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 C₁₈-modified surface. The results show that surface diffusion on the planar C₁₈ 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 C₁₈-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 (C₁₈)-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 C₁₈-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-phospho­choline (DMPC) with the surface C₁₈ chains. The DMPC surface density was quantified from the relative Raman scattering intensities of C₁₈ 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 C₁₈ chain scattering, showing that the lipid acyl and C₁₈ chains melt simultaneously in a single phase transition. The surface density of lipid in the hybrid bilayer, the ordering of both C₁₈ and lipid acyl chains upon bilayer formation, and decoupling of C₁₈ 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 C₁₈ 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 ν­(C–S), ν_s(COO^–), and ν­(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^– 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) × 10⁵ 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′-diocta­decyl-3,3,3′3′-tetramethyl­indo­carbo­cyanine (DiI) on planar C₁₈- and C₁-modified surfaces