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.

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.

We report excited-state lifetime modification of diffusing molecules by Al nanoapertures in the UV. Lifetime reductions of ∼3.5× have been observed for the high quantum yield laser dye p-terphenyl in a 60 nm diameter aperture. The lifetime reduction is smaller for the low quantum yield molecule tryptophan, for which a maximum reduction of ∼1.7 is observed. Lifetime reduction as a function of aperture size and native quantum yield is accurately predicted by simulation. Simulation further predicts greater net fluorescence enhancement for tryptophan compared to p-terphenyl, which is consistent with the expectation that low quantum yield emitters experience greater enhancement in the effective quantum yield. T here has been a recent surge of interest in UV plasmonics. 1−5 One of the motivating factors is accessing the electronic resonances of organic molecules, which lie in the UV part of the spectrum. Biomolecules such as peptides and proteins contain residues that absorb in the 220−280 nm range. 6,7 However, these aromatic residues have relatively low fluorescence quantum yields and molar extinction coefficients, 6,8 as do nucleic acids. 9 Achieving significant emission enhancement via plasmonic structures 10 could be a key enabling factor in the label-free detection of proteins 11 or DNA molecules. 12,13 Furthermore, there are numerous organic dye labels that absorb and fluoresce in the UV. 14 To date, there have been no reports of UV plasmonicenhanced fluorescence of freely diffusing molecules, nor has lifetime modification in the UV been reported. Arguably the most successful plasmonic nanostructure for analyzing freely diffusing molecules is the simple nanoaperture (of various shapes), which has been used extensively with visible fluorescence 15−20 as well as for the basis for novel label-free methods. 21,22 While several of these studies used Al nanoapertures, others adopted Au in order to realize greater fluorescence and local field enhancements in the visible. 23−25 However, conventional "plasmonic" metals such as Au suffer from the influence of interband transitions near the blue part of the spectrum. Therefore, studies to date of plasmonic structures in the UV have employed other metals, such as Al. 10,14,26−37 Aluminum has an interband transition near 800 nm with a Drude-like free-electron response from the visible to UV wavelengths. 38 Here, we use round Al nanoapertures to investigate UV fluorescence lifetime reduction of diffusing molecules, which is a first step toward more quantitative fluorescence analysis. We further show that the lifetime reduction depends on the native quantum yield of the molecule and is sensitive to the physical details of the nanoaperture, including undercutting of the nanoaperture into the substrate. ■ SIMULATION Fluorescence Model. A fluorescent molecule can be treated as a system of three energy levels: a singlet ground state S 0 , a first excited singlet state S 1 , and a first excited triplet state T 1 . The fluorescence count rate per molecule (CRM) in steady state is ...

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.

Fluorescence imaging and counting of single molecules adsorbed or bound to surfaces are being employed in a number of quantitative analysis applications. Reliable molecular counts with knowledge of counting uncertainties, both false-positive and false-negative probabilities, are critical to these applications. By counting stationary single molecules on a surface, spatial criteria may be applied to the image analysis to improve confidence in detection, which is especially critical when detecting single fluorescent labels. In this work, we describe a simple approach to incorporating spatial criteria for counting single molecules by using an intensity threshold to locate regions with multiple, adjacent intense pixels, where the size of these regions is guided by the point-spread function of the microscope. By requiring multiple, spatially correlated bright pixels, false-positive events resulting from random samples of background noise are minimized. The reliability of detection is established by quantitative knowledge of the distributions of background and signals. By measuring and modeling both the background and single-molecule intensity distributions, false-positive and false-negative detection probabilities are estimated for arbitrary threshold parameters by using combinatorial statistics. From this theory, detection parameters can be optimized to minimize false-positive and false-negative probabilities, which can be calculated explicitly. For detection of single rhodamine 6G molecules at a threshold set at 2.5 times the standard deviation above background, the false-negative probability was only 1.5%, determined from distributions of single-molecule intensities on well-populated surfaces, and the false-positive probability from background noise was 2.8 spots per 50 x 50 microm image. The false-positive events compare favorably with theoretical probabilities calculated using combinatorial statistical analysis and simulated false-positive events counted in images of random noise.