Simultaneous Multipass Resistive-Pulse Sensing and Fluorescence Imaging of Liposomes.

Simultaneous multipass resistive-pulse sensing and fluorescence imaging have been used to correlate the size and fluorescence intensity of individual E. coli lipid liposomes composed of E. coli polar lipid extracts labeled with membrane-bound 3,3-dioctadecyloxacarbocyanine (DiO) fluorescent molecules. Here, a nanopipet serves as a waveguide to direct excitation light to the resistive-pulse sensing zone at the end of the nanopipet tip. Individual DiO-labeled liposomes (>50 nm radius) were multipassed back and forth through the orifices of glass nanopipets' 110-150 nm radius via potential switching to obtain subnanometer sizing precision, while recording the fluorescence intensity of the membrane-bound DiO molecules. Fluorescence was measured as a function of liposome radius and found to be approximately proportional to the total membrane surface area. The observed relationship between liposome size and fluorescence intensity suggests that multivesicle liposomes emit greater fluorescence compared to unilamellar liposomes, consistent with all lipid membranes of the multivesicle liposomes containing DiO. Fluorescent and nonfluorescent liposomes are readily distinguished from each other in the same solution using simultaneous multipass resistive-pulse sensing and fluorescence imaging. A fluorescence "dead zone" of ∼1 µm thickness just outside of the nanopipet orifice was observed during resistive-pulse sensing, resulting in "on/off" fluorescent behavior during liposome multipassing.

Bee Pollinator Mortality Due to Pesticide-Laden Particulate Matter from Beef Cattle Feedyards.

Wild and managed bees are critical for the stability of trophic webs, angiosperm reproduction, and agricultural productivity. Unfortunately, as many as 40% of crop pollinators are in a steep decline due to habitat loss and exposure to agrochemicals. Pyrethroids, neonicotinoids, and macrocyclic lactones are among the many agrochemicals toxic to pollinating insects that are used extensively in industrial beef cattle feeding operations throughout the world. Fugitive feedyard particulate matter (PM) transports agrochemicals into the surrounding environs. To determine the impact of agrochemical-laden feedyard particulate matter on bee pollinators, we conducted in situ experiments wherein honeybees and mason bees were placed downwind and upwind of feedyards (N = 40). Concurrent, colocated total suspended particulate matter samples contained multiple insecticides and parasiticides including pyrethroids, neonicotinoids, and macrocyclic lactones, in significantly higher concentrations downwind of feedyards (bifenthrin, 8.45 ± 4.92; permethrin, 1032.34 ± 740.76; clothianidin, 3.61 ± 1.48; imidacloprid, 73.32 ± 47.52; thiamethoxam, 5.81 ± 3.16; abamectin, 0.45 ± 0.29; ivermectin, 8.88 ± 5.06 ng/g). Honeybees and mason bees sited downwind of feedyards always experienced higher mortality than those correspondingly sited upwind, and male mason bees experienced significantly higher mortality compared to females when both were sited downwind. Bees occurring downwind of beef cattle feedyards for 1 h are 232-260% more likely to die than those occurring upwind. Thus, agrochemicals used on and emitted from beef cattle feedyards are significant threats to bee pollinators.

A novel laboratory method for simulating pollinator exposure to agrochemical-laden particulate matter.

Environmental transport and deposition of particulate matter (PM) associated with toxic chemicals has begun to receive attention as a source of risk to pollinators. For example, dust arising from manipulations of insecticide-treated seed has potential to exert toxic effects among non-target insects. Similarly, synthetic steroid growth promoters, antibiotics and multiple insecticides and parasiticides detected in fugitive beef cattle feedyard PM may also negatively impact pollinators since many of these chemicals have been detected on wildflowers and pollinators collected near beef cattle feedyards. Therefore, there is a need to assess risk to pollinators posed by deposition of agrochemical-laden PM, both in the field and the laboratory. Unfortunately, established laboratory methods for simulating PM exposure or toxicity associated with contaminated PM are few and highly situation-specific. Herein we describe development and use of a PM circulation system that can be employed to evaluate toxicity of agrochemical-contaminated PM in the laboratory under controlled conditions. Two model organisms (honeybees (Apis mellifera) and mason bees (Osmia lignaria)) were exposed to agrochemical-free PM in the circulator system, and post-exposure mortality was compared with controls. No significant differences in mortality between exposed and control bees were observed. Next, honeybees and mason bees were exposed to PM spiked with an insecticide known to exert toxic effects to pollinators (thiamethoxam). Bees experienced significantly higher mortality when exposed to thiamethoxam-laden PM at environmentally relevant concentrations as compared to bees exposed to agrochemical-free PM. These results confirm the validity of these methods for use in controlled laboratory PM toxicity tests and offer a source of positive and negative control groups for laboratory and field experiments examining exposure of pollinators to potentially toxic agrochemical-laden PM. This method facilitates generation of more realistic toxicity data than standard contact toxicity tests when pollinator exposure scenarios involve particulate-based agrochemicals or other toxic chemicals.

Agrochemical occurrence on colocated wildflowers and wild bees collected near beef cattle feed yards and row crops.

It is well established that agrochemicals can pose significant threats to native pollinators; however, relatively little is known about pollinator risks associated with agrochemicals that are used on beef cattle feed yards. Recently, feed yard-derived agrochemicals and those from row crop agriculture were quantified on wildflowers growing on the High Plains, USA. To better characterize pollinator risks on the High Plains, we collected colocated wildflowers and foraging bees across three field seasons for analytical determination of residual agrochemicals. Agrochemicals were detected and quantified on the majority of wildflowers (85%) and nearly half of bees (49%). Permethrin was the most frequently detected analyte on wildflowers (32%) and bees (17%). Flower hazard quotients and flower hazard indices were calculated to deterministically evaluate risk to foraging pollinators. Mean flower hazard quotients exceeded one for 5/16 analytes (31%), and flower hazard quotients calculated for 30% of wildflowers were greater than 50. Flower hazard quotients for clothianidin exceeded 400 for 14% of wildflowers, which portends conditions conducive to frequent bee mortalities. Flower hazard indices were greater on wildflowers from mid-July to mid-September as compared with wildflowers collected earlier in the summer, which coincides with row crop planting and increased prevalence of feed yard flies. Hazard quotients and hazard index values calculated from agrochemical residue data suggest that pollinators frequenting wildflowers near beef cattle feed yards and row crops on the High Plains are at risk from both individual sources, and more so when considered in combination. Integr Environ Assess Manag 2022;18:163-173. © 2021 SETAC.

Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics.

Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.

Use of nest bundles to monitor agrochemical exposure and effects among cavity nesting pollinators.

Cavity nesting bees are proficient and important pollinators that can augment or replace honey bee pollination services for some crops. Relatively little is known about specific pesticide concentrations present in cavity nesting insect reed matrices and associated potential risks to cavity nesting bees. Nesting substrates (Phragmites australis reeds in bundles) were deployed in an agriculturally intensive landscape to evaluate colonization and agrochemical exposure among cavity nesting pollinators over two consecutive field seasons. Composition of insect species colonizing reeds within nest bundles varied considerably; those placed near beef cattle feed yards were dominated by wasps (93% of the total number of individuals occupying reed nest bundles), whereas nest bundles deployed in cropland-dominated landscapes were colonized primarily by leaf cutter bees (71%). All nesting/brood matrices in reeds (mud, leaves, brood, pollen) contained agrochemicals. Mud used in brood chamber construction at feed yard sites contained 21 of 23 agrochemicals included in analysis and >70% of leaf substrate stored in reeds contained at least one agrochemical. Moxidectin was most frequently detected across all reed matrices from feed yard sites, and moxidectin concentrations in nonviable larvae were more than four times higher than those quantified in viable larvae. Agrochemical concentrations in leaf material and pollen were also quantified at levels that may have induced toxic effects among developing larvae. To our knowledge, this is the first study to characterize agrochemical concentrations in multiple reed matrices provisioned by cavity-nesting insects. Use of nest bundles revealed that cavity nesting pollinators in agriculturally intensive regions are exposed to agrochemicals during all life stages, at relatively high frequencies, and at potentially lethal concentrations. These results demonstrate the utility of nest bundles for characterizing risks to cavity nesting insects inhabiting agriculturally intensive regions.

Confocal Raman Microscopy Enables Label-Free, Quantitative, and Structurally Informative Detection of DNA Hybridization at Porous Silica Surfaces.

Characterization of DNA at solid/liquid interfaces remains a challenge because most surface-sensitive techniques are unable to provide quantitative insight into the base content, length, or structure. Surface-enhanced Raman scattering measurements of DNA hybridization on plasmonic-metal substrates have been used to overcome small Raman-scattering cross-sections; however, surface-enhanced Raman spectroscopy measurements are not generally quantitative due to the fall-off in the scattering signal with the decay of the electric field enhancement from the surface, which also limits the length of oligonucleotides that can be investigated. In this work, we introduce an experimental methodology in which confocal Raman microscopy is used to characterize hybridization reactions of ssDNA immobilized at the solid/liquid interface of porous silica particles. By focusing the femtoliter confocal probe volume within a single porous particle, signal enhancement arises from the ∼1500-times greater surface area detected compared to a planar substrate. Because the porous support is a purely dielectric material, the scattering signal is independent of the proximity of the oligonucleotide to the silica surface. With this technique, we characterize a 19-mer capture strand and determine its hybridization efficiency with 9-mer and 16-mer target sequences from the scattering of a structurally insensitive phosphate-stretching mode. Changes in polarizability and frequency of scattering from DNA bases were observed, which are consistent with Watson-Crick base pairing. Quantification of base content from their duplex scattering intensities allows us to discriminate between hybridization of two target strands of equivalent length but with different recognition sequences. A duplex having a single-nucleotide polymorphism could be distinguished from hybridization of a fully complementary strand based on differences in base content and duplex conformation.

Toxic responses of blue orchard mason bees (Osmia lignaria) following contact exposure to neonicotinoids, macrocyclic lactones, and pyrethroids.

Analysis of particulate matter originating from beef cattle feed yards on the High Plains of the United States has revealed occurrence of multiple pesticides believed to potentially impact non-Apis pollinators. Among these pesticides are those that are highly toxic to Apis mellifera (honey bees). However, little non-Apis bee species toxicity data exist; especially pertaining to beef cattle feed yard-derived pesticides. Therefore, we conducted a series of 96-h contact toxicity tests with blue orchard mason bees (Osmia lignaria) using three neonicotinoids, two pyrethroids, and two macrocyclic lactones. Neonicotinoids (thiamethoxam, imidacloprid, and clothianidin) were most toxic with LD50 values ranging from 2.88 to 26.35 ng/bee, respectively. Macrocyclic lactones (abamectin and ivermectin) were also highly toxic to O. lignaria with LD50 estimates of 5.51-32.86 ng/bee. Pyrethroids (permethrin and bifenthrin) were relatively less toxic with LD50 values greater than 33 ng/bee. Sensitivity ratios for each pesticide were calculated to relate O. lignaria LD50 values to existing honey bee toxicity data. All three neonicotinoids were more toxic to O. lignaria than A. mellifera, but pyrethroids and abamectin were relatively less toxic. Additionally, three of seven pesticides (43%) resulted in significantly different mass normalized LD50 values for male and female O. lignaria. These results indicate that non-Apis pollinators may be highly susceptible to pesticides originating from beef cattle feed yards, necessitating consideration of more stringent regulatory protections than those based on A. mellifera pesticide sensitivity.

Pesticides Used on Beef Cattle Feed Yards Are Aerially Transported into the Environment Via Particulate Matter.

Considering the recent discovery of veterinary pharmaceutical aerial transport from industrial cattle feeding operations via particulate matter, the objective of this study is to determine the extent to which insecticides are also transported into the environment by total suspended particulates emanating from beef cattle feed yards. Of 16 different pesticides quantified in particulate matter samples collected from beef cattle feed yards, permethrin was detected most frequently at >67% of particulate matter samples and at a mean concentration of 1211.7 ± 781.0 (SE) ng/m3. Imidacloprid was detected at a mean concentration of 62.8 ± 38.2 (SE) ng/m3 or equivalent to published concentrations in dust from treated seed planting activities. When insecticide concentrations observed in this study are projected to all United States of America feed yards, the resulting particulate matter (669,000 kg) could contain enough insecticides (active ingredient mass basis) to kill over a billion honeybees daily. Furthermore, a novel transport pathway for macrocyclic lactone entry into the environment was identified. These data raise concern that nontarget organisms may be exposed to potentially toxic levels of pesticides from beef cattle feed yards.

Probing the Mechanism of Structure-Switching Aptamer Assembly by Super-Resolution Localization of Individual DNA Molecules.

Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, l-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring the rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.

Exposure of Foraging Bees (Hymenoptera) to Neonicotinoids in the U.S. Southern High Plains.

Exposure to pesticides is a major threat to insect pollinators, potentially leading to negative effects that could compromise pollination services and biodiversity. The objectives of this study were to quantify neonicotinoid concentrations among different bee genera and to examine differences attributable to body size and surrounding land use. During the period of cotton planting (May-June), 282 wild bees were collected from habitat patches associated with cropland, grassland, and urban land cover and analyzed for three neonicotinoids (thiamethoxam, clothianidin, and imidacloprid). Twenty bees among eight genera contained one or more of the neonicotinoid compounds and detections occurred in all landscape types, yet with the most detections occurring in cropland-associated habitats. Apis Linnaeus (Hymenoptera: Apidae), Melissodes Latreille (Apidae), Perdita Smith (Andrenidae), and Lasioglossum Curtis (Halictidae) had multiple individuals with neonicotinoid detections. Two of the largest bees (Apis and Melissodes) had the greatest number of detections within genera, yet the relatively small-bodied genus Perdita had the three highest neonicotinoid concentrations reported. The number of detections within a genus and average generic body mass showed a marginally significant trend towards larger bees having a greater frequency of neonicotinoid detections. Overall, the relatively low percentage of detections across taxa suggests practices aimed at conserving grassland remnants in intensified agricultural regions could assist in mitigating exposure of wild bees to agrochemicals, while differences in bee traits and resource use could in part drive exposure. Further work is needed to address variable agrochemical exposures among pollinators, to support strategies for conservation and habitat restoration in affected landscapes.

Single-Molecule Kinetics Show DNA Pyrimidine Content Strongly Affects RNA:DNA and TNA:DNA Heteroduplex Dissociation Rates.

The heteroduplex hybridization thermodynamics of DNA with either RNA or TNA are greatly affected by DNA pyrimidine content, where increased DNA pyrimidine content leads to significantly increased duplex stability. Little is known, however, about the effect that purine or pyrimidine content has on the hybridization kinetics of these duplexes. In this work, single-molecule imaging is used to measure the hybridization kinetics of oligonucleotides having varying DNA pyrimidine content with complementary DNA, RNA, and TNA sequences. Results suggest that the change in duplex stability from DNA pyrimidine content (corresponding to purine content in the complementary TNA or RNA) is primarily due to changes in the dissociation rate, and not single-strand ordering or other structural changes that increase the association rate. Decreases in heteroduplex hybridization rates with pyrimidine content are similar for RNA and TNA, indicating that TNA behaves as a kinetic analogue for RNA.

Super-Resolution Imaging of Competitive Unlabeled DNA Hybridization Reveals the Influence of Fluorescent Labels on Duplex Formation and Dissociation Kinetics.

Single-molecule fluorescence imaging is a powerful method to measure reversible reaction kinetics, allowing one to monitor the bound state of individual probe molecules with fluorescently labeled targets. In the case of DNA hybridization, previous studies have shown that the presence of a fluorescent label on a target strand can exhibit significant influence on the stability of a DNA duplex that is formed. In this work, we have developed a super-resolution imaging method to measure the hybridization kinetics of unlabeled target DNA that compete with a fluorescently labeled tracer DNA strand to hybridize with an unlabeled probe DNA immobilized at a surface. The hybridization of an unlabeled DNA target cannot be detected directly, but its presence blocks the immobilized probe DNA, influencing the measured time intervals between labeled DNA hybridization events. We derive a model for competitive hybridization kinetics to extract the association and dissociation rate constants of the unlabeled species from the distribution of time intervals between hybridization events of the labeled tracer DNA at individual localized DNA probe sites. We use this methodology to determine the hybridization kinetics of a model 11-mer unlabeled target DNA strand and then determine how five different fluorescent labels attached to the same target DNA strand impact the hybridization kinetics. Compared to the unlabeled target, these labels can slow the association and dissociation rates by as much as a factor of 5. The super-resolution time-interval methodology provides a unique approach to determining fundamental (label-free) rates of DNA hybridization, revealing the significant influence of fluorescent labels on these kinetics. This measurement concept can be extended to studies of other reversible reaction systems, where kinetics of unlabeled species can be determined from their influence on the reaction of a labeled species with localized probe molecules on a surface.

Toxicity of Agrochemicals Among Larval Painted Lady Butterflies (Vanessa cardui).

In the Southern High Plains of the United States, beef cattle feed yards and row crop agriculture are predominant sources of agrochemical usage. Beef cattle feed yards use large quantities of veterinary pharmaceuticals to promote cattle growth and health, along with insecticides to control insect pests, whereas row crop-based agriculture relies on herbicides, fungicides, and insecticides to increase yields. Previous studies have documented the occurrence of agrochemicals beyond feed yard and row crop agriculture boundaries in uncultivated, marginal areas, raising concern that migratory pollinators and pollinators indigenous to the Southern High Plains frequenting these remaining habitat corridors may become exposed to toxic agrochemicals. Larvae of the painted lady butterfly (Vanessa cardui) were used to investigate the potential toxicity of agrochemicals used on feed yards and in row crop agriculture among pollinators. Moxidectin, an antiparasiticide used on beef cattle feed yards, was determined to be extremely toxic to V. cardui larvae, with a lethal dose at which 50% of larvae died of 2.1 ± 0.1 ng/g. Pyraclostrobin, clothianidin, and permethrin all delayed V. cardui development. However, moxidectin was the only chemical that produced significant toxic effects at environmentally relevant concentrations. These results indicate that agrochemicals originating from feed yards have the potential to adversely impact the development of pollinator larvae occurring in the Southern High Plains. Environ Toxicol Chem 2019;38:2629-2636. © 2019 SETAC.

Thermostability Trends of TNA:DNA Duplexes Reveal Strong Purine Dependence.

The development of high fidelity polymerases and streamlined synthesis of threose nucleic acid (TNA) triphosphates and phosphoramidites has made TNA accessible as a motif for generating nuclease-resistant high-affinity aptamers, antisense oligos, and synthetic genetic biopolymers. Little is known, however, about the thermostability trends of TNA:DNA duplexes. Here we investigate the thermostability of 14 TNA:DNA duplexes with the goal of elucidating the fundamental factors governing TNA:DNA duplex stability. We find that purine content in TNA significantly influences the stability and conformation of TNA:DNA duplexes. Low TNA purine content destabilizes duplexes, with Tm values often 5 °C lower than analogous DNA:DNA and RNA:DNA duplexes. By contrast, TNA:DNA duplexes having high TNA purine content display greater stability than DNA:DNA or RNA:DNA duplexes having the same sequences. High TNA purine content leads TNA:DNA duplexes to adopt conformations similar to RNA:RNA (A-form) configuration, whereas duplexes with low TNA purine content have conformations more similar to DNA:DNA (B-form) configuration. These insights provide a basis for understanding and predicting TNA:DNA duplex stability, which is anticipated to guide the practical use of TNA in biotechnology applications.

Single-Molecule Kinetic Investigation of Cocaine-Dependent Split-Aptamer Assembly.

Aptamers are short nucleic-acid biopolymers selected to have high affinity and specificity for protein or small-molecule target analytes. Aptamers can be engineered into split-aptamer biosensors comprising two nucleic acid strands that coassemble as they bind to a target, resulting in a large signal change from attached molecular probes (e.g., molecular beacons). The kinetics of split-aptamer assembly and their dependence on target recognition are largely unknown; knowledge of these kinetics could help in design and optimization of split-aptamer biosensors. In this work, we measure assembly kinetics of cocaine-dependent split-aptamer molecules using single-molecule fluorescence imaging. Assembly is monitored between a DNA strand tethered to a glass substrate and solutions containing the other strand tagged with a fluorescent label, with varying concentrations of the cocaine analyte. Dissociation rates are measured by tracking individual molecules and measuring their bound lifetimes. Dissociation-time distributions are biexponential, possibly indicating different folded states of the aptamer. The dissociation rate of only the longer-lived complex decreases with cocaine concentration, suggesting that cocaine stabilizes the long-lived aptamer complex. The variation in the slow dissociation rate with cocaine concentration is well described with an equilibrium-binding model, where the dissociation rate approaches a saturation limit consistent with the dissociation-equilibrium constant for cocaine-binding to the split aptamer. This single-molecule methodology provides a sensitive readout of cocaine-binding based on the dissociation kinetics of the split aptamer, allowing one to distinguish target-dependent aptamer assembly from background assembly. This methodology could be used to study other systems where target association affects the stability of aptamer duplexes.

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.

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 Ka 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.

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, K(a) = 38 (±1) µM(-1), k(on) = 1.64 (±0.06) × 10(6) M(-1) s(-1), and k(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.

Super-Resolution Imaging and Quantitative Analysis of Membrane Protein/Lipid Raft Clustering Mediated by Cell-Surface Self-Assembly of Hybrid Nanoconjugates.

Super-resolution imaging was used to quantify organizational changes in the plasma membrane after treatment with hybrid nanoconjugates. The nanoconjugates crosslinked CD20 on the surface of malignant B cells, thereby inducing apoptosis. Super-resolution images were analyzed by using pair-correlation analysis to determine cluster size and to count the average number of molecules in the clusters. The role of lipid rafts was investigated by pre-treating cells with a cholesterol chelator and actin destabilizer to prevent lipid raft formation. Lipid raft cluster size correlated with apoptosis induction after treatment with the nanoconjugates. Lipid raft clusters had radii of ∼ 200 nm in cells treated with the hybrid nanoconjugates. Super-resolution images provided precise molecule location coordinates that could be used to determine density of bound conjugates, cluster size, and number of molecules per cluster.