization. Containing more mass, larger samples exhibited lower concentration gradients, making them less sensitive to diffusion and well-suited to transport once dispensed. In the loading process, however, larger samples were found to be more sensitive to pressure effects than more focused samples. This was investigated by imaging sample geometries under various applied fields in the presence of a constant pressure gradient. Laplace pressure originating from differential meniscus curvatures in the reservoirs was found to be the most significant source of such pressure disturbances in these geometries.

Journal Article↗
PubMed2003

A dynamic loading method for controlling on-chip microfluidic sample injection.

A new technique for controlling discrete sample injection in straight-cross microfluidic chips is presented here. This technique involves a three-part process with a dynamic loading step in between the steady-state loading step and the dispensing step. During the intermediate step, sample is pumped into the intersection and into the three connecting channels. The key features of this technique are the ability to dynamically control the sample size and the ability to inject well-defined samples at the original sample concentration. Injections of these samples with lengths varying from 2 channel widths (100 microm) to 20 channel widths (millimeter-sized) are demonstrated. The sample concentration profiles obtained are compared with those of focused and less-focused pinched-valve injections. In applications such as high-speed capillary zone electrophoresis, this technique can provide an increase in signal with a small increase in sample length. This technique is especially applicable to many large-sample applications in which the offset twin-T microchip has been previously employed.

Journal Article↗
PubMed2000

An illustrated identification key to four different species of adult Dinoderus (Coleoptera: Bostrichidae), commonly attacking dried cassava chips in West Africa.

Bostrichids of the genus Dinoderus are commonly found as pests of dried cassava chips in West Africa. An illustrated key facilitating the identification of the four most frequent African species, i.e. D. bifoveolatus (Wollaston), D. minutus (Fabricius), D. porcellus Lesne, and D. oblongopunctatus Lesne is presented.

Journal Article↗
PubMed1999

Detection of salmonella in poultry using a silicon chip-based biosensor.

Salmonella typhimurium was detected to levels as low as 119 CFUs using the Threshold Immunoassay System. This immunoassay system utilizes solution-based binding of the biotin and fluorescein labeled antibodies to salmonella, followed by filtration-capture of the immunocomplex on a biotin-coated nitrocellulose membrane. Lastly, an anti-fluorescein urease conjugate is bound to the immunocomplex. Detection of the bound immunocomplex is made possible via the silicon chip-based light-addressable potentiometric sensor. In the presence of the urea, urease converts the substrate to ammonia and CO2 and this results in a pH change at the silicon surface. The resultant pH change is monitored with time and the signal output is reported in microV s(-1). An experiment whereby chicken carcass washings were fortified with salmonella showed a recovery of 90%, indicating that the technique can be used to test for salmonella under these conditions. Precautions must be used with this instrument as sample debris will affect sample flow through the membrane and hence the signal output.

Biosensing Techniques↗
PubMed2000

Antibodies in diagnostics - from immunoassays to protein chips.

Antibodies are used extensively as diagnostic tools in a wide array of different analyses. Monoclonal and recombinant antibodies provide a never ending source of molecules and can produce endless possibilities for novel genetic constructs. Antibodies are still very much in vogue and are now also being used in microarray analysis of the proteome using protein chips. Here, recent opportunities presented by antibodies as diagnostic tools are reviewed.

Antibodies↗
PubMed2001

Use of surface-enhanced laser desorption ionization protein chip system to analyze streptococcal exotoxin B activity secreted by Streptococcus pyogenes.

Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr approximately 41,000 and the fully active enzyme Mr approximately 28,500. SpeB production in culture supernatants was demonstrated to be growth-phase regulated and SpeB positive and negative variants of a blood passaged S. pyogenes isolate could readily be distinguished. In kinetic studies of the autoactivation of the zymogen form of SpeB, the sequential generation of four intermediates was detected before the accumulation of the fully active enzyme. The methods described enabled enhanced speed, use of lower sample volumes and concentrations, and a more complete molecular characterization of SpeB than allowed by existing methods of analysis using SDS-PAGE and Western immunoblotting.

Bacterial Proteins↗
PubMed2001

Solution and chip arrays in protein profiling.

The industrialization of proteomics demands reproducible, robust and high-throughput profiling technologies that current two-dimensional gel electrophoresis cannot achieve. New technologies in protein arrays, either on chips or with self-encoded elements in solution, hold much promise for interrogating the diverse and immense proteome.

Biosensing Techniques↗
PubMed1999

Chromatography and electrophoresis on chips: critical elements of future integrated, microfluidic analytical systems for life science.

Liquid chromatography and electrophoresis played a major role in the life-science revolution, most strikingly in protein purification, peptide fractionation and sequencing, amino acid analysis, and DNA sequencing. The objective of this article is to examine the potential role of separation systems in the continuing evolution of biochemistry, biotechnology and molecular biology. Very small chip-based systems may change how chemical analyses in biology, medical research and health care evolve over the next decade.

Biological Science Disciplines↗