共查询到20条相似文献,搜索用时 15 毫秒
1.
A new biosensor platform for the detection of bacterial pathogens based on long-range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) is presented. The resonant excitation of LRSP modes provides an enhanced intensity of the electromagnetic field, which is directly translated to an increased strength of fluorescence signal measured upon the capture of target analyte at the sensor surface. LRSPs originate from a coupling of surface plasmons across a thin metallic film embedded in dielectrics with similar refractive indices. With respect to regular surface plasmon-enhanced fluorescence spectroscopy, the excitation of LRSPs offers the advantage of a larger enhancement of the evanescent field intensity and a micrometer probing depth that is comparable to the size of target bacterial pathogens. The potential of the developed sensor platform is demonstrated in an experiment in which the detection of E. coli O157:H7 was carried out using sandwich immunoassays. The limit of detection below 10 cfu mL(-1) and detection time of 40 min were achieved. 相似文献
2.
When properly implemented, fluorescence correlation spectroscopy (FCS) reveals numerous static and dynamic properties of molecules in solution. However, complications arise whenever the measurement scenario is complex. Specific limitations occur when the detection region does not match the ideal Gaussian geometry ubiquitously assumed by FCS theory, or when properties of multiple fluorescent species are assessed simultaneously. A simple binary solution of diffusers, where both mole fraction and diffusion constants are sought, can face interpretive difficulty. In order to better understand the limits of FCS, this study systematically explores the relationship between detection-volume distortion, diffusion constants, species mole fraction, and fitting methodology in analyses that utilize a two-component autocorrelation model. FCS measurements from solution mixtures of dye-labeled protein and free dye are compared to simulations, which predict the performance of FCS under a variety of experimental circumstances. The results reveal a range of conditions necessary for performing accurate measurements and describe experimental scenarios that should be avoided. The findings also provide guidelines for obtaining meaningful measurements when grossly distorted detection volumes are utilized and generally assess the latent information contained in FCS datasets. 相似文献
3.
Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for the study of molecular dynamics at the single-molecule level. Because photophysical dynamics often dramatically influence FCS measurements, the role of various photophysical processes in FCS measurements must be understood to accurately interpret FCS data. We describe the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the effects of excitation saturation on the size and shape of the two-photon fluorescence observation volume and derive a new analytical expression for fluorescence correlation functions that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers. 相似文献
4.
We developed a tunable (220-285-nm) UV and fixed 266-nm laser-induced fluorescence (LIF) system using a spectrometer and a cooled CCD imaging detector to measure the excitation-emission matrix spectra of various compounds in water, including quinine sulfate and plastic compound bisphenol-A. The LIF instrument was used for the fast, nonspecific determination of trace amounts of dissolved organic compounds present in natural water supplies and various brand name bottled distilled water and bottled drinking water. Plastic-related compounds that leached out of plastic utensils and containers were also detected with this instrument. The sensitivity of the system was approximately 1-2 orders of magnitude better than that for a commercial system. 相似文献
5.
In fluorescence correlation spectroscopy (FCS), an accurate evaluation of the probe volume is the basis of correct interpretation of experimental data and solution of an appropriate diffusion model. Poor fitting convergence has been a problem in the determination of the dimensional parameters, the beam radius, omega, and the distance along the optical axis of the probe volume, l. In this work, the instability of fitting during the calibration process is investigated by examining the chi(2) surfaces. We demonstrate that the minimum of chi(2) in the omega dimension is well defined for both converging and diverging data. The difficulty of fitting comes from the l dimension. The uncertainty in l could be significantly larger than that in omega, as determined by F-statistics. A modified calibration process is recommended based on examining two data treatment methods, combining several short data sets into a single long run and averaging the correlation functions of several short data sets. It is found that by using the mean of several converging correlation functions from short data sets instead of a long time correlation, more stable and consistent dimensional parameters are extracted to define the probe volume. 相似文献
6.
In this paper we present and demonstrate a technique for mapping fluid flow rates in microfluidic systems with sub-micrometer resolution using confocal microscopy in conjunction with fluorescence correlation spectroscopy (FCS). Flow velocities ranging from approximately 50 microm/s to approximately 10 cm/s can be recorded using fluorescent polymer nanospheres as fluid motion tracers. Velocity profiles and images of the flow in poly(dimethylsiloxane)-glass microchannels are presented and analyzed. Using the method, velocity images along the horizontal (top view) and vertical planes within a microdevice can be obtained. This is, to our knowledge, the first report of FCS for producing velocity maps. The high-resolution velocity maps can be used to characterize and optimize microdevice performance and to validate simulation efforts. 相似文献
7.
Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion coefficients in an open detection volume. However, in several practical cases, when FCS measurements are carried out in small compartments like microchannels, neglecting boundary effects could lead to erroneous results. Here, a close form solution is proposed to explicitly account for the presence of walls located at a distance comparable with the characteristic detection volume lengths. We derive a one-dimensional diffusion constrained model and then generalize the solution to the two- and the three-dimensional constrained cases. We further indicate within which limits the standard autocorrelation function (ACF) model gives reliable results in microconfinement. Our model relies just on the assumption of elastic hits at the system walls and succeeds in describing the ACF of fluorescent probes confined along one direction. Through the analysis of FCS experimental data, we are able to predict the correct shape of the ACF in channels of micrometric and submicrometric width and measure the extent of lateral confinement. In addition, it permits the investigation of microstructured material features such as cages and cavities having dimensions on the micrometric range. On the basis of the proposed model, we also show in which conditions confinement could generate an apparent time dependent probe mobility, thus allowing a proper interpretation of the transport process taking place in submicrometric compartments. 相似文献
8.
Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam. 相似文献
9.
Determination of nitrite in waters by microplate fluorescence spectroscopy and HPLC with fluorescence detection. 总被引:4,自引:0,他引:4
A selective and versatile fluorescence spectroscopic method for the determination of nitrite in waters has been developed. Nitrite reacts in the presence of mineral acids with the nonfluorescent N-methyl-4-hydrazino-7-nitrobenzofurazan forming N-methyl-4-amino-7-nitrobenzofurazan, which can be detected by fluorescence spectroscopy with an excitation maximum at lambda = 468 nm and an emission maximum at lambda = 537 nm in acetonitrile. Three new methods based on this reaction have been developed: Direct fluorescence spectroscopy, HPLC/fluorescence, or HPLC with UV/vis detector may be selected as detection techniques. On microplates, high-throughput fluorescence spectroscopy is achieved, while HPLC/fluorescence provides lower limits of detection, and HPLC with UV/vis detection enables evaluation of the reaction with standard instrumentation. Different water samples were investigated using all detection modes, and a photometric standard procedure was successfully employed to validate the new methods with an independent technique. 相似文献
10.
Afterpulsing arises from feedback in a photon detector. This means that each real signal pulse can be followed by an afterpulse at a later time. This effect is particularly troubling in photon correlation experiments. Few treatments of this effect have appeared in the literature, and few software programs to solve the problem have been written. We demonstrate the afterpulsing effect in fluorescence correlation spectroscopy by using different avalanche photodiodes. We prove theoretically that under simple and reasonable conditions afterpulsing in autocorrelation can be eliminated to the leading order; we have found it easy to program software for the correction. We compare our results with those from cross correlation. We also discuss some experimental parameters that may affect the afterpulsing. 相似文献
11.
We explore the combination of a latex microsphere with a low NA lens to form a high performance optical system, and enable the detection of single molecules by fluorescence correlation spectroscopy (FCS). Viable FCS experiments at concentrations 1-1000 nM with different objectives costing less than $40 are demonstrated. This offers a simple and low-cost alternative to the conventional complex microscope objectives. 相似文献
12.
In this report, a combined imaging and fluorescence correlation spectroscopy (FCS) method is described and its ability to characterize microsecond fluctuations in the fluorescence emission of a sample is demonstrated. A sample scanning laser confocal microscope is operated in the customary way while recording the time that each photon is detected with a time resolution of 50 ns using a low-cost counting board. The serial data stream of photon detection times allows access to fluorescence signal fluctuations that can be used to characterize dynamics using correlation methods. The same data stream is used to generate images of the sample. Using the technique, we demonstrate that it is possible to characterize the kinetics of transitions to and from nonemitting or "dark" states of the fluorescent dyes DiIC16 and ATTO 520. Results are similar to, but deviate slightly from, a model that has been frequently used for extracting singlet-triplet: conversion rates using conventional solution-based FCS. Like conventional FCS, the concentration, or in our case the areal density of coverage, of fluorescent species can also be obtained. Many single-molecule fluorescence experiments aim to extract kinetics from intensity trajectories; this method may be used as a rapid and convenient technique for characterization of surface-linked or thin-film samples prior to performing the more time and effort intensive single-molecule measurements. Besides the capacity to measure photophysical phenomena, the surface-sensitive FCS method could also be applied for measuring conformational changes or interaction kinetics for species immobilized on a surface. One possible scenario is measurements of the frequency and duration of association of ligand-receptor pairs where a fluorescently labeled component is freely diffusing and the other is surface immobilized. Given that microarrays of custom-designed, surface-immobilized peptides and nucleic acids are now readily available, the ability to sensitively measure association and dissociation rates of the surface-linked species with a freely diffusing species could be a useful extension to what has already become an extremely important tool for characterizing the interactions of biomolecules. 相似文献
13.
In fluorescence correlation spectroscopy (FCS) analysis it is generally assumed that molecular species diffuse freely in volumes much larger than the three-dimensional FCS observation volume. However, this standard assumption is not valid in many measurement conditions, particularly in tubular structures with diameters in the micrometer range, such as those found in living cells (organelles, dendrites) and microfluidic devices (capillaries, reaction chambers). As a result the measured autocorrelation functions (ACFs) deviate from those predicted for free diffusion, and this can shift the measured diffusion coefficient by as much as ~50% when the tube diameter is comparable with the axial extension of the FCS observation volume. We show that the range of validity of the FCS measurements can be drastically improved if the tubular structures are located in the close vicinity of a mirror on which FCS is performed. In this case a new fluctuation time in the ACF, arising from the diffusion of fluorescent probes in optical fringes, permits measurement of the real diffusion coefficient within the tubular structure without assumptions about either the confined geometry or the FCS observation volume geometry. We show that such a measurement can be done when the tubular structure contains at least one pair of dark and bright fringes resulting from interference between the incoming and the reflected excitation beams on the mirror surface. Measurement of the diffusion coefficient of the enhanced green fluorescent protein (EGFP) and IscS-EGFP in the cytoplasm of living Escherichia coli illustrates the capabilities of the technique. 相似文献
14.
Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs. 相似文献
15.
Mn2+-doped ZnS nanocrystals with an amine-capping layer have been synthesized and used for the fluorescence detection of ultratrace 2,4,6-trinitrotoluene (TNT) by quenching the strong orange Mn2+ photoluminescence. The organic amine-capped nanocrystals can bind TNT species from solution and atmosphere by the acid-base pairing interaction between electron-rich amino ligands and electron-deficient aromatic rings. The resultant TNT anions bound onto the amino monolayer can efficiently quench the Mn2+ photoluminescence through the electron transfer from the conductive band of ZnS to the lowest unoccupied molecular orbital (LUMO) of TNT anions. The amino ligands provide an amplified response to the binding events of nitroaromatic compounds by the 2- to approximately 5-fold increase in quenching constants. Moreover, a large difference in quenching efficiency was observed for different types of nitroaromatic analytes, dependent on the affinity of nitro analytes to the amino monolayer and their electron-accepting abilities. The amine-capped nanocrystals can sensitively detect down to 1 nM TNT in solution or several parts-per-billion of TNT vapor in atmosphere. The ion-doped nanocrystal sensors reported here show a remarkable air/solution stability, high quantum yield, and strong analyte affinity and, therefore, are well-suited for detecting the ultratrace TNT and distinguishing different nitro compounds. 相似文献
16.
A gas sensor based on the gas-correlation technique has been developed using a multimode diode laser (MDL) in a dual-beam detection scheme. Measurement of CO(2) mixed with CO as an interfering gas is successfully demonstrated using a 1570 nm tunable MDL. Despite overlapping absorption spectra and occasional mode hops, the interfering signals can be effectively excluded by a statistical procedure including correlation analysis and outlier identification. The gas concentration is retrieved from several pair-correlated signals by a linear-regression scheme, yielding a reliable and accurate measurement. This demonstrates the utility of the unsophisticated MDLs as novel light sources for gas detection applications. 相似文献
17.
Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis. 相似文献
18.
X-ray fluorescence spectroscopy is demonstrated here as a novel, element-specific detector for capillary electrophoresis. Monochromatic 10 keV X-rays from a synchrotron light source are used to excite core electrons, causing emission of characteristic Kalpha X-ray fluorescence (XRF) lines. Using this technique, XRF energies provide elemental identification, while XRF intensities can be used to quantitate the metal composition of each eluent. An X-ray transparent polymer coupling is used to create a window for the on-line, X-ray detection. This coupling contributes no measurable extra-column variance, and electrophoretic mobilities for the metal complexes used as model solutes are highly reproducible. The combination of XRF detection with capillary electrophoresis (CE-XRF) creates the first on-line detection system that is element-specific, nondestructive, and directly applicable to a broad range of applications including nonelectroactive species. CE-XRF is successfully demonstrated here for high binding-constant complexes of Fe(III), Co(II), Cu(II), and Zn(II). Within a single injection, electropherograms are obtained for each element of interest, with the element identity obtained directly from the emission energy. In contrast with ICPMS, this detection technique is directly on-line and does not require volatilization of the eluent. As a result, element-specific detection is not limited by the sample or the buffer volatility or atomization efficiency. Simultaneous XRF and UV absorbance detection can be used to provide an on-line determination of metal/chelate ratios. Although XRF detection limits are presently only in the 0.1 mM (0.5 ng) range, both collection geometry and incident intensity have yet to be optimized. Further optimization is expected to enhance this detection limit by another 2-3 orders of magnitude. As a result, the advent of XRF detection combined with the separating power of CE presents new possibilities for on-line, element-specific analysis. 相似文献
19.
Correlation coefficient mapping has been applied to intrinsic fluorescence spectra of colonic tissue for the purpose of cancer diagnosis. Fluorescence emission spectra were collected of 57 colonic tissue sites in a range of 4 physiological conditions: normal (29), hyperplastic (2), adenomatous (5), and cancerous tissues (21). The sample-sample correlation was used to examine the ability of correlation coefficient mapping to determine tissue disease state. The correlation coefficient map indicates two main categories of samples. These categories were found to relate to disease states of the tissue. Sensitivity, selectivity, predictive value positive, and predictive value negative for differentiation between normal tissue and all other categories were all above 92%. This was found to be similar to, or higher than, tissue classification using existing methods of data reduction. Wavelength-wavelength correlation among the samples highlights areas of importance for tissue classification. The two-dimensional correlation map reveals absorption by NADH and hemoglobin in the samples as negative correlation, an effect not obvious from the one-dimensional fluorescence spectra alone. The integrity of tissue was examined in a time series of spectra of a single tissue sample taken after tissue resection. The wavelength-wavelength correlation coefficient map shows the areas of significance for each fluorophore and their relation to each other. NADH displays negative correlation to collagen and FAD, from the absorption of emission or fluorescence resonance energy transfer. The wavelength-wavelength correlation map for the decay set also clearly shows that there are only three fluorophores of importance in the samples, by the well-defined pattern of the map. The sample-sample correlation coefficient map reveals the changes over time and their impact on tissue classification. Correlation coefficient mapping proves to be an effective method for sample classification and cancer detection. 相似文献
20.
Culbertson MJ Williams JT Cheng WW Stults DA Wiebracht ER Kasianowicz JJ Burden DL 《Analytical chemistry》2007,79(11):4031-4039
The suitability of mathematical models used to extract kinetic information from correlated data constitutes a significant issue in fluorescence correlation spectroscopy (FCS). Standard FCS equations are derived from a simple Gaussian approximation of the optical detection volume, but some investigations have suggested this traditional practice can lead to inaccurate and misleading conclusions under many experimental circumstances, particularly those encountered in one-photon confocal measurements. Furthermore, analytical models cannot be derived for all measurement scenarios. We describe a novel numerical approach to FCS that circumvents conventional analytical models, enabling meaningful analyses even under extraordinarily unusual measurement conditions. Numerical fluorescence correlation spectroscopy (NFCS) involves quantitatively matching experimental correlation curves with synthetic curves generated via diffusion simulation or direct calculation based on an experimentally determined 3D map of the detection volume. Model parameters are adjusted iteratively to minimize the residual differences between synthetic and experimental correlation curves. In order to reduce analysis time, we distribute calculations across a network of processors. As an example of this new approach, we demonstrate that synthetic autocorrelation curves correspond well with experimental data and that NFCS diffusion measurements of Rhodamine B remain constant, regardless of the distortion present in a confocal detection volume. 相似文献