26 research outputs found
Fluorescence-Correlation Spectroscopy Study of Molecular Transport within Reversed-Phase Chromatographic Particles Compared to Planar Model Surfaces
Reversed-phase liquid chromatography
(RPLC) is a widely used technique
for molecular separations. Stationary-phase materials for RPLC generally
consist of porous silica-gel particles functionalized with <i>n</i>-alkane ligands. Understanding motions of molecules within
the interior of these particles is important for developing efficient
chromatographic materials and separations. To characterize these dynamics,
time-resolved spectroscopic methods (photobleach recovery, fluorescence
correlation, single-molecule imaging) have been adapted to measure
molecular diffusion rates, typically at <i>n</i>-alkane-modified
planar silica surfaces, which serve as models of chromatographic interfaces.
A question arising from these studies is how dynamics of molecules
on a planar surface relate to motions of molecules within the interior
of a porous chromatographic particle. In this paper, imaging-fluorescence-correlation
spectroscopy is used to measure diffusion rates of a fluorescent probe
molecule 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine
perchlorate (DiI) within authentic RPLC porous silica particles and
compared with its diffusion at a planar C<sub>18</sub>-modified surface.
The results show that surface diffusion on the planar C<sub>18</sub> substrate is much faster than the diffusion rate of the probe molecule
through a chromatographic particle. Surface diffusion within porous
particles, however, is governed by molecular trajectories along the
tortuous contours of the interior surface of the particles. By accounting
for the greater surface area that a molecule must explore to diffuse
macroscopic distances through the particle, the molecular-scale diffusion
rates on the two surfaces can be compared, and they are virtually
identical. These results provide support for the relevance of surface-diffusion
measurements made on planar model surfaces to the dynamic behavior
of molecules on the internal surfaces of porous chromatographic particles
Single-Molecule Fluorescence Imaging of DNA at a Potential-Controlled Interface
Many interfacial
chemical phenomena are governed in part by electrostatic
interactions between polyelectrolytes and charged surfaces; these
phenomena can influence the performance of biosensors, adsorption
of natural polyelectrolytes (humic substances) on soils, and production
of polyelectrolyte multilayer films. In order to understand electrostatic
interactions that govern these phenomena, we have investigated the
behavior of a model polyelectrolyte, 15 kbp fluorescently labeled
plasmid DNA, near a polarized indium tin oxide (ITO) electrode surface.
The interfacial population of DNA was monitored in situ by imaging
individual molecules through the transparent electrode using total-internal-reflection
fluorescence microscopy. At applied potentials of +0.8 V versus Ag/AgCl,
the DNA interfacial population near the ITO surface can be increased
by 2 orders of magnitude relative to bulk solution. The DNA molecules
attracted to the interface do not adsorb to ITO, but rather they remain
mobile with a diffusion coefficient comparable to free solution. Ionic
strength strongly influences the sensitivity of the interfacial population
to applied potential, where the increase in the interfacial population
over a +300 mV change in potential varies from 20% in 30 mM ionic
strength to over 25-fold in 300 μM electrolyte. The DNA accumulation
with applied potential was interpreted using a simple Boltzmann model
to predict average ion concentrations in the electrical double layer
and the fraction of interfacial detection volume that is influenced
by applied potential. A Gouy–Chapman model was also applied
to the data to account for the dependence of the ion population on
distance from the electrode surface, which indicates that the net
charge on DNA responsible for interactions with the polarized surface
is low, on the order of one excess electron. The results are consistent
with a small fraction of the DNA plasmid being resident in the double-layer
and with counterions screening much of the DNA excess charge
Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C<sub>18</sub>-Functionalized Chromatographic Particles
Measuring lipid-membrane
partitioning of small molecules is critical
to predicting bioavailability and investigating molecule–membrane
interactions. A stable model membrane for such studies has been developed
through assembly of a phospholipid monolayer on <i>n</i>-alkane-modified surfaces. These hybrid bilayers have recently been
generated within <i>n</i>-alkyl-chain (C<sub>18</sub>)-modified
porous silica and used in chromatographic retention studies of small
molecules. Despite their successful application, determining the structure
of hybrid bilayers within chromatographic silica is challenging because
they reside at buried interfaces within the porous structure. In this
work, we employ confocal Raman microscopy to investigate the formation
and temperature-dependent structure of hybrid–phospholipid
bilayers in C<sub>18</sub>-modified, porous-silica chromatographic
particles. Porous silica provides sufficient surface area within a
confocal probe volume centered in an individual particle to readily
measure, with Raman microscopy, the formation of an ordered hybrid
bilayer of 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphoÂcholine
(DMPC) with the surface C<sub>18</sub> chains. The DMPC surface density
was quantified from the relative Raman scattering intensities of C<sub>18</sub> and phospholipid acyl chains and found to be ∼40%
of a DMPC vesicle membrane. By monitoring Raman spectra acquired versus
temperature, the bilayer main phase transition was observed to be
broadened and shifted to higher temperature compared to a DMPC vesicle,
in agreement with differential scanning calorimetry (DSC) results.
Raman scattering of deuterated phospholipid was resolved from protonated
C<sub>18</sub> chain scattering, showing that the lipid acyl and C<sub>18</sub> chains melt simultaneously in a single phase transition.
The surface density of lipid in the hybrid bilayer, the ordering of
both C<sub>18</sub> and lipid acyl chains upon bilayer formation,
and decoupling of C<sub>18</sub> methylene C–H vibrations by
deuterated lipid acyl chains all suggest an interdigitated acyl chain
structure. The simultaneous melting of both layers is also consistent
with an interdigitated structure, where immobility of surface-grafted
C<sub>18</sub> chains decreases the cooperativity and increases the
melting temperature compared to a vesicle bilayer
Surface-Enhanced Raman Scattering Study of the Kinetics of Self-Assembly of Carboxylate-Terminated <i>n</i>-Alkanethiols on Silver
Adsorption of 11-mercaptoundecanoic acid (MUA) on silver
from methanol and aqueous solutions was monitored <i>in situ</i> by surface-enhanced Raman scattering (SRES) spectroscopy. While
adsorption of MUA from methanol is a one-step formation of a thiol-bound
monolayer, SERS spectra reveal that monolayer formation from aqueous
solution involves interactions of both carboxylate and thiol groups
of MUA with the silver surface. Several Raman scattering bands, including
the νÂ(C–S), ν<sub>s</sub>(COO<sup>–</sup>), and νÂ(C–C), were used to investigate the evolution
of the structure of adsorbed MUA on silver surfaces. The time-dependent
profiles of these bands for assembly from aqueous solution indicate
a multistep process, which is initiated by the binding of both carboxylate
and thiol groups to silver, producing a mixture of gauche and trans
conformations. In a subsequent step, the COO–Ag interactions
are displaced by stronger S–Ag bonds, leading to ordering of
the resulting monolayer with formation of a complete SAM with all-trans
conformations. The results also showed that the adsorption process
depended strongly on the solution pH and surface potential of the
metal. These factors can significantly affect the participation and
displacement of −COO<sup>–</sup> during self-assembly
of MUA from aqueous solution
Stable Immobilization of DNA to Silica Surfaces by Sequential Michael Addition Reactions Developed with Insights from Confocal Raman Microscopy
The
immobilization of DNA to surfaces is required for
numerous
biosensing applications related to the capture of target DNA sequences,
proteins, or small-molecule analytes from solution. For these applications
to be successful, the chemistry of DNA immobilization should be efficient,
reproducible, and stable and should allow the immobilized DNA to adopt
a secondary structure required for association with its respective
target molecule. To develop and characterize surface immobilization
chemistry to meet this challenge, it is invaluable to have a quantitative,
surface-sensitive method that can report the interfacial chemistry
at each step, while also being capable of determining the structure,
stability, and activity of the tethered DNA product. In this work,
we develop a method to immobilize DNA to silica, glass, or other oxide
surfaces by carrying out the reactions in porous silica particles.
Due to the high specific surface area of porous silica, the local
concentrations of surface-immobilized molecules within the particle
are sufficiently high that interfacial chemistry can be monitored
at each step of the process with confocal Raman microscopy, providing
a unique capability to assess the molecular composition, structure,
yield, and surface coverage of these reactions. We employ this methodology
to investigate the steps for immobilizing thiolated-DNA to thiol-modified
silica surfaces through sequential Michael addition reactions with
the cross-linker 1,4-phenylene-bismaleimide. A key advantage of employing
a phenyl-bismaleimide over a comparable alkyl coupling reagent is
the efficient conversion of the initial phenyl-thiosuccinimide to
a more stable succinamic acid thioether linkage. This transformation
was confirmed by in situ Raman spectroscopy measurements, and the
resulting succinamic acid thioether product exhibited greater than
95% retention of surface-immobilized DNA after 12 days at room temperature
in aqueous buffer. Confocal Raman microscopy was also used to assess
the conformational freedom of surface-immobilized DNA by comparing
the structure of a 23-mer DNA hairpin sequence under duplex-forming
and unfolding conditions. We find that the immobilized DNA hairpin
can undergo reversible intramolecular duplex formation based on the
changes in frequencies and intensities of the phosphate backbone and
base-specific vibrational modes that are informative of the hybridization
state of DNA
Confocal Raman Microscopy for pH-Gradient Preconcentration and Quantitative Analyte Detection in Optically Trapped Phospholipid Vesicles
The ability of a vesicle membrane
to preserve a pH gradient, while
allowing for diffusion of neutral molecules across the phospholipid
bilayer, can provide the isolation and preconcentration of ionizable
compounds within the vesicle interior. In this work, confocal Raman
microscopy is used to observe (<i>in situ</i>) the pH-gradient
preconcentration of compounds into individual optically trapped vesicles
that provide sub-femtoliter collectors for small-volume samples. The
concentration of analyte accumulated in the vesicle interior is determined
relative to a perchlorate-ion internal standard, preloaded into the
vesicle along with a high-concentration buffer. As a guide to the
experiments, a model for the transfer of analyte into the vesicle
based on acid–base equilibria is developed to predict the concentration
enrichment as a function of source-phase pH and analyte concentration.
To test the concept, the accumulation of benzyldimethylamine (BDMA)
was measured within individual 1 μm phospholipid vesicles having
a stable initial pH that is 7 units lower than the source phase. For
low analyte concentrations in the source phase (100 nM), a concentration
enrichment into the vesicle interior of (5.2 ± 0.4) × 10<sup>5</sup> was observed, in agreement with the model predictions. Detection
of BDMA from a 25 nM source-phase sample was demonstrated, a noteworthy
result for an unenhanced Raman scattering measurement. The developed
model accurately predicts the falloff of enrichment (and measurement
sensitivity) at higher analyte concentrations, where the transfer
of greater amounts of BDMA into the vesicle titrates the internal
buffer and decreases the pH gradient. The predictable calibration
response over 4 orders of magnitude in source-phase concentration
makes it suitable for quantitative analysis of ionizable compounds
from small-volume samples. The kinetics of analyte accumulation are
relatively fast (∼15 min) and are consistent with the rate
of transfer of a polar aromatic molecule across a gel-phase phospholipid
membrane
Single-Molecule Fluorescence Imaging of DNA at a Potential-Controlled Interface
Many interfacial
chemical phenomena are governed in part by electrostatic
interactions between polyelectrolytes and charged surfaces; these
phenomena can influence the performance of biosensors, adsorption
of natural polyelectrolytes (humic substances) on soils, and production
of polyelectrolyte multilayer films. In order to understand electrostatic
interactions that govern these phenomena, we have investigated the
behavior of a model polyelectrolyte, 15 kbp fluorescently labeled
plasmid DNA, near a polarized indium tin oxide (ITO) electrode surface.
The interfacial population of DNA was monitored in situ by imaging
individual molecules through the transparent electrode using total-internal-reflection
fluorescence microscopy. At applied potentials of +0.8 V versus Ag/AgCl,
the DNA interfacial population near the ITO surface can be increased
by 2 orders of magnitude relative to bulk solution. The DNA molecules
attracted to the interface do not adsorb to ITO, but rather they remain
mobile with a diffusion coefficient comparable to free solution. Ionic
strength strongly influences the sensitivity of the interfacial population
to applied potential, where the increase in the interfacial population
over a +300 mV change in potential varies from 20% in 30 mM ionic
strength to over 25-fold in 300 μM electrolyte. The DNA accumulation
with applied potential was interpreted using a simple Boltzmann model
to predict average ion concentrations in the electrical double layer
and the fraction of interfacial detection volume that is influenced
by applied potential. A Gouy–Chapman model was also applied
to the data to account for the dependence of the ion population on
distance from the electrode surface, which indicates that the net
charge on DNA responsible for interactions with the polarized surface
is low, on the order of one excess electron. The results are consistent
with a small fraction of the DNA plasmid being resident in the double-layer
and with counterions screening much of the DNA excess charge
Single-Molecule Fluorescence Imaging of DNA at a Potential-Controlled Interface
Many interfacial
chemical phenomena are governed in part by electrostatic
interactions between polyelectrolytes and charged surfaces; these
phenomena can influence the performance of biosensors, adsorption
of natural polyelectrolytes (humic substances) on soils, and production
of polyelectrolyte multilayer films. In order to understand electrostatic
interactions that govern these phenomena, we have investigated the
behavior of a model polyelectrolyte, 15 kbp fluorescently labeled
plasmid DNA, near a polarized indium tin oxide (ITO) electrode surface.
The interfacial population of DNA was monitored in situ by imaging
individual molecules through the transparent electrode using total-internal-reflection
fluorescence microscopy. At applied potentials of +0.8 V versus Ag/AgCl,
the DNA interfacial population near the ITO surface can be increased
by 2 orders of magnitude relative to bulk solution. The DNA molecules
attracted to the interface do not adsorb to ITO, but rather they remain
mobile with a diffusion coefficient comparable to free solution. Ionic
strength strongly influences the sensitivity of the interfacial population
to applied potential, where the increase in the interfacial population
over a +300 mV change in potential varies from 20% in 30 mM ionic
strength to over 25-fold in 300 μM electrolyte. The DNA accumulation
with applied potential was interpreted using a simple Boltzmann model
to predict average ion concentrations in the electrical double layer
and the fraction of interfacial detection volume that is influenced
by applied potential. A Gouy–Chapman model was also applied
to the data to account for the dependence of the ion population on
distance from the electrode surface, which indicates that the net
charge on DNA responsible for interactions with the polarized surface
is low, on the order of one excess electron. The results are consistent
with a small fraction of the DNA plasmid being resident in the double-layer
and with counterions screening much of the DNA excess charge
Identification of Individual Immobilized DNA Molecules by Their Hybridization Kinetics Using Single-Molecule Fluorescence Imaging
Single-molecule
fluorescence methods can count molecules without
calibration, measure kinetics at equilibrium, and observe rare events
that cannot be detected in an ensemble measurement. We employ total
internal reflection fluorescence microscopy to monitor hybridization
kinetics between individual spatially resolved target DNA molecules
immobilized at a glass interface and fluorescently labeled complementary
probe DNA in free solution. Using super-resolution imaging, immobilized
target DNA molecules are located with 36 nm precision, and their individual
duplex formation and dissociation kinetics with labeled DNA probe
strands are measured at site densities much greater than the diffraction
limit. The purpose of this study is to evaluate uncertainties in identifying
these individual target molecules based on their duplex dissociation
kinetics, which can be used to distinguish target molecule sequences
randomly immobilized in mixed-target samples. Hybridization kinetics
of individual target molecules are determined from maximum likelihood
estimation of their dissociation times determined from a sample of
hybridization events at each target molecule. The dissociation time
distributions thus estimated are sufficiently narrow to allow kinetic
discrimination of different target sequences. For example, a single-base
thymine-to-guanine substitution on immobilized strands produces a
2.5-fold difference in dissociation rates of complementary probes,
allowing for the identification of individual target DNA molecules
by their dissociation rates with 95% accuracy. This methodology represents
a step toward high-density single-molecule DNA microarray sensors
and a powerful tool to investigate the kinetics of hybridization at
surfaces at the molecular level, providing information that cannot
be acquired in ensemble measurements
Imaging Fluorescence-Correlation Spectroscopy for Measuring Fast Surface Diffusion at Liquid/Solid Interfaces
The
development of techniques to probe interfacial molecular transport
is important for understanding and optimizing surface-based analytical
methods including surface-enhanced spectroscopies, biological assays,
and chemical separations. Single-molecule-fluorescence imaging and
tracking has been used to measure lateral diffusion rates of fluorescent
molecules at surfaces, but the technique is limited to the study of
slower diffusion, where molecules must remain relatively stationary
during acquisition of an image in order to build up sufficient intensity
in a spot to detect and localize the molecule. Although faster time
resolution can be achieved by fluorescence-correlation spectroscopy
(FCS), where intensity fluctuations in a small spot are related to
the motions of molecules on the surface, long-lived adsorption events
arising from surface inhomogeneity can overwhelm the correlation measurement
and mask the surface diffusion of the moving population. Here, we
exploit a combination of these two techniques, imaging-FCS, for measurement
of fast interfacial transport at a model chromatographic surface.
This is accomplished by rapid imaging of the surface using an electron-multiplied-charged-coupled-device
(CCD) camera, while limiting the acquisition to a small area on the
camera to allow fast framing rates. The total intensity from the sampled
region is autocorrelated to determine surface diffusion rates of molecules
with millisecond time resolution. The technique allows electronic
control over the acquisition region, which can be used to avoid strong
adsorption sites and thus minimize their contribution to the measured
autocorrelation decay and to vary the acquisition area to resolve
surface diffusion from adsorption and desorption kinetics. As proof
of concept, imaging-FCS was used to measure surface diffusion rates,
interfacial populations, and adsorption–desorption rates of
1,1′-dioctaÂdecyl-3,3,3′3′-tetramethylÂindoÂcarboÂcyanine
(DiI) on planar C<sub>18</sub>- and C<sub>1</sub>-modified surfaces