Biomedical subjects

Mary J Wirth

Publications and source records attributed to Mary J Wirth.

12 recordsLinked to original sources

PubMed2007

Single-molecule probing of adsorption and diffusion on silica surfaces.

Single-molecule spectroscopy has emerged as a valuable tool in probing kinetics and dynamic equilibria in adsorption because advances in instrumentation and technology have enabled researchers to obtain high signal-to-noise ratios for common dyes at room temperature. Single-molecule spectroscopy was applied to the study of an important problem in chromatography: peak broadening and asymmetry in the chromatograms of pharmaceuticals, peptides, and proteins. Using DiI, a cationic dye that exhibits the same problematic chromatographic behavior, investigators showed that the adsorption sites that cause chromatographic problems are located at defects on the silica crystal surface.

Journal Article↗

PubMed2006

Probing topography and tailing for commercial stationary phases using AFM, FT-IR, and HPLC.

Atomic force microscopy was used to study surface characteristics of three chromatographic silica products: Agilent Zorbax SB300, Waters Symmetry 300, and Merck Chromolith. Each is modified with a monomeric C18 monolayer. Both topographic and adhesive force measurements were made for each product. Topographical images revealed that all three materials are as smooth as glass on the scale of 100 nm and below. Adhesive forces for all three materials were much lower and much more uniform than for chemically modified fused silica. FT-IR spectra for all three materials showed a low abundance of isolated silanols, thus explaining the low adhesion. Chromatograms of a cationic dye, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), ranging in concentration from 1 to 300 microM were obtained for each column. All three materials exhibited classic nonlinear tailing; the Zorbax exhibited fronting as well. Chromatographic simulations were performed for the Symmetry and Chromolith products to determine the number of strong adsorption sites. The AFM, FT-IR, and HPLC were all consistent in indicating that the Chromolith material had half as many strong adsorption sites as the Symmetry material. The Zorbax material exhibited a number of isolated silanols that was comparable to the other materials, yet its adhesive force suggested a less adsorptive material, and its chromatographic performance suggested a more adsorptive material. Its topography is discussed as a possible reason for its anomalous chromatographic behavior.

Chromatography, Gel↗

PubMed2006

High-speed electroseparations inside silica colloidal crystals.

Crystals made from 200 nm silica colloids are hardened and chemically modified with chlorodimethyloctadecylsilane for use in electrically driven, reversed-phase separations. A van Deemter plot reveals extremely narrow peak widths for the separation of a cationic hydrophobic dye, DiI, with both the A and C terms 10-fold smaller than those for a conventional HPLC column. Electrically driven separations are demonstrated to be achieved in less than 10 s for three dyes differing in hydrophobicity and also for three peptides differing in electrophoretic mobility. The results show that these media are promising for high-speed separations.

Colloids↗

PubMed2006

Frequency domain analysis for fluorescence recovery after photobleaching.

Fourier transformation is evaluated as a means of improving precision in the analysis of fluorescence-recovery-after-photobleaching (FRAP) data. Simulations of FRAP data of 2m points, where m is an integer, are Fourier transformed to obtain the frequency domain data. Analogous to frequency domain techniques in nanosecond spectroscopy, frequency domain analysis of FRAP data is shown to provide more precise results. For a single exponential decay acquired over a time window of five decay constants, frequency domain analysis increases the precision by six fold without requiring that any more data be acquired. For a double exponential decay with decay constants that differ by a factor of two and noise of 5% relative standard deviation, time domain analysis is unable to distinguish this from a single exponential decay (chi2=1.1), whereas frequency domain analysis reveals that it does not fit to a single exponential decay (chi2=2.5). For a double exponential decay with five-fold differing decay constants, improved precision is obtained in the frequency domain for both of the decay constants, as well as the fractional amount of each. In contrast to nanosecond spectroscopy, the FRAP analysis described here combines the higher precision of the frequency domain with the direct observation in the time domain to facilitate the assessment of artifacts.

Algorithms↗

PubMed2005

Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins.

The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-d-penicillamine, 5-d-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D(1) = 2.0(+/-1.2) x 10(-7) and D(2) = 1.2(+/-0.5) x 10(-6) cm(2)/s) and also the largest electrophoretic mobility for the transmembrane protein (3 x 10(-8) cm(2)/V.s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface.

Acrylic Resins↗

PubMed2005

Electromigration of single molecules of DNA in a crystalline array of 300-nm silica colloids.

The velocities and conformations of single DNA chains were probed as they electromigrated at varying electric field strength through a crystalline array of silica colloids. An optically transparent film consisting of 300-nm silica colloids was formed on glass as a transparent crystalline layer of 7 mum thickness with an effective pore size of 45 nm. The behaviors of individual lambda-DNA molecules (48,502 base pairs) electromigrating through this material were observed to be analogous to the behaviors of long DNA chains in electrophoresis gels, including chain extension, hooking of chains around the matrix, and hernia formation. The electrophoretic mobility of lambda-DNA in this dense, narrow-pore material is surprisingly high: 1.8 cm2/Vs at 10 V/cm, which is at least as high as for much wider-pore gels. Imaging of the single molecules revealed that higher field strength caused increased chain extension and increased mobility, which reached an apparent plateau just above 2.0 cm2/Vs at 200 V/cm. Pulsed, crossed electric fields of 200 V/cm at 120 degrees to one another were applied to the material. The DNA chains were observed by imaging to electromigrate in an orderly fashion, and the migration rate was found to be length-dependent. The results indicate that these thin, robust, self-assembling inorganic materials are interesting as possible alternatives to polymeric gels for higher speed electrophoresis.

Bacteriophage lambda↗

PubMed2004

pH dependence of tailing in reversed-phase chromatography of a cationic dye: measurement of the strong adsorption site surface density.

A question that has interested Dr. J.J. Kirkland is addressed: what is the nature of the silanols that cause tailing to persist at low pH in reversed-phase chromatography? Chromatograms for a cationic dye, 1,1'-didodecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI), were studied at varying pH using an Agilent SB-C8 column and 80% ACN/water for six DiI concentrations ranging from 0.9 to 316 microM. The chromatograms showed increased retention and tailing from pH 1 to 5, as expected. Simulations of the chromatograms agreed well with experiment for a bi-Langmuir isotherm with weak (C8) and strong (silica) adsorption sites. The simulation parameters revealed that the number of strong adsorption sites decreases by 40% from pH 1 to 5, which indicates that the silanols causing tailing are in the SiOH, not the SiO-, form. This seems paradoxical because tailing increases with increasing pH. The simulation parameters reveal that this increased tailing from pH 1 to 5 owes to doubling of the partition coefficient for DiI to the strong adsorption site, which more than compensates for the decreasing number of sites. We attribute this increased partition coefficient to increased long-range coulombic interactions with the increasingly abundant SiO- groups at higher pH, which boosts DiI's partition coefficient for both the C8 and SiOH sites. The picture thus emerges that for DiI, higher pH causes increased tailing because the SiO- groups exacerbate tailing that actually originates from adsorption to SiOH groups.

Adsorption↗

PubMed2004

Origin of enhanced chiral selectivity by acidic additives for a polysaccharide-based stationary phase.

The effects of ethanesulfonic acid (ESA) and n-butylamine as additives were studied for a wide variety of chiral compounds using the polysaccharide chiral stationary phase (CSP), Chiralpak AD. The mobile phase consisted of hexane-ethanol (90:10, v/v). The additives typically had small effects, with one exception: the acidic additive had an enormous effect on the chiral selectivity of amino acid esters. The improved chiral selectivity was largely due to the longer retention of the later eluting enantiomer. Retention behavior of amines indicated that the higher selectivity for amino acid esters owes to increased hydrogen-bonding donation by the amine group of the analyte. Computation establishes the feasibility of a planar complex between the analyte and the cliral stationary phase, involving a pair of complementary hydrogen-bonding groups on each species, enabled by protonation of the analyte. Retention behaviors for a range of structures point to steric hindrance as the third interaction to comprise the requisite three interactions in chiral recognition.

Acids↗

PubMed2004

Measurement and simulation of tailing zones of a cationic dye in analytical-scale reversed phase chromatography.

A quantitative physical description of tailing is reported here for analytical-scale reversed phase chromatography with Type B silica. Simulations of experimental chromatograms for a cationic dye, 1,1'-didodecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) were performed as a function of DiI concentration and flow rate, revealing nonlinear tailing due to a bi-Langmuir adsorption isotherm. The strong site comprises less than 0.1% of the residual silanols, the desorption rate constant of DiI from this type of site is (85 +/- 8 ms)(-1), and the free energy of the silanophilic interaction is 16 kJ/mol, indicating hydrogen bonding or another strong electrostatic interaction.

Carbocyanines↗

PubMed2004

Surface modification of the channels of poly(dimethylsiloxane) microfluidic chips with polyacrylamide for fast electrophoretic separations of proteins.

The electrophoresis of proteins was investigated using poly(dimethylsiloxane) (PDMS) microfluidic chips whose surfaces were modified with polyacrylamide through atom-transfer radical polymerization. PDMS microchips were made using a glass replica to mold channels 10 microm high and 30 microm wide, with a T-intersection. The surface modification of the channels involved surface oxidation, followed by the formation of a self-assembled monolayer of benzyl chloride initiators, and then atom-transfer radical polymerization to grow a thin layer of covalently bonded polyacrylamide. The channels filled spontaneously with aqueous buffer due to the hydrophilicity of the coating. The resistance to protein adsorption was studied by open-channel electrophoresis for bovine serum albumin labeled with fluorophor. A plate height of 30 microm, corresponding to an efficiency of 33 000 plates/m, was obtained for field strengths from 18 to 889 V/cm. The lack of dependence of plate height on field strength indicates that there is no detectable contribution to broadening from adsorption. A 2- to 3-fold larger plate height was obtained for electrophoresis in a 50-cm polyacrylamide-coated silica capillary, and the shape of the electropherogram indicated the efficiency is limited by a distribution of species. The commercial capillary exhibited both reversible and irreversible adsorption of protein, whereas the PDMS microchip exhibited neither. A separation of lysozyme and cytochrome c in 35 s was demonstrated for the PDMS microchip.

Acrylic Resins↗

PubMed2003

Fluorescence imaging of the desorption of dye from fused silica versus silica gel.

The desorption rate constants for a cationic dye from strong adsorption sites are compared for the same chromatographic interface but for two different substrates, fused silica and chromatographic silica gel. The dye is 1,1'-didodecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI). The interface consists of acetonitrile and a hydrocarbon monolayer (C8) covalently bound to the silica substrate. To measure slow desorption from fused silica, fluorescence imaging combined with correlation spectroscopy is used. To measure slow desorption from silica gel, fluorescence movies of silica gel particles are used. In both cases, the results show that there are two types of slow desorption processes on time scales exceeding 1 s. The desorption time from one type of site is within an experimental error of 7 s for both silica substrates. The adsorption kinetics for this type of site are slow, and the equilibrium population of DiI on these sites is comparable to that for DiI weakly adsorbed to the hydrocarbon monolayer. For the second type of site, for fused silica, the population of DiI is even higher than that of weakly adsorbed DiI, and the desorption time constant is approximately 2 min, although this is likely shortened by photobleaching. For silica gel, the relative population of DiI on this ultrastrong site is more than an order of magnitude lower, and the desorption time constant is 4.0 +/- 0.1 min. Both silica substrates thus show two types of sites whose time constants agree within experimental error, suggesting that the strong adsorption sites on fused silica are chemically the same as those on chromatographic silica gel.

Journal Article↗

PubMed2002

Single-molecule resolution and fluorescence imaging of mixed-mode sorption of a dye at the interface of C18 and acetonitrile/water.

Single-molecule imaging is used for the first time to study the cationic dye, 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI), at the chromatographic interface consisting of acetonitrile/water and a hydrocarbon monolayer (C18) covalently bound to silica. Autocorrelations of burst data agree with our previous single-molecule counting results, showing that most dye molecules are diffusing and that there is a rare specific adsorption site associated with a 0.07-s desorption time. These autocorrelations go further in detecting an even rarer specific adsorption event associated with a 2.6-s desorption time. The latter desorption time would contribute much more significantly to peak tailing in chromatography. In water, the populations of DiI at these two specific adsorption sites are shown to be 11% and 4%, respectively, for the weaker and stronger sites, relative to the diffusing population of DiI. In 60% acetonitrile/water, the relative populations of the specific adsorption sites are 11% and 17%, showing that acetonitrile enhances the population of the stronger specific adsorption site. Fluorescence movies of single and multiple molecules link the stronger specific adsorption sites to specific locations on the surface. The imaging makes rare observations frequent by pinpointing where the events occur spatially. This ability to observe rare events by imaging reveals the presence of a third type of specific adsorption site, for which DiI has a desorption time in excess of 20 s.

Journal Article↗