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Biomedical subjects

L J Kricka

Publications and source records attributed to L J Kricka.

At least 37 records · Page 2Linked to original sources
PubMed1998

Degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human DNA on microchip-based devices.

Random amplification of the human genome using the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) was performed in a silicon-glass chip. An aliquot of the DOP-PCR amplified genomic DNA was then introduced into another silicon-glass chip for a locus-specific, multiplex PCR of the dystrophin gene exons in order to detect deletions causing Duchenne/Becker muscular dystrophy. Amplicons were analyzed by both conventional capillary electrophoresis and microchip electrophoresis and results were compared to those obtained using standard non-chip-based PCR assays. Results from microchip electrophoresis were consistent with those from conventional capillary electrophoresis. Whole genome amplification products obtained by DOP-PCR proved to be a suitable template for multiplex PCR as long as amplicon size was < 250 bp. Successful detection and resolution of all PCR products from the multiplex PCR clearly shows the feasibility of performing complex PCR assays using microfabricated devices.

DNA↗
PubMed1998

Miniaturization of analytical systems.

Miniaturization has been a long-term trend in clinical diagnostics instrumentation. Now a range of new technologies, including micromachining and molecular self-assembly, are providing the means for further size reduction of analyzers to devices with micro- to nanometer dimensions and submicroliter volumes. Many analytical techniques (e.g., mass spectrometry and electrophoresis) have been successfully implemented on microchips made from silicon, glass, or plastic. The new impetus for miniaturization stems from the perceived benefits of faster, easier, less costly, and more convenient analyses and by the needs of the pharmaceutical industry for microscale, massively parallel drug discovery assays. Perfecting a user-friendly interface between a human and a microchip and determining the realistic lower limit for sample volume are key issues in the future implementation of these devices. Resolution of these issues will be important for the long-term success of microminiature analyzers; in the meantime, the scope, diversity, and rate of progress in the development of these devices promises products in the near future.

Clinical Laboratory Techniques↗
PubMed1997

Healthcare in the United States and the practice of laboratory medicine.

US healthcare and the impact of the changes in the healthcare system on the current and future practice of laboratory medicine are analyzed. Factors considered include--population and government, healthcare expenditure, organization of healthcare delivery (institutions, personnel, healthcare industry, knowledge-personnel production, financing), the impact of managed care, and political and public health issues. The effect of the changing healthcare scene on laboratories and the consequent cost-containment measures for laboratory medicine are examined, including centralization, consolidation, supplier relationships, reengineering and automation (total lab automation), expert systems, CQI, and the move to point-of-care testing.

Clinical Laboratory Techniques↗
PubMed1997

N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free intracellular calcium.

N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium concentrations were examined in response to activation of recombinant NMDA receptors expressed in human embryonic kidney 293 cells. Intracellular calcium was measured in transfected cell populations by cotransfection with the calcium-sensitive, bioluminescent protein aequorin and by single cell imaging with the fluorescent calcium indicator fluo-3. Agonist application to NR1/2A or NR1/2B-transfected cells elicited robust rises in intracellular calcium. NR1/2A responses were inhibited by the noncompetitive antagonists MK-801 and dextromethorphan and were dependent on extracellular calcium but not on intracellular calcium stores. In contrast, no detectable intracellular calcium responses were observed in NR1/2C-transfected cells. These findings indicate that NMDA receptors in the absence of other neuron-specific factors can mediate increases in intracellular calcium with subunit specificity and extracellular calcium dependence.

Calcium↗
PubMed1997

The use of capillary electrophoresis for point-mutation screening.

Advances in capillary electrophoresis technology over the past three years have been rapid. Capillary electrophoresis offers high-throughput, high-resolution, automatic operation and on-line detection with automatic data acquisition, and this has stimulated its application to the analysis of DNA mutations. Many different PCR-based DNA-mutation assays have been developed for unknown and known mutations. This article compares conventional PCR-based mutation-detection assays with the methods developed for use with capillary electrophoresis. Future trends for mutation detection using capillary electrophoresis are also assessed, with a special emphasis on totally integrated, microchip capillary-electrophoresis-based mutation-detection systems.

DNA↗
PubMed1997

Micromachined analytical devices: microchips for semen testing.

Micromachined devices (microchips) have been designed and tested for a range of clinically important assays. In this study we compare sperm motility determined using disposable glass microchips and a conventional Makler chamber. The 17 x 14 mm glass microchips contained three etched test structures each comprising either duplicate or quadruplicate analytical microchannels. Semen samples with sperm counts ranging from 21 to 78 million sperm per ml and forward progression scores of from 1+ to 3+ were evaluated and swimming times ranging from 360 s (3.3+ progression) to 770 s (1+,2 forward progression) observed in the microchips. Motility determined by the time taken for sperm to swim to the end of a microchannel (100 microns wide x 40 microns deep x 10 mm long) in the microchip correlated with forward progression of the sperm determined by the conventional Makler chamber method. This study demonstrates the feasibility of microchips for sperm motility testing and suggests that this technique would be applicable to the study of other types of motile cells.

Biotechnology↗
PubMed1997

Diagnosis of Duchenne/Becker muscular dystrophy and quantitative identification of carrier status by use of entangled solution capillary electrophoresis.

Use of capillary electrophoresis, a new and useful analytical tool, offers a variety of advantages for nucleic acid analyses, including rapid analysis, automation, high resolution, qualitative and quantitative results, and low consumption of both sample and reagents. We report the first example of the use of entangled solution capillary electrophoresis (ESCE) and laser-induced fluorescence detection (LIF) for separation-based diagnostics in the quantitative analysis of multiplex PCR products for determination of carrier status of Duchenne/ Becker muscular dystrophy (DMD/BMD). This approach greatly improved the speed, resolution, and sensitivity of information needed for the diagnosis of DMD/BMD compared with that from conventional diagnostic methods, and is of general utility for diagnosis of genetic diseases.

Electrophoresis, Capillary↗
PubMed1996

Synthesis and characterization of 4-iodophenylboronic acid: a new enhancer for the horseradish peroxidase-catalyzed chemiluminescent oxidation of luminol.

4-Iodophenylboronic acid has been synthesized and shown to be a potent enhancer of the chemiluminescent horseradish peroxidase (Type VI-A)-catalyzed oxidation of luminol and isoluminol. The enhancer was effective (>100-fold enhancement) in the concentration range 10-1500 microM. Light emission in the presence of the enhancer peaked at 5-10 min after initiation of the reaction and then decayed very slowly. 4-Iodophenylboronic acid also enhanced reactions catalyzed by horseradish peroxidase Type IX. The detection limit for Type VI-A horseradish peroxidase was 509 amol, and optimum signal enhancement was obtained at 769 microM compared to 154 microM for 4-iodophenol under the same conditions.

Blotting, Western↗
PubMed1996

Analysis of ligase chain reaction products amplified in a silicon-glass chip using capillary electrophoresis.

Ligase chain reaction (LCR) is a useful molecular technique for detecting known point mutations. We report the first example of the use of a disposable silicon-glass micro-chip for LCR and the first application of capillary electrophoresis (CE) to analyze samples amplified by LCR in a chip. Silicon-glass chips were manufactured using conventional photolithography and anodic bonding. The chips provide three distinct advantages for LCR: excellent thermal conductivity, a micro reaction volume ( < 10 microliters), and reproducible, low-cost manufacturing. Investigation and quantitation of amplification efficiency of LCR in a chip or in a tube requires an analytical technique that is faster and more convenient than the conventional slab gel methods. Slab gel electrophoresis uses relatively large amounts of sample and is labor-intensive and time-consuming, and thus is unsuitable for the separation and detection of LCR products. In contrast CE requires sample volume (original LCR products) of less than 1 microliter and is therefore well-suited to analysis of the micro-volume reaction mixture from chips. We combined CE with a sensitive laser induced fluorescence (LIF) detection system for the rapid separation and quantitative detection of LCR products amplified from the lacI gene in a silicon-glass chip. Comparative studies were made with LCR between tubes and silicon-glass chips. CE-LIF analysis is ideally suited to examination of micro-LCR amplification with high throughput. The technologies may find medical uses in disease diagnosis and research.

Base Sequence↗
PubMed1996

Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR.

The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify 'PCR friendly' surfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube.

Base Sequence↗
PubMed1996

Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips.

We examined PCR in silicon dioxide-coated silicon-glass chips (12 microl in volume with a surface to volume ratio of approximately 17.5 mm(2)/microl) using two PCR reagent systems: (i) the conventional reagent system using Taq DNA polymerase; (ii) the hot-start reagent system based on a mixture of TaqStart antibody and Taq DNA polymerase. Quantitative results obtained from capillary electrophoresis for the expected amplification products showed that amplification in microchips was reproducible (between batch coefficient of variation 7.71%) and provided excellent yields. We also used the chip for PCR directly from isolated intact human lymphocytes. The amplification results were comparable with those obtained using extracted human genomic DNA. This investigation is fundamental to the integration of sample preparation, polynucleotide amplification and amplicate detection on a microchip.

Antibodies↗
PubMed1996

Comparison of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione and luminol as co-substrates for detection of horseradish peroxidase in enhanced chemiluminescent reactions.

The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).

Boronic Acids↗