Single-molecule detection and identification of multiple species by multiparameter fluorescence detection

Two general strategies are introduced to identify and quantify single molecules in dilute solutions by employing a spectroscopic method for data registration and specific burst analysis, denoted multiparameter fluorescence detection (MFD). MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows for selection of specific events for subsequent analysis. Using the multiple fluorescence dimensions, we demonstrate a dye labeling scheme of oligonucleotides, by which it is possible to identify and separate 16 different compounds in the mixture via their characteristic pattern by MFD. Such identification procedures and multiplex assays with single-molecule sensitivity may have a great impact on screening of species and events that do not lend themselves so easily to amplification, such as disease-specific proteins and their interactions.     

Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate

We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species: Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.     

DNA damage detection technique applying time-resolved fluorescence measurements

A novel DNA damage detection technique based on the characteristic fluorescence lifetimes exhibited by Pico-Green-single-stranded DNA and -double-stranded DNA complexes is employed to establish the damage produced on DNA isolated from sheep white blood cells following gamma radiation. This technique, which incorporates key concepts such as alkaline unwinding buffers and higher unwinding rates of damaged DNA compared to undamaged DNA, allows for the differentiation of DNA damage resulting from doses of gamma radiation in the 0-100-Gy range, with the potential of analyzing samples consisting of as little as 10(4) cells. Experiments were carried out using commercial DNA sources as well as DNA isolated from sheep white blood cells, suggesting its potential for use with isolated DNA from virtually any eukaryotic cell.     

Manganese-doped ZnSe quantum dots as a probe for time-resolved fluorescence detection of 5-fluorouracil

Quantum dots (QDs) are generally used for the conventional fluorescence detection. However, it is difficult for the QDs to be applied in time-resolved fluorometry due to their short-lived emission. In this paper, high-quality Mn-doped ZnSe QDs with long-lived emission were prepared using a green and rapid microwave-assisted synthetic approach in aqueous solution. Fluorescence lifetime of the Mn-doped ZnSe QDs was extended as long as 400 μs, which was 10,000 times higher than that of conventional QDs such as CdS, CdSe, and CdTe. The QDs exhibited an excellent photostability over 35 h under continuous irradiation at 260 nm. Capped with mercaptopropionic acid (MPA), the Mn-doped ZnSe QDs were used for the time-resolved fluorescence detection of 5-fluorouracil (5-FU) with the detection limit of 128 nM. The relative standard deviation for seven independent measurements of 1.5 μM 5-FU was 3.8%, and the recovery ranged from 93% to 106%. The results revealed that the Mn-doped ZnSe QDs could be a good candidate as a luminescence probe for highly sensitive time-resolved fluorometry.     

Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami

DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with <30 nm resolution. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.     

Nanochannels on a fused-silica microchip and liquid properties investigation by time-resolved fluorescence measurements

We have fabricated nanometer-sized channels, demonstrated a technique for the introduction of liquid into the channels, and carried out time-resolved fluorescence measurements of aqueous solutions. In this study, 330-nm- and 850-nm-sized channels were fabricated on fused-silica substrates by fast atom beam etching and hydrofluoric acid bonding methods. A liquid introduction method utilizing capillary action was demonstrated. The liquid introduction was observed under an optical microscope, and the liquid velocity during the introduction was analyzed by surface energy and macroscale hydrodynamics. The liquid velocity due to capillary action in the nanometer-sized channel seemed more than four times slower than the estimation. Then, aqueous solutions of rhodamine 6G (R6G), sulforhodamine 101 (SR101), and rhodamine B (RB) in the channels were measured by time-resolved fluorescence spectroscopy; spectra of the same solution in a 250-microm-sized channel were also measured as a reference for the macrospace. Although the fluorescence spectra in the 330-nm-, 850-nm- and 250-microm-sized channels agreed with one another, the fluorescent decays in the nanometer-sized channels were faster for R6G and SR101 and slower for RB than the respective decays in the 250-microm-sized channels. The results suggested the solutions had lower dielectric constants and higher viscosities in the nanometer-sized channels.     

Separation and identification of DNA-carcinogen adduct conformers by polyacrylamide gel electrophoresis with laser-induced fluorescence detection.

We have developed a separation protocol utilizing high-resolution polyacrylamide gel electrophoresis (PAGE) to isolate stable anti-benzo[a]pyrene diol epoxide adducts of oligodeoxynucleotides. Both enantiomers produced multiple adduct species. The distribution of adduct types could be quantitated by densitometry of autoradiograms or Cerenkov counting of eluted oligomers modified by anti-BPDE isomers. Laser-induced fluorescence (LIF) spectra of eluted adducts at 4.2 K (fluorescence line-narrowing spectroscopy) and 77 K revealed that bands corresponded to pure conformers of pyrene chromophore. Carcinogen-modified oligodeoxynucleotides were single-stranded, but there were often considerable stacking interactions between the pyrenyl residues and the oligonucleotide bases, indicating that electrophoresed oligomers were single-stranded but in a native, versus random coil, conformation. The ability to identify and quantitate adducts by PAGE-LIF, coupled with the high resolution and sensitivity of both techniques, makes PAGE and LIF in tandem a potentially powerful tool in the study of chemical carcinogenesis or other ligand-DNA interactions.     

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.     

Single-molecule detection and mismatch discrimination of unlabeled DNA targets

We report on a single-molecule readout scheme on total internal reflection fluorescence microscopy (TIRFM) demonstrating a detection limit in the low fM regime for short (30-mer) unlabeled DNA strands. Detection of unlabeled DNA targets is accomplished by letting them mediate the binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (? approximately 100 nm) to a DNA-modified substrate. On top of rapid and sensitive detection, the technique is also shown capable of extracting kinetics data from statistics of the residence time of the binding reaction in equilibrium, that is, without following neither the rate of binding upon injection nor release upon rinsing. The potential of this feature is demonstrated by discriminating a single mismatch from a fully complementary sequence. The success of the method is critically dependent on a surface modification that provides sufficiently low background. This was achieved through self-assembly of a biotinylated copolymer, Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) on a silicon dioxide surface, followed by subsequent addition of streptavidin and biotinylated DNA. The proposed detection scheme is particularly appealing due to the simplicity of the sensor, which relies on self-assembly principles and conventional TIRFM. Therefore, we foresee a great potential of the concept to serve as an important component in future multiplexed sensing schemes. This holds in particular true in cases when information about binding kinetics is valuable, such as in single nucleotide polymorphism diagnostics.     

Single-molecule detection: applications to ultrasensitive biochemical analysis

Recent developments in laser-based detection of fluorescent molecules have made possible the implementation of very sensitive techniques for biochemical analysis. We present and discuss our experiments on the applications of our recently developed technique of single-molecule detection to the analysis of molecules of biological interest. These newly developed methods are capable of detecting and identifying biomolecules at the single-molecule level of sensitivity. In one case, identification is based on measuring fluorescence brightness from single molecules. In another, molecules are classified by determining their electrophoretic velocities.     

Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.     

Nondestructive varnish identification by ultraviolet fluorescence spectroscopy

The identification of the chemical nature of varnish is essential for art restorers in order to choose a suitable solvent during its removal. Until today, such identification has been performed using chemical analysis after sampling. An innovative technique is presented here, using ultraviolet (UV) fluorescence spectroscopy. The method is nondestructive, workable in situ, and leads to results in real time. It is based on the comparison between the emission spectrum of an unknown varnish with those of fresh, artificially aged, or old reference resins and varnishes, for different monochromatic excitation wavelengths. The resin and the nature of the varnish as spirit, oil, or mixed can thus be identified. Various examples are presented on home-made samples applied on fluorescent backgrounds and on real works of art.     

2,4,6-Trinitrotoluene detection by laser-photofragmentation-laser-induced fluorescence

Photofragmentation (PF) and subsequent nitric oxide (NO) laser-induced fluorescence (LIF) is being developed to measure the concentration of energetic materials (EM's) in soil and other media. Laser radiation near 226 nm photodissociates gas-phase EM to NO(2), which predissociates into NO that gives an intense luminescence. The EM concentration is inferred from the intensity of the NO fluorescence. We have studied the factors that affect the PF-LIF signal intensity, including the effect of buffer gas on the LIF spectrum of pure NO, the effect of 2,4,6-trinitrotoluene (TNT) pressure on the PF-LIF spectrum, the effect of buffer-gas pressure on the PF-LIF signal intensity of pure TNT, and the effect of temperature on the PF-LIF spectra of pure TNT and of TNT in simulated soil. Heating of the TNT sample above 343 K was found to increase the magnitude of the PF-LIF signal intensity significantly, but also was found to cause physical and chemical changes in the TNT sample. The effects of heating and evacuating on the TNT sample were investigated. TNT concentration calibration curves were obtained for TNT in simulated soil mixtures. The limit of detection of TNT in soil was estimated to be 40 parts in 10(9).     

Single-molecule detection using surface-enhanced resonance Raman scattering and Langmuir-Blodgett monolayers

The Langmuir-Blodgett (LB) technique has been used to obtain spatially resolved surface-enhanced resonance Raman scattering (SERRS) spectra of single dye molecules dispersed in the matrix of a fatty acid. The experimental results presented here mimic the original electrochemical surface-enhanced Raman scattering (SERS) work where the background bulk water did not interfere with the detection of the SERS signal of molecules adsorbed onto the rough silver electrode. LB monolayers of the dye in fatty acid have been fabricated on silver island films with a concentration, in average, of one probe molecule per micrometer square. The properties of single-molecule spectroscopy were investigated using micro-Raman including mapping and global images. Blinking of the SERRS signal was also observed.     

A flow injection kinase assay system based on time-resolved fluorescence resonance energy-transfer detection in the millisecond range

A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 micros and a gate time of 75 micros to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC(50) value of 5.1 +/- 0.4 nM obtained.     

Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates.

We report an ultrasensitive, enzymatically amplified, time-resolved fluorescence immunoassay with a terbium chelate as the detectable moiety. In this immunoassay, the primary label is the enzyme alkaline phosphatase (ALP). ALP cleaves phosphate out of a fluorogenic substrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid (FSA). 5-Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FSA-Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. In this assay, exceptional sensitivity is achieved because of the enzymatic amplification introduced by ALP and the quantification by laser-induced microsecond time-resolved fluorometry. Time-resolved fluorometry is applicable because of the long fluorescence lifetime of the Tb3+ complexes. It is shown that in a model AFP assay 10(6) or 1.5 x 10(5) molecules can be detected (final assay volume, 100 microL) by using monoclonal or polyclonal detection antibodies, respectively. The assay demonstrates excellent precision (approximately 4%), and it seems to be highly suited for automated, sensitive, and rapid immunoassays.     

Single-molecule detection with axial flow into a micrometer-sized capillary

We characterize a new geometry for single-molecule detection with flow for use with a submilliliter drop of sample on an inverted confocal microscope. The solution is sucked into a glass capillary positioned above the ellipsoidal confocal volume so that molecules traverse the longest axis of the ellipsoid for greatest photon yield. Decreased spacing between the capillary tip and laser focus gives increased flow speed, as measured by fluorescence correlation spectroscopy, but also increased background from capillary autofluorescence. Flow can alleviate localized triplet and photobleaching effects and speed single-molecule sampling rates for fluorescence fluctuation spectroscopy determinations of slowly diffusing biomolecules in pharmaceutical drug discovery research.     

Mathematical model for time-resolved and frequency-domain fluorescence spectroscopy in biological tissues

In general it is not possible to write an analytic expression for the fluorescence signal generated by a fluorophore distributed in a scattering medium such as tissue. However, by assuming that the scattering properties of the tissue are the same at the excitation and emission wavelengths, we have derived a simple relation between the fluorescence and the scatter signals. Along with diffusion theory, this was used to write expressions for the fluorescence signal detected at the tissue surface in both the time and the frequency domains. Experiments using the fluorophore aluminum chlorosulfonated phthalocyanine in tissue-simulating materials confirmed the accuracy of the model. Applications to in vivo spectroscopy are discussed.