The Experts below are selected from a list of 17319 Experts worldwide ranked by ideXlab platform
A D Mirzabekov - One of the best experts on this subject based on the ideXlab platform.
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massive parallel analysis of the binding specificity of histone like protein hu to single and double stranded dna with generic oligodeoxyribonucleotide Microchips
Nucleic Acids Research, 2001Co-Authors: Alexander Krylov, O A Zasedateleva, Dmitry Prokopenko, Josette Rouviereyaniv, A D MirzabekovAbstract:A generic hexadeoxyribonucleotide Microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the Microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to Microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded Microchip oligonucleotides; (iii) the effect of HU binding on melting curves of Microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic Microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded Microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (Tm) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower Tm. The results demonstrate that generic Microchips could be an efficient approach in analysis of sequence specificity of proteins.
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protein Microchips use for immunoassay and enzymatic reactions
Analytical Biochemistry, 2000Co-Authors: Pavel Arenkov, Alexander Kukhtin, Anne Gemmell, Sergey Voloshchuk, Valentina Vladimirovna Chupeeva, A D MirzabekovAbstract:Abstract Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 × 100 × 20-μm gel pads of protein Microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the Microchip reaction chamber speeded up antigen–antibody interactions within the gel. Protein Microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein Microchip can be used several times in different immunoassays and enzymatic kinetic measurements.
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massive parallel analysis of dna hoechst 33258 binding specificity with a generic oligodeoxyribonucleotide Microchip
Nucleic Acids Research, 1999Co-Authors: Aleksei Drobyshev, A D Mirzabekov, A S Zasedatelev, Gennadiy YershovAbstract:A generic oligodeoxyribonucleotide Microchip was used to determine the sequence specificity of Hoechst 33258 binding to double-stranded DNA. The generic Microchip contained 4096 oxctadeoxynucleo-tides in which all possible 4(6)= 4096 hexadeoxy-nucleotide sequences are flanked on both the 3'- and 5'-ends with equimolar mixtures of four bases. The Microchip was manufactured by chemical immobilization of presynthesized 8mers within polyacrylamide gel pads. A selected set of immobilized 8mers was converted to double-stranded form by hybridization with a mixture of fluorescently labeled complementary 8mers. Massive parallel measurements of melting curves were carried out for the majority of 2080 6mer duplexes, in both the absence and presence of the Hoechst dye. The sequence-specific affinity for Hoechst 33258 was calculated as the increase in melting temperature caused by ligand binding. The dye exhibited specificity for A:T but not G:C base pairs. The affinity is low for two A:T base pairs, increases significantly for three, and reaches a plateau for four A:T base pairs. The relative ligand affinity for all trinucleotide and tetranucleotide sequences (A/T)(3)and (A/T)(4)was estimated. The free energy of dye binding to several duplexes was calculated from the equilibrium melting curves of the duplexes formed on the oligonucleotide Microchips. This method can be usedmore » as a general approach for massive screening of the sequence specificity of DNA-binding compounds.« less
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parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide Microchips
Nucleic Acids Research, 1998Co-Authors: Alexander Fotin, A D Mirzabekov, Aleksei Drobyshev, Dmitri Proudnikov, Alexander N PerovAbstract:A Microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the Microchips. The equilibrium melting curves for all Microchip duplexes were measured in real time in parallel for all Microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the Microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in Microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide Microchips.
Adam T Woolley - One of the best experts on this subject based on the ideXlab platform.
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phase changing sacrificial materials for interfacing microfluidics with ion permeable membranes to create on chip preconcentrators and electric field gradient focusing Microchips
Analytical Chemistry, 2006Co-Authors: Ryan T Kelly, Adam T WoolleyAbstract:We have developed a novel approach for interfacing ionically conductive membranes with microfluidic systems using phase-changing sacrificial layers. Imprinted microchannels in a polymer substrate are filled with a heated liquid that solidifies at room temperature, a monomer solution is placed over the protected channels and polymerized to form a rigid semipermeable copolymer, and then the protective layer is melted and removed, leaving an open microchannel interfaced with a polymer membrane. We have applied this method in miniaturizing electric field gradient focusing (EFGF) and carrying out on-chip protein preconcentration. A semipermeable copolymer in the EFGF Microchips fills a region of changing cross-sectional area, which allows a gradient in electric field to be established when an electrical potential is applied. Our technique provides Microchip EFGF devices that offer 3-fold improved resolution in protein focusing compared with capillary-based systems. In addition, these EFGF Microchips can separate peptide samples with resolution similar to what is obtained in capillary electrophoresis microdevices, and the micro-EFGF systems enrich analytes by a factor of >150. Finally, we have fabricated membrane-integrated microfluidic devices that can concentrate protein samples (R-phycoerythrin) over 10 000-fold to facilitate Microchip capillary electrophoresis. Interfacing microchannels with ion-permeable membranes has great potential to enhance Microchip analysis of biomolecules.
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phase changing sacrificial materials for interfacing microfluidics with ion permeable membranes to create on chip preconcentrators and electric field gradient focusing Microchips
Analytical Chemistry, 2006Co-Authors: Ryan T Kelly, Yi Li, Adam T WoolleyAbstract:We have developed a novel approach for interfacing ionically conductive membranes with microfluidic systems using phase-changing sacrificial layers. Imprinted microchannels in a polymer substrate are filled with a heated liquid that solidifies at room temperature, a monomer solution is placed over the protected channels and polymerized to form a rigid semipermeable copolymer, and then the protective layer is melted and removed, leaving an open microchannel interfaced with a polymer membrane. We have applied this method in miniaturizing electric field gradient focusing (EFGF) and carrying out on-chip protein preconcentration. A semipermeable copolymer in the EFGF Microchips fills a region of changing cross-sectional area, which allows a gradient in electric field to be established when an electrical potential is applied. Our technique provides Microchip EFGF devices that offer 3-fold improved resolution in protein focusing compared with capillary-based systems. In addition, these EFGF Microchips can separa...
James P. Landers - One of the best experts on this subject based on the ideXlab platform.
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fundamentals and practice for ultrasensitive laser induced fluorescence detection in microanalytical systems
Electrophoresis, 2004Co-Authors: Mitchell E Johnson, James P. LandersAbstract:Laser-induced fluorescence is an extremely sensitive method for detection in chemical separations. In addition, it is well-suited to detection in small volumes, and as such is widely used for capillary electrophoresis and Microchip-based separations. This review explores the detailed instrumental conditions required for sub-zeptomole, sub-picomolar detection limits. The key to achieving the best sensitivity is to use an excitation and emission volume that is matched to the separation system and that, simultaneously, will keep scattering and luminescence background to a minimum. We discuss how this is accomplished with confocal detection, 90 degrees on-capillary detection, and sheath-flow detection. It is shown that each of these methods have their advantages and disadvantages, but that all can be used to produce extremely sensitive detectors for capillary- or Microchip-based separations. Analysis of these capabilities allows prediction of the optimal means of achieving ultrasensitive detection on Microchips.
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hydroxyethylcellulose as an effective polymer network for dna analysis in uncoated glass Microchips optimization and application to mutation detection via heteroduplex analysis
Analytical Biochemistry, 2002Co-Authors: Huijun Tian, James P. LandersAbstract:Abstract The nature of the sieving matrix for DNA fragment separation is of immense importance in capillary and Microchip electrophoresis. The chemical nature of the surface of the capillary or microchannel wall is equally as important, particularly with DNA electrophoresis where a substantial electroosmotic flow (EOF) may be detrimental to the separation. Although DNA analysis has been carried out successfully in both coated and uncoated capillaries, analysis of unpurified polymerase chain reaction products has been carried out primarily with covalently coated surfaces, especially with Microchip electrophoresis. In this report, double-stranded (ds) DNA fragment analysis using hydroxyethylcellulose (HEC) buffered in 1 × Tris–borate–EDTA is demonstrated both in uncoated capillaries and in Microchips. EOF was suppressed 20% in the presence of 1.5% HEC, and the effectiveness of HEC as a polymer for dsDNA fragment analysis was dependent on the pH, with pH 8.6 being optimal. Using separation efficiency (number of theoretical plates) and resolution to gauge the effectiveness of a variety of polymers for the capillary separation of dsDNA fragments in the size range 60–587 bp, HEC was found to be comparable in performance to polydimethylacrylamide (PDMA), and superior to linear polyacrylamide and polyethylene oxide for DNA analysis. With respect to longevity and robust performance, HEC could be used effectively in an uncoated capillary for more than 40 runs and for more than 90 runs (without replenishing the polymer) in an uncoated Microchip. Application of the optimized HEC conditions is demonstrated through its ability to facilitate heteroduplex analysis.
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A method for UV-bonding in the fabrication of glass electrophoretic Microchips
Electrophoresis, 2001Co-Authors: Zhili Huang, Joshua C. Sanders, Celeste Dunsmor, Hossein Ahmadzadeh, James P. LandersAbstract:This paper presents an approach for the development of methodologies amenable to simple and inexpensive Microchip fabrication, potentially applicable to dissimilar materials bonding and chip integration. The method involves a UV-curable glue that can be used for glass Microchip fabrication bonding at room temperature. This involves nothing more than fabrication of glue "guide channels" into the Microchip architecture that upon exposure to the appropriate UV light source, bonds the etched plate and cover plate together. The Microchip performance was verified by capillary zone electrophoresis (CZE) of small fluorescent molecules with no microchannel surface modification carried out, as well as with a DNA fragment separation following surface modification. The performance of these UV-bonded electrophoretic Microchips indicates that this method may provide an alternative to high temperature bonding.
Ryan T Kelly - One of the best experts on this subject based on the ideXlab platform.
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phase changing sacrificial materials for interfacing microfluidics with ion permeable membranes to create on chip preconcentrators and electric field gradient focusing Microchips
Analytical Chemistry, 2006Co-Authors: Ryan T Kelly, Adam T WoolleyAbstract:We have developed a novel approach for interfacing ionically conductive membranes with microfluidic systems using phase-changing sacrificial layers. Imprinted microchannels in a polymer substrate are filled with a heated liquid that solidifies at room temperature, a monomer solution is placed over the protected channels and polymerized to form a rigid semipermeable copolymer, and then the protective layer is melted and removed, leaving an open microchannel interfaced with a polymer membrane. We have applied this method in miniaturizing electric field gradient focusing (EFGF) and carrying out on-chip protein preconcentration. A semipermeable copolymer in the EFGF Microchips fills a region of changing cross-sectional area, which allows a gradient in electric field to be established when an electrical potential is applied. Our technique provides Microchip EFGF devices that offer 3-fold improved resolution in protein focusing compared with capillary-based systems. In addition, these EFGF Microchips can separate peptide samples with resolution similar to what is obtained in capillary electrophoresis microdevices, and the micro-EFGF systems enrich analytes by a factor of >150. Finally, we have fabricated membrane-integrated microfluidic devices that can concentrate protein samples (R-phycoerythrin) over 10 000-fold to facilitate Microchip capillary electrophoresis. Interfacing microchannels with ion-permeable membranes has great potential to enhance Microchip analysis of biomolecules.
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phase changing sacrificial materials for interfacing microfluidics with ion permeable membranes to create on chip preconcentrators and electric field gradient focusing Microchips
Analytical Chemistry, 2006Co-Authors: Ryan T Kelly, Yi Li, Adam T WoolleyAbstract:We have developed a novel approach for interfacing ionically conductive membranes with microfluidic systems using phase-changing sacrificial layers. Imprinted microchannels in a polymer substrate are filled with a heated liquid that solidifies at room temperature, a monomer solution is placed over the protected channels and polymerized to form a rigid semipermeable copolymer, and then the protective layer is melted and removed, leaving an open microchannel interfaced with a polymer membrane. We have applied this method in miniaturizing electric field gradient focusing (EFGF) and carrying out on-chip protein preconcentration. A semipermeable copolymer in the EFGF Microchips fills a region of changing cross-sectional area, which allows a gradient in electric field to be established when an electrical potential is applied. Our technique provides Microchip EFGF devices that offer 3-fold improved resolution in protein focusing compared with capillary-based systems. In addition, these EFGF Microchips can separa...
Reidun Sirevag - One of the best experts on this subject based on the ideXlab platform.
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parallel nanoliter detection of cancer markers using polymer Microchips
Lab on a Chip, 2005Co-Authors: Anja Gulliksen, Lars Anders Solli, Frank Karlsen, Henrik Rogne, Eivind Hovig, Klaus Stefan Drese, Olaf Sorensen, Reidun SirevagAbstract:A general multipurpose Microchip technology platform for point-of-care diagnostics has been developed. Real-time nucleic acid sequence-based amplification (NASBA) for detection of artificial human papilloma virus (HPV) 16 sequences and SiHa cell line samples was successfully performed in cyclic olefin copolymer (COC) Microchips, incorporating supply channels and parallel reaction channels. Samples were distributed into 10 parallel reaction channels, and signals were simultaneously detected in 80 nl volumes. With a custom-made optical detection unit, the system reached a sensitivity limit of 10−6 µM for artificial HPV 16 sequences, and 20 cells µl−1 for the SiHa cell line. This is comparable to the detection limit of conventional readers, and clinical testing of biological samples in polymer Microchips using NASBA is therefore possible.
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real time nucleic acid sequence based amplification in nanoliter volumes
Analytical Chemistry, 2004Co-Authors: Anja Gulliksen, Lars Anders Solli, Frank Karlsen, Henrik Rogne, Eivind Hovig, Trine Nordstrom, Reidun SirevagAbstract:Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a Microchip with oligonucleotides at a concentration of 1.0 and 0.1 μM, in 10- and 50-nL reaction chambers, respectively. The Microchip was developed in a silicon−glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in Microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.
