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At least 595 records · Page 33Linked to original sources
PubMed2005

Protein microarray using alpha-amino acids as metal tags on chips.

Procedures for synthesizing alpha-amino acids on a chip for coordination with transitional metal ions and a His-Tagged protein have been successfully developed as a stable protein microarray. Using the recombinant His-Tagged 3CL-protease (3CLpro) as a model for attachment to chips containing D-/L-Glu, Asp, Orn, Ser via different transitional metal ions, it was found that the Orn chip was the best of affinity binding and stability by which Zn2+ was the best metal ion for affinity while Co2+ was the best metal ion for stability. Thus, this protein microarray can be alternatively used as a high throughput screening method for rapid detection against SARS CoV 3CLpro and/or efficient purification of other Tagged proteins.

Amino Acids↗
PubMed2004

Chip electrochromatography.

Electrochromatography (EC) in microfluidic chips is emerging as an attractive alternative to capillary electrophoresis (CE) for on-chip separations. This review summarizes recent developments in the rapidly growing area of chip electrochromatography with a focus on "column" technologies. Relevant achievements are summarized according to the types of stationary phase used for the separations including open channels, microfabricated structures, and channels packed with beads or containing a porous monolith. The advantages and disadvantages of each, as well as practical aspects of their application, are discussed. The analytical performance of these devices is demonstrated with separations involving various families of compounds mostly in the reversed-phase chromatographic mode.

Animals↗
PubMed2005

Oligo-chip based detection of tick-borne bacteria.

We have developed an oligonucleotide-chip based assay for detection of 16S ribosomal PCR products from tick-borne bacteria. This chip contains 14 specific probes, which target variable regions of 16S rDNA of tick-borne bacteria including Borrellia spp., Rickettsia spp., Anaplasma spp., Coxiella burnetii and Francisella tularensis. The specificity of these probes was tested by hybridization of the chip with fluorescently labeled PCR products amplified from the genomic DNA of selected tick-borne bacteria. The assay was also tested for detection of tick-borne bacteria in single ticks.

Anaplasma↗
PubMed2004

Identification and characterization of Clostridium perfringens using single target DNA microarray chip.

A DNA microarray method was developed to identify the presence of toxin genes: encoding beta toxin (cpb), epsilon toxin (etx), enterotoxin (cpe), alpha toxin (cpa), and iota toxin (iA) in Clostridium perfringens. To build the DNA chip, each gene sequence was represented by one approximately 22-bp amino-modified oligonucleotide printed twice on aldehyde-coated slides. Multiplex PCR with Cy3 and Cy5-dCTP derivatized fluorescent nucleotides was used to label five genes and fluorescent probes were prepared. The PCR probes were denatured and single-strand-labeled DNAs were separated and purified using magnetic beads. The presence of toxin genes in C. perfringens was detected by hybridization of amplified ssDNA probes to oligonucleotides on the chip representing one target sequence of each toxin gene. The DNA chip was able to identify eight strains of C. perfringens.

Clostridium perfringens↗
PubMed2004

Fully-automated chip-based nanoelectrospray tandem mass spectrometry of gangliosides from human cerebellum.

The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is described.

Adult↗
PubMed2005

A method for fabricating uni-dsDNA microarray chip for analyzing DNA-binding proteins.

This paper describes an approach for preparing unimolecular double-stranded DNA (uni-dsDNA) microarray chip. In this method, the various target oligonucleotides containing a reverse complementary sequence at 5' end were firstly annealed to a same universal oligonucleotide with amino group at 5' end and immobilized on aldehyde-derivatized glass slide. An on-chip DNA polymerization reaction was then performed to elongate the universal oligonucleotides. After a denaturation and a followed intra-strand annealing, a hairpin structure was formed at the free 3' end of the immobilized oligonucleotides. Finally, another on-chip DNA polymerization was done to synthesize the uni-dsDNA microarray. Combining with a PCR amplification of chemically synthesized target oligonucleotides, this method was much cost-effective for production of the uni-dsDNA microarray. The uni-dsDNA microarray was verified applicable for detecting the presence and monitoring the DNA-binding activity of the sequence-specific DNA-binding proteins.

Cost-Benefit Analysis↗
PubMed2006

Effect of antifungal agents on protein composition of Candida albicans studied by capillary electrophoresis and chip technology.

In the present study protein profile of a Candida albicans strain had been examined by chip technology and conventional capillary electrophoresis (CE). Profiles could be characterised by the presence of ten dominating protein peaks. These proteins could be distinguished by both techniques, but their quantity showed significant differences in the electropherograms obtained by CE and chip method. Changes in the protein profile were induced by administration of different antifungal agents. Fluconazole and amphotericin B treatment was able to induce similar changes in the pattern, appearance of a 40-kDa protein and up-regulation of a 60-kDa protein was observed by chip technology. Increase in the quantity of these proteins under stress effect (antifungal treatment) might refer to their stress function in the fungal cell. Treatment of C. albicans cells with MK 94 (fused cyclic Mannich ketone) antifungal compound induced not only the previously mentioned changes, but further specific alterations, appearance of a 19-kDa protein and up-regulation of the low molecular weight proteins. This might refer to the different mode of action of this agent on the fungal cells. Conventional capillary electrophoresis was suitable to detect the appearance of the 19-kDa peak, and up-regulation of the 60 kDa protein, but the other changes could not be detected by this technique. Shorter running time, more effective and baseline separation of proteins refer to the advantages of microchip-based method in the analysis of complex biological samples.

Amphotericin B↗
PubMed2004

Transfection microarray of human mesenchymal stem cells and on-chip siRNA gene knockdown.

The transfection efficiency of primary cells is the bottleneck for their use with miniaturized formats for gene validation assays. We have found that when formulations containing various reporter plasmids were microarrayed on glass slides (chips), hMSCs cultivated on the chip incorporated and expressed the microarrayed plasmid DNAs with high efficiency and virtually total spatial resolution. Fibronectin, as the key formulation component, was found to significantly increase the on-chip transfection efficiency in hMSCs as well as many other cells. Further, we have conclusively proven that when siRNA was co-arrayed with the target plasmid DNA, a concentration-dependent gene knockdown was observed. Thus, massively miniaturized RNAi gene knockdown experiments can now be performed in primary cells, previously unusable with transfection microarrays (TMA).

Animals↗
PubMed2004

Novel assay format permitting the prolonged use of regeneration-based sensor chip technology.

A polyclonal antibody raised against morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine) was used in the development of a novel assay format using a surface plasmon resonance (SPR)-based biosensor. Previously developed assays have generated calibration curves based on differences in the quantity of response units binding to the surface of a chip coated with the analyte. The novel assay described here was based on the development of a standard curve using the slope of a series of consecutive binding interactions. Using this format, regeneration between each assay cycle was no longer required. This increased the useable life span of the chip surface and, as a result, decreased the cost associated with the assay. Thus, at least 15 binding interactions could be carried out before the saturation of antibody on the surface of the chip caused the response to deviate significantly from linearity. After 15 nonregenerated binding interactions, the slope still remained within 1.5% of the slope after a single binding event. Analysis time, and the sample volumes required were also markedly decreased while sensitivity was enhanced. The inhibition assay developed had a detection range of 270 to 17,500 pg ml(-1).

Antibodies↗
PubMed2005

Molecular identification of Yersinia enterocolitica isolated from pasteurized whole milk using DNA microarray chip hybridization.

A DNA microarray chip of four virulence genes and 16S ribosomal DNA gene conserved region among all Gram negative species, including Yersinia, as a positive control was developed and evaluated using 22 Yersinia enterocolitica isolates. Eight different oligonucleotide probes (oligoprobes) with an average size of 22 bp, complementary to the unique sequences of each gene, were designed and immobilized on the surface of chemically modified slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all five genes, and single stranded DNA (ssDNA) samples for microarray analysis were prepared by using a primer extension of amplicons in the presence of one primer of all genes. The presence of genes in Y. enterocolitica was established by hybridization of the fluorescently labeled ssDNA representing different samples of the microarray gene-specific oligoprobes and confirmed by PCR. Results of the study showed specificity of genotyping Y. enterocolitica using multiple microarray-based assays. Final validation of the chip's ability to identify Y. enterocolitica genes from adulterated pasteurized whole milk was confirmed and successful. The limit of chip detection of virulence genes in pasteurized whole milk was found to be 1000 CFU per hybridization.

Animals↗
PubMed2006

Using PCR amplification to increase the confidence level of Salmonella typhimurium DNA microarray chip hybridization.

In order to design and validate a method to identify virulence genes of Salmonella typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to validate the hybridization signals. Therefore, a DNA microarray chip of 71 virulence genes of S. typhimurium was developed and evaluated using 10 isolates. Each gene was represented by 65bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hinf1 and Sau3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in 10 strains of S. typhimurium was established by measuring a fluorescent signal above the background noise of the chip. PCR amplification of 10 genes (orgA, ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, rRNA, and purR) of S. typhimurium was used as a standard to verify the confidence level of the DNA microarray chip. In conclusion, using PCR amplification to increase the confidence level of the microarray hybridization data was successful.

Base Sequence↗
PubMed2005

"Fluorescent Cell Chip" for immunotoxicity testing: development of the c-fos expression reporter cell lines.

The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals.

Animal Testing Alternatives↗
PubMed2007

Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip.

Ultrasonic-standing-wave (USW) technology has potential to become a standard method for gentle and contactless cell handling in microfluidic chips. We investigate the viability of adherent cells exposed to USWs by studying the proliferation rate of recultured cells following ultrasonic trapping and aggregation of low cell numbers in a microfluidic chip. The cells form 2-D aggregates inside the chip and the aggregates are held against a continuous flow of cell culture medium perpendicular to the propagation direction of the standing wave. No deviations in the doubling time from expected values (24 to 48 h) were observed for COS-7 cells held in the trap at acoustic pressure amplitudes up to 0.85 MPa and for times ranging between 30 and 75 min. Thus, the results demonstrate the potential of ultrasonic standing waves as a tool for gentle manipulation of low cell numbers in microfluidic systems.

Animals↗
PubMed2004

Sensitivity, specificity, and the hybridization isotherms of DNA chips.

Competitive hybridization, at the surface and in the bulk, lowers the sensitivity of DNA chips. Competitive surface hybridization occurs when different targets can hybridize with the same probe. Competitive bulk hybridization takes place when the targets can hybridize with free complementary chains in the solution. The effects of competitive hybridization on the thermodynamically attainable performance of DNA chips are quantified in terms of the hybridization isotherms of the spots. These relate the equilibrium degree of the hybridization to the bulk composition. The hybridization isotherm emerges as a Langmuir isotherm modified for electrostatic interactions within the probe layer. The sensitivity of the assay in equilibrium is directly related to the slope of the isotherm. A simpler description is possible, in terms of c(50) values specifying the bulk composition corresponding to 50% hybridization at the surface. The effects of competitive hybridization are important for the quantitative analysis of DNA chip results, especially when used to study point mutations.

Base Sequence↗
PubMed2001

On-chip definition of picolitre sample injection plugs for miniaturised liquid chromatography.

The fabrication of components for a miniaturised liquid chromatography system on silicon has recently been reported by our research group [J. Cap. Electrophoresis Microchip Technol. 6 (1999) 33; Analyst 125 (2000) 25]. To date, inlet and outlet connection ports, separation micro-channels (20-200 microm in width, 0.5-10 microm in depth, 15-60 cm in length), and an intersection for picolitre injection have been etched on a silicon wafer and then sealed with a Pyrex cover plate on which platinum electrodes for on-chip detection have been patterned. The platinum electrodes have been used for the amperometric detection of phenol, using 20 nl off-chip injection. In this work we present our latest results obtained with on-chip pressure driven picolitre injection, designed to realize the full capabilities of this micro-LC system. The injection volume is dependent on the micro-channel depth, width, and also on the intersection length, allowing injection in the low nanolitre to picolitre range.

Chromatography, High Pressure Liquid↗
PubMed1982

Incidental carcinoma of the prostate at the time of transurethral resection: importance of evaluating every chip.

Incidental adenocarcinoma of the prostate has been divided into stage A1--less than 3 foci of well differentiated adenocarcinoma present and stage A2--3 or more foci of poorly differentiated tumor present. The clinical significance of these 2 stages has been well documented, with stage A1 lesions causing no increased mortality, while up to 30 per cent of patients with clinical stage A2 disease will have positive pelvic lymph nodes at exploration and, thus, will have surgical stage D1 tumor. Most pathology laboratories submit only a fraction of the transurethral resection chips for permanent blocks. In an effort to evaluate the over-all incidence and distribution of stages A1 and A2 lesions were began a prospective study in 1978 whereby all prostatic chips were submitted for permanent sections. A review of 500 consecutive cases of transurethral resection for clinically benign prostates before 1978 revealed 43 cases of adenocarcinoma: 10 (23 per cent) stage A1 and 33 (77 per cent) stage A2. A review of a similar series of 500 consecutive patients since 1978 revealed 71 cases of adenocarcinoma: 17 (24 per cent) clinical stage A1 and 54 (76 per cent) clinical stage A2. Thus, we found that since 178 incidental adenocarcinoma of the prostate has increased by 65 per cent and the distribution of stages A1 and A2 lesions has remained unchanged, 76 per cent of these lesions being clinical stage A2 with its much greater clinical significance. Evaluation of every chip does make a clinically significant difference in the subsequent management of patients with incidental adenocarcinoma of the prostate.

Adenocarcinoma↗
PubMed2002

Towards quantitative assays with peptide chips: a surface engineering approach.

The development of peptide and protein microarrays has created enormous opportunities in biomedical research. Current chip-based assays are well suited for identifying candidate protein or enzyme activities but still require conventional solution phase experiments to validate hits. Here, three surface-engineering strategies for microarray design are described and are illustrated in the development of a peptide chip for the quantitative analysis of kinase activity on solid support. These strategies promise to widen the application of microarrays by permitting the evaluation of hits in a chip-based format.

Amino Acid Sequence↗
PubMed1997

Corneal endothelial changes after divide and conquer versus chip and flip phacoemulsification.

PURPOSE: To evaluate changes in central corneal endothelial cells and corneal thickness after divide and conquer phacoemulsification and chip and flip phacoemulsification. SETTING: Houston Eye Clinic, Houston, Texas, USA. METHODS: Forty-four eyes were randomly assigned to one of two groups to have divide and conquer (D/C Group) or chip and flip (C/F Group) phacoemulsification with implantation of a posterior chamber, foldable, silicone intraocular lens performed by one surgeon. Of these, 41 eyes of 37 patients (22 D/C Group, 19 C/F Group) met the inclusion criteria. All surgeries were uneventful. A complete eye examination including specular microscopy and pachymetry was performed preoperatively and 1 week and 1 and 3 months postoperatively. RESULTS: There were no significant between-group differences in any of the following means: sex; age; time at 70% phaco power; total phacoemulsification time. No correlation was found between ultrasound time and increased corneal thickness except for a significant increase in percentage of hexagonal cells in the C/F Group at 1 month. The only statistically significant between-group differences were greater endothelial cell loss in the C/F Group at 1 month and the percentage change in hexagonal cells at 1 week (1.1% increase, D/C Group; 10.7% decrease, C/F Group). CONCLUSION: The divide and conquer technique led to less endothelial loss and hexagonal cell change than the chip and flip technique, although at 3 months the differences were not significant.

Aged↗