Biomedical subjects

Richard A Mathies

Publications and source records attributed to Richard A Mathies.

At least 37 records · Page 2Linked to original sources

PubMed2005

An integrated microfluidic processor for single nucleotide polymorphism-based DNA computing.

An integrated microfluidic processor is developed that performs molecular computations using single nucleotide polymorphisms (SNPs) as binary bits. A complete population of fluorescein-labeled DNA "answers" is synthesized containing three distinct polymorphic bases; the identity of each base (A or T) is used to encode the value of a binary bit (TRUE or FALSE). Computation and readout occur by hybridization to complementary capture DNA oligonucleotides bound to magnetic beads in the microfluidic device. Beads are loaded into sixteen capture chambers in the processor and suspended in place by an external magnetic field. Integrated microfluidic valves and pumps circulate the input DNA population through the bead suspensions. In this example, a program consisting of a series of capture/rinse/release steps is executed and the DNA molecules remaining at the end of the computation provide the solution to a three-variable, four-clause Boolean satisfiability problem. The improved capture kinetics, transfer efficiency, and single-base specificity enabled by microfluidics make our processor well-suited for performing larger-scale DNA computations.

Biotinylation↗

PubMed2005

Femtosecond stimulated Raman study of excited-state evolution in bacteriorhodopsin.

Femtosecond time-resolved stimulated Raman spectroscopy (FSRS) is used to examine the photoisomerization dynamics in the excited state of bacteriorhodopsin. Near-IR stimulated emission is observed in the FSRS probe window that decays with a 400-600-fs time constant. Additionally, dispersive vibrational lines appear at the locations of the ground-state vibrational frequencies and decay with a 260-fs time constant. The dispersive line shapes are caused by a nonlinear effect we term Raman initiated by nonlinear emission (RINE) that generates vibrational coherence on the ground-state surface. Theoretical expressions for the RINE line shapes are developed and used to fit the spectral and temporal evolution of the spectra. The rapid 260-fs decay of the RINE peak intensity, compared to the slower evolution of the stimulated emission, indicates that the excited-state population moves in approximately 260 fs to a region on the potential energy surface where the RINE signal is attenuated. This loss of RINE signal is best explained by structural evolution of the excited-state population along multiple low-frequency modes that carry the molecule out of the harmonic photochemically inactive Franck-Condon region and into the photochemically active geometry.

Bacteriorhodopsins↗

PubMed2005

Development and evaluation of a microdevice for amino acid biomarker detection and analysis on Mars.

The Mars Organic Analyzer (MOA), a microfabricated capillary electrophoresis (CE) instrument for sensitive amino acid biomarker analysis, has been developed and evaluated. The microdevice consists of a four-wafer sandwich combining glass CE separation channels, microfabricated pneumatic membrane valves and pumps, and a nanoliter fluidic network. The portable MOA instrument integrates high voltage CE power supplies, pneumatic controls, and fluorescence detection optics necessary for field operation. The amino acid concentration sensitivities range from micromolar to 0.1 nM, corresponding to part-per-trillion sensitivity. The MOA was first used in the lab to analyze soil extracts from the Atacama Desert, Chile, detecting amino acids ranging from 10-600 parts per billion. Field tests of the MOA in the Panoche Valley, CA, successfully detected amino acids at 70 parts per trillion to 100 parts per billion in jarosite, a sulfate-rich mineral associated with liquid water that was recently detected on Mars. These results demonstrate the feasibility of using the MOA to perform sensitive in situ amino acid biomarker analysis on soil samples representative of a Mars-like environment.

Amino Acids↗

PubMed2005

Dependence of line shapes in femtosecond broadband stimulated Raman spectroscopy on pump-probe time delay.

The effect of the time delay between the picosecond Raman pump and the femtosecond Stokes probe pulse on the Raman gain line shape in femtosecond broadband stimulated Raman spectroscopy (FSRS) is presented. Experimental data are obtained for cyclohexane to investigate the dependence of the FSRS line shape on this time delay. Theoretical simulations of the line shapes as a function of the time delay using the coupled wave theory agree well with experimental data, recovering broad line shapes at positive time delays and narrower bands with small Raman loss side wings at negative time delays. The analysis yields the lower bounds of the vibrational dephasing times of 2.0 ps and 0.65 ps for the 802 and 1027 cm(-1) modes for cyclohexane, respectively. The theoretical description and simulation using the coupled wave theory are also consistent with the observed Raman gain intensity profile over time delay, reaching the maximum at a slightly negative time delay (approximately -1 ps), and show that the coupled wave theory is a good model for describing FSRS.

Journal Article↗

PubMed2005

High-throughput single-strand conformation polymorphism analysis on a microfabricated capillary array electrophoresis device.

A high-density 384-lane microfabricated capillary array electrophoresis device is evaluated for high-throughput single-strand conformation polymorphism (SSCP) analysis. A delayed back bias direct electrokinetic injection scheme is used to provide better than 10-bp resolution with an 8.0-cm effective separation length. Separation of a HaeIII digest of PhiX174 yielded theoretical plate numbers of 4.0 x 10(6). Using 5% PDMA containing 10% glycerol and 15% urea, 21 single-nucleotide polymorphisms (SNPs) from HFE, MYL2, MYL3, and MYH7 genes associated with hereditary hemochromatosis (HHC) and hereditary hypertrophic cardiomyopathy (HCM) are discriminated at two running temperatures (25 degrees C and 40 degrees C), providing 100% sensitivity. The data in this study demonstrate that the 384-lane microCAE device provides the resolution and detection sensitivity required for SSCP analysis, showing its potential for ultrahigh-throughput mutation detection.

Cardiomyopathies↗

PubMed2004

Sparsely cross-linked "nanogel" matrixes as fluid, mechanically stabilized polymer networks for high-throughput microchannel DNA sequencing.

We have developed sparsely cross-linked "nanogels", subcolloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel replaceable DNA sequencing matrixes for capillary and microchip electrophoresis. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and a low percentage (approximately 10(-4) mol %) of N,N-methylene bisacrylamide (Bis). Nanogels and nanogel networks were characterized by multiangle laser light scattering and rheometry, respectively, and tested for DNA sequencing in both capillaries and chips with four-color LIF detection. Typical nanogels have an average radius of approximately 230 nm, with approximately 75% of chains incorporating a Bis cross-linker. The properties and performance of nanogel matrixes are compared here to those of a linear polyacrylamide (LPA) network, matched for both polymer weight-average molar mass (M(w)) and the extent of interchain entanglements (c/c). At sequencing concentrations, the two matrixes have similar flow characteristics, important for capillary and microchip loading. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, substantially longer average read lengths are obtained under standard conditions with the nanogel matrix at a 98.5% accuracy of base-calling (for CE: 680 bases, an 18.7% improvement over LPA, with the best reads as long as 726 bases, compared to 568 bases for the LPA matrix). We further investigated the use of the nanogel matrixes in a high-throughput microfabricated DNA sequencing device consists of 96 separation channels densely fabricated on a 6-in. glass wafer. Again, preliminary DNA sequencing results show that the nanogel matrixes are capable of delivering significantly longer average read length, compared to an LPA matrix of comparable properties. Moreover, nanogel matrixes require 30% less polymer per unit volume than LPA. The addition of a small amount of low molar mass LPA or ultrahigh molar mass LPA to the optimized nanogel sequencing matrix further improves read length as well as the reproducibility of read length (RSD < 1.6%). This is the first report of a replaceable DNA sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrixes appropriately matched for molar mass and the extent of interchain entanglements. These results could have significant implications for the improvement of microchip-based DNA sequencing technology.

Acrylic Resins↗

PubMed2004

Resonance Raman analysis of the mechanism of energy storage and chromophore distortion in the primary visual photoproduct.

The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C(11)=C(12) hydrogen-out-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do not cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing approximately 40 degrees twists in the same direction about the C(11)=C(12) and C(12)-C(13) bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9- and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.

Animals↗

PubMed2004

Theory of femtosecond stimulated Raman spectroscopy.

Femtosecond broadband stimulated Raman spectroscopy (FSRS) is a new technique that produces high-resolution (time-resolved) vibrational spectra from either the ground or excited electronic states of molecules, free from background fluorescence. FSRS uses simultaneously a narrow bandwidth approximately 1-3 ps Raman pump pulse with a continuum approximately 30-50 fs Stokes probe pulse to produce sharp Raman gains, at positions corresponding to vibrational transitions in the sample, riding on top of the continuum Stokes probe spectrum. When FSRS is preceded by a femtosecond actinic pump pulse that initiates the photochemistry of interest, time-resolved Raman spectroscopy can be carried out. We present two theoretical approaches to FSRS: one is based on a coupling of Raman pump and probe light waves with the vibrations in the medium, and another is a quantum-mechanical description. The latter approach is used to discuss the conditions of applicability and limitations of the coupled-wave description. Extension of the quantum-mechanical description to the case where the Raman pump beam is on resonance with an excited electronic state, as well as when FSRS is used to probe a nonstationary vibrational wave packet prepared by an actinic pump pulse, is also discussed.

Journal Article↗

PubMed2004

Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis.

Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).

Amino Acid Sequence↗

PubMed2004

Femtosecond Time-Resolved Stimulated Raman Spectroscopy of the S(2) (1B(u)) Excited State of beta-Carotene.

The electronic and vibrational structure of beta-carotene's early excited states are examined using femtosecond time-resolved stimulated Raman spectroscopy. The vibrational spectrum of the short-lived ( approximately 160 fs) second excited singlet state (S(2),1B(u) (+))of beta-carotene is obtained. Broad, resonantly enhanced vibrational features are observed at approximately 1100, 1300, and 1650 cm(-1) that decay with a time constant corresponding to the electronic lifetime of S(2). The temporal evolution of the vibrational spectra are consistent with significant population of only two low-lying excited electronic states (1B(u) (+) and 2A(g) (-)) in the ultrafast relaxation pathway of beta-carotene.

Journal Article↗

PubMed2004

Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system.

Microfabricated capillary electrophoresis chips containing an integrated sheath-flow electrochemical detector are developed with the goal of minimizing the influence of separation voltages on end-column detection while maintaining optimum performance. The microdevice consists of an upper glass wafer carrying the etched separation, injection, and sheath-flow channels and a lower glass wafer on which gold- and silver-plated electrodes have been fabricated. The sheath-flow channels join the end of the separation channel from each side, and gravity-driven flow carries the analytes to the electrochemical detector placed at working distances of 100, 150, 200, and 250 microm from the separation channel exit. The performance of this detector is evaluated using catechol and a detection limit of 4.1 microM obtained at a working distance of 250 microm. Detection of DNA restriction fragments and PCR product sizing is demonstrated using the electroactive intercalating dye, iron phenanthroline. Additionally, an allele-specific, PCR-based single-nucleotide polymorphism typing assay for the C282Y substitution diagnostic for hereditary hemochromatosis is developed and evaluated using ferrocene-labeled primers. This study advances the feasibility of high-speed, high-throughput chemical and genetic analysis using microchip electrochemical detection.

Electrochemistry↗

PubMed2004

Resonance Raman spectra of electrons solvated in liquid alcohols.

Resonance Raman spectra of electrons solvated in liquid methanol, ethanol, and n-propanol are presented. At least five distinct solvent modes exhibit resonantly enhanced scattering, including the OH torsion, CO/CC stretches, the OH in-plane bend, methyl deformations, and the OH stretch. The 200-350 cm-1 frequency downshift of the OH stretch indicates a strong H-bond interaction between the electron and the hydroxyl group. The multiple modes including alkyl vibrations that are coupled to the electronic transition of the solvated electron reveal the extension of the electron's wavefunction into the alkyl solvent environment.

Journal Article↗

PubMed2004

Multichannel homogeneous immunoassay for detection of 2,4,6-trinitrotoluene (TNT) using a microfabricated capillary array electrophoresis chip.

A high-throughput homogeneous immunoassay for the sensitive detection of 2,4,6-trinitrotoluene (TNT) has been developed using radial capillary array electrophoresis microdevices. Samples consisting of equilibrium mixtures of anti-TNT antibody (Ab), fluorescein-labeled TNT, and various concentrations of unlabeled TNT were electrokinetically injected into 48 channels of a radial capillary array electrophoresis microchannel plate. The rapid electrophoretic separation allows us to analyze the equilibrium ratio formed by the competition between the labeled and the unlabeled TNT for Ab binding. The simultaneous parallel TNT separations facilitate determination of a calibration curve for the TNT assay, which has high sensitivity (LOD, 1 ng/mL) and a wide dynamic range (1-300 ng/mL).

Antibodies↗

PubMed2004

Time-resolved resonance raman structural studies of the pB' intermediate in the photocycle of photoactive yellow protein.

Time-resolved resonance Raman spectroscopy is used to obtain chromophore vibrational spectra of the pR, pB', and pB intermediates during the photocycle of photoactive yellow protein. In the pR spectrum, the C8-C9 stretching mode at 998 cm(-1) is approximately 60 cm(-1) lower than in the dark state, and the combination of C-O stretching and C7H=C8H bending at 1283 cm(-1) is insensitive to D2O substitution. These results indicate that pR has a deprotonated, cis chromophore structure and that the hydrogen bonding to the chromophore phenolate oxygen is preserved and strengthened in the early photoproduct. However, the intense C7H=C8H hydrogen out-of-plane (HOOP) mode at 979 cm(-1) suggests that the chromophore in pR is distorted at the vinyl and adjacent C8-C9 bonds. The formation of pB' involves chromophore protonation based on the protonation state marker at 1174 cm(-1) and on the sensitivity of the COH bending at 1148 cm(-1) as well as the combined C-OH stretching and C7H=C8H bending mode at 1252 cm(-1) to D2O substitution. The hydrogen out-of-plane Raman intensity at 985 cm(-1) significantly decreases in pB', suggesting that the pR-to-pB' transition is the stage where the stored photon energy is transferred from the distorted chromophore to the protein, producing a more relaxed pB' chromophore structure. The C=O stretching mode downshifts from 1660 to 1651 cm(-1) in the pB'-to-pB transition, indicating the reformation of a hydrogen bond to the carbonyl oxygen. Based on reported x-ray data, this suggests that the chromophore ring flips during the transition from pB' to pB. These results confirm the existence and importance of the pB' intermediate in photoactive yellow protein receptor activation.

Bacterial Proteins↗

PubMed2004

Femtosecond broadband stimulated Raman spectroscopy: Apparatus and methods.

The laser, detection system, and methods that enable femtosecond broadband stimulated Raman spectroscopy (FSRS) are presented in detail. FSRS is a unique tool for obtaining high time resolution (<100 fs) vibrational spectra with an instrument response limited frequency resolution of <10 cm(-1). A titanium:Sapphire-based laser system produces the three different pulses needed for FSRS: (1) A femtosecond visible actinic pump that initiates the photochemistry, (2) a narrow bandwidth picosecond Raman pump that provides the energy reservoir for amplification of the probe, and (3) a femtosecond continuum probe that is amplified at Raman resonances shifted from the Raman pump. The dependence of the stimulated Raman signal on experimental parameters is explored, demonstrating the expected exponential increase in Raman intensity with concentration, pathlength, and Raman pump power. Raman spectra collected under different electronic resonance conditions using highly fluorescent samples highlight the fluorescence rejection capabilities of FSRS. Data are also presented illustrating our ability: (i) To obtain spectra when there is a large transient absorption change by using a shifted excitation difference technique and (ii) to obtain high time resolution vibrational spectra of transient electronic states.

Journal Article↗

PubMed2003

Chiral separation of fluorescamine-labeled amino acids using microfabricated capillary electrophoresis devices for extraterrestrial exploration.

Chiral separations of fluorescamine-labeled amino acids are characterized and optimized on a microfabricated capillary electrophoresis (CE) device. A standard mixture of acidic and neutral amino acids is labeled with fluorescamine in less than 5 min and the hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentration, temperature, and pH are optimized (15 mM HPbetaCD, 6 degrees C, pH < 9) to achieve high-quality and low background chiral separations in less than 200 s. All four stereoisomers formed in the labeling reaction of the chiral dye with the chiral amino acids are typically resolved. At pH > 9, isomerization of the dye chiral center is observed that occurs on the time scale of the chip separation. Typical limits of detection are approximately 50 nM. These results demonstrate the feasibility of combining fluorescamine labeling of amino acids with microfabricated CE devices to develop low-volume, high-sensitivity apparatus and methods for extraterrestrial exploration.

Electrophoresis, Capillary↗

PubMed2003

Integrated hydrogenated amorphous Si photodiode detector for microfluidic bioanalytical devices.

Hydrogenated amorphous silicon (a-Si:H) PIN photodiodes have been developed and characterized as fluorescence detectors for microfluidic analysis devices. A discrete a-Si:H photodiode is first fabricated on a glass substrate and used to detect fluorescent dye standards using conventional confocal microscopy. In this format, the limit of detection for fluorescein flowing in a 50-microm deep channel is 680 pM (S/N = 3). A hybrid integrated detection system consisting of a half-ball lens, a ZnS/YF3 multilayer optical interference filter with a pinhole, and an annular a-Si:H photodiode is also developed that allows the laser excitation to pass up through the central aperture in the detector. Using this integrated detection device, the limit of detection for fluorescein is 17 nM, and DNA fragment sizing and chiral analysis of glutamic acid are successfully performed. The a-Si:H detector exhibits high sensitivity at the emission wavelengths of commonly used fluorescent dyes and is readily microfabricated and integrated at low cost making it ideal for portable microfluidic bioanalyzers and emerging large scale integrated microfluidic technologies.

Bacteriophage phi X 174↗

PubMed2003

Femtosecond Time-Resolved Stimulated Raman Spectroscopy: Application to the Ultrafast Internal Conversion in beta-Carotene.

We have developed the technique of femtosecond stimulated Raman spectroscopy (FSRS), which allows the rapid collection of high-resolution vibrational spectra on the femtosecond time scale. FSRS combines a sub-50 fs actinic pump pulse with a two-pulse stimulated Raman probe to obtain vibrational spectra whose frequency resolution limits are uncoupled from the time resolution. This allows the acquisition of spectra with <100 fs time resolution and <30 cm(-1) frequency resolution. Additionally, FSRS is unaffected by background fluorescence, provides rapid (100 ms) acquisition times, and exhibits traditional spontaneous Raman line shapes. FSRS is used here to study the relaxation dynamics of beta-carotene. Following optical excitation to S(2) (1B(u) (+)) the molecule relaxes in 160 fs to S(1) (2A(g) (-)) and then undergoes two distinct stages of intramolecular vibrational energy redistribution (IVR) with 200 and 450 fs time constants. These processes are attributed to rapid (200 fs) distribution of the internal conversion energy from the S(1) C=C modes into a restricted bath of anharmonically coupled modes followed by complete IVR in 450 fs. FSRS is a valuable new technique for studying the vibrational structure of chemical reaction intermediates and transition states.

Journal Article↗