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

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

Fluorescence Imaging of Single-Molecule Retention Trajectories in Reversed-Phase Chromatographic Particles

Due to its high specific surface area and chemical stability, porous silica is used as a support structure in numerous applications, including heterogeneous catalysis, biomolecule immobilization, sensors, and liquid chromatography. Reversed-phase liquid chromatography (RPLC), which uses porous silica support particles, has become an indispensable separations tool in quality control, pharmaceutics, and environmental analysis requiring identification of compounds in mixtures. For complex samples, the need for higher resolution separations requires an understanding of the time scale of processes responsible for analyte retention in the stationary phase. In the present work, single-molecule fluorescence imaging is used to observe transport of individual molecules within RPLC porous silica particles. This technique allows direct measurement of intraparticle molecular residence times, intraparticle diffusion rates, and the spatial distribution of molecules within the particle. On the basis of the localization uncertainty and characteristic measured diffusion rates, statistical criteria were developed to resolve the frame-to-frame behavior of molecules into moving and stuck events. The measured diffusion coefficient of moving molecules was used in a Monte Carlo simulation of a random-walk model within the cylindrical geometry of the particle diameter and microscope depth-of-field. The simulated molecular transport is in good agreement with the experimental data, indicating transport of moving molecules in the porous particle is described by a random-walk. Histograms of stuck-molecule event times, locations, and their contributions to intraparticle residence times were also characterized

Fluorescence Imaging of Single-Molecule Retention Trajectories in Reversed-Phase Chromatographic Particles

Due to its high specific surface area and chemical stability, porous silica is used as a support structure in numerous applications, including heterogeneous catalysis, biomolecule immobilization, sensors, and liquid chromatography. Reversed-phase liquid chromatography (RPLC), which uses porous silica support particles, has become an indispensable separations tool in quality control, pharmaceutics, and environmental analysis requiring identification of compounds in mixtures. For complex samples, the need for higher resolution separations requires an understanding of the time scale of processes responsible for analyte retention in the stationary phase. In the present work, single-molecule fluorescence imaging is used to observe transport of individual molecules within RPLC porous silica particles. This technique allows direct measurement of intraparticle molecular residence times, intraparticle diffusion rates, and the spatial distribution of molecules within the particle. On the basis of the localization uncertainty and characteristic measured diffusion rates, statistical criteria were developed to resolve the frame-to-frame behavior of molecules into moving and stuck events. The measured diffusion coefficient of moving molecules was used in a Monte Carlo simulation of a random-walk model within the cylindrical geometry of the particle diameter and microscope depth-of-field. The simulated molecular transport is in good agreement with the experimental data, indicating transport of moving molecules in the porous particle is described by a random-walk. Histograms of stuck-molecule event times, locations, and their contributions to intraparticle residence times were also characterized

Single-Molecule Fluorescence Imaging of Interfacial DNA Hybridization Kinetics at Selective Capture Surfaces

Accurate knowledge of the kinetics of complementary oligonucleotide hybridization is integral to the design and understanding of DNA-based biosensors. In this work, single-molecule fluorescence imaging is applied to measuring rates of hybridization between fluorescently labeled target ssDNA and unlabeled probe ssDNA immobilized on glass surfaces. In the absence of probe site labeling, the capture surface must be highly selective to avoid the influence of nonspecific adsorption on the interpretation of single-molecule imaging results. This is accomplished by increasing the probe molecule site densities by a factor of ∼100 compared to optically resolvable sites so that nonspecific interactions compete with a much greater number of capture sites and by immobilizing sulfonate groups to passivate the surface between probe strands. The resulting substrates exhibit very low nonspecific adsorption, and the selectivity for binding a complementary target sequence exceeds that of a scrambled sequence by nearly 3 orders of magnitude. The population of immobilized DNA probe sites is quantified by counting individual DNA duplexes at low target concentrations, and those results are used to calibrate fluorescence intensities on the same sample at much higher target concentrations to measure a full binding isotherm. Dissociation rates are determined from interfacial residence times of individual DNA duplexes. Equilibrium and rate constants of hybridization, <i>K</i>_a = 38 (±1) μM^–1, <i>k</i>_on = 1.64 (±0.06) × 10⁶ M^–1 s^–1, and <i>k</i>_off = 4.3 (±0.1) × 10^–2 s^–1, were found not to change with surface density of immobilized probe DNA, indicating that hybridization events at neighboring probe sites are independent. To test the influence of probe-strand immobilization on hybridization, the kinetics of the probe target reaction at the surface were compared with the same reaction in free solution, and the equilibrium constants and dissociation and association rates were found to be nearly equivalent. The selectivity of these capture surfaces should facilitate sensitive investigations of DNA hybridization at the limit of counting molecules. Because the immobilized probe DNA on these surfaces is unlabeled, photobleaching of a probe label is not an issue, allowing capture substrates to be used for long periods of time or even reused in multiple experiments

Competitive Assays of Label-Free DNA Hybridization with Single-Molecule Fluorescence Imaging Detection

Single-molecule imaging of fluorescently labeled biomolecules is a powerful technique for measuring association interactions; however, care must be taken to ensure that the fluorescent labels do not influence the system being probed. Label-free techniques are needed to understand biomolecule interactions free from the influence of an attached label, but these techniques often lack sensitivity and specificity. To solve these challenges, we have developed a competitive assay that uses single-molecule detection to track the population of unlabeled target single-stranded DNA (ssDNA) hybridized with probe DNA immobilized at a glass interface by detecting individual duplexes with a fluorescently labeled “tracer” ssDNA. By labeling a small fraction (<0.2%) of target molecules, the “tracer” DNA tracks the available probe DNA sites without significant competition with the unlabeled target population. Single-molecule fluorescence imaging is a good read-out scheme for competitive assays, as it is sufficiently sensitive to detect tracer DNA on substrates with relatively low densities of probe DNA, ∼10^–3 of a monolayer, so that steric interactions do not hinder DNA hybridization. Competitive assays are used to measure the association constant of complementary strand DNA hybridization of 9- and 10-base pair targets, where the tracer assay predicts the same association constant as a traditional displacement competitive assay. This methodology was used to compare the <i>K</i>_a of hybridization for identical DNA strands differing only by the presence of a fluorescent label tethered to the 5′ end of the solution-phase target. The addition of the fluorescent label significantly stabilizes the DNA duplex by 3.6 kJmol^–1, adding more stability than an additional adenine-thymine base-pairing interaction, 2.7 kJmol^–1. This competitive tracer assay could be used to screen a number of labeled and unlabeled target DNA strands to measure the impact of fluorescent labeling on duplex stability. This single-molecule competitive hybridization scheme could be easily adapted into a sensitive assay, where competition between tracer and target oligonucleotides for probe sites could be used to measure concentrations of unlabeled DNA or RNA

Fluorescence Imaging of Single-Molecule Retention Trajectories in Reversed-Phase Chromatographic Particles

Due to its high specific surface area and chemical stability, porous silica is used as a support structure in numerous applications, including heterogeneous catalysis, biomolecule immobilization, sensors, and liquid chromatography. Reversed-phase liquid chromatography (RPLC), which uses porous silica support particles, has become an indispensable separations tool in quality control, pharmaceutics, and environmental analysis requiring identification of compounds in mixtures. For complex samples, the need for higher resolution separations requires an understanding of the time scale of processes responsible for analyte retention in the stationary phase. In the present work, single-molecule fluorescence imaging is used to observe transport of individual molecules within RPLC porous silica particles. This technique allows direct measurement of intraparticle molecular residence times, intraparticle diffusion rates, and the spatial distribution of molecules within the particle. On the basis of the localization uncertainty and characteristic measured diffusion rates, statistical criteria were developed to resolve the frame-to-frame behavior of molecules into moving and stuck events. The measured diffusion coefficient of moving molecules was used in a Monte Carlo simulation of a random-walk model within the cylindrical geometry of the particle diameter and microscope depth-of-field. The simulated molecular transport is in good agreement with the experimental data, indicating transport of moving molecules in the porous particle is described by a random-walk. Histograms of stuck-molecule event times, locations, and their contributions to intraparticle residence times were also characterized

Fluorescence Imaging of Single-Molecule Retention Trajectories in Reversed-Phase Chromatographic Particles

Due to its high specific surface area and chemical stability, porous silica is used as a support structure in numerous applications, including heterogeneous catalysis, biomolecule immobilization, sensors, and liquid chromatography. Reversed-phase liquid chromatography (RPLC), which uses porous silica support particles, has become an indispensable separations tool in quality control, pharmaceutics, and environmental analysis requiring identification of compounds in mixtures. For complex samples, the need for higher resolution separations requires an understanding of the time scale of processes responsible for analyte retention in the stationary phase. In the present work, single-molecule fluorescence imaging is used to observe transport of individual molecules within RPLC porous silica particles. This technique allows direct measurement of intraparticle molecular residence times, intraparticle diffusion rates, and the spatial distribution of molecules within the particle. On the basis of the localization uncertainty and characteristic measured diffusion rates, statistical criteria were developed to resolve the frame-to-frame behavior of molecules into moving and stuck events. The measured diffusion coefficient of moving molecules was used in a Monte Carlo simulation of a random-walk model within the cylindrical geometry of the particle diameter and microscope depth-of-field. The simulated molecular transport is in good agreement with the experimental data, indicating transport of moving molecules in the porous particle is described by a random-walk. Histograms of stuck-molecule event times, locations, and their contributions to intraparticle residence times were also characterized