Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.     

Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants.

A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These peptide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.     

Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays

In fluorescence resonance energy transfer (FRET)-based assays, spectral separation of acceptor emission from donor emission is a common problem affecting the assay sensitivity. The challenge derives from small Stokes shifts characteristic to conventional fluorescent dyes resulting in leakage of donor emission to the measurement window intended only to collect the acceptor emission. We have studied a FRET-based homogeneous bioaffinity assay utilizing a tandem dye acceptor with a large pseudo-Stokes shift (139 nm). The tandem dye was constructed using B-phycoerythrin as an absorber and multiple Alexa Fluor 680 dyes as emitters. As a donor, we employed upconverting phosphor particles, which uniquely emit at visible wavelengths under low-energy infrared excitation enabling the fluorescence measurements free from autofluorescence even without time-resolved detection. With the tandem dye, it was possible to achieve four times higher signal from a single binding event compared to the conventional Alexa Fluor 680 dye alone. Tandem dyes are widely used in cytometry and other multiplex purposes, but their applications can be expanded to fluorescence-based homogeneous assays. Both the optimal excitation and emission wavelengths of tandem dye can be tuned to a desired region by choosing appropriate fluorophores enabling specifically designed acceptor dyes with large Stokes shift.     

Efficient detection of single DNA fragments in flowing sample streams by two-photon fluorescence excitation

This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes was accomplished using a mode-locked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 labeled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our flow cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximately 75%. We also demonstrate the ability to distinguish between different sized DNA fragments in a mixture by their individual fluorescence burst sizes by TPE. These studies indicate that using TPE for single molecule flow cytometry experiments lowers the intensity of the background radiation by approximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE.     

Platinum plasmonic nanostructure arrays for massively parallel single-molecule detection based on enhanced fluorescence measurements

We fabricated platinum bowtie nanostructure arrays producing fluorescence enhancement and evaluated their performance using two-photon photoluminescence and single-molecule fluorescence measurements. A comprehensive selection of suitable materials was explored by electromagnetic simulation and Pt was chosen as the plasmonic material for visible light excitation near 500 nm, which is preferable for multicolor dye-labeling applications like DNA sequencing. The observation of bright photoluminescence (λ = 500-600 nm) from each Pt nanostructure, induced by irradiation at 800 nm with a femtosecond laser pulse, clearly indicates that a highly enhanced local field is created near the Pt nanostructure. The attachment of a single dye molecule was attempted between the Pt triangles of each nanostructure by using selective immobilization chemistry. The fluorescence intensities of the single dye molecule localized on the nanostructures were measured. A highly enhanced fluorescence, which was increased by a factor of 30, was observed. The two-photon photoluminescence intensity and fluorescence intensity showed qualitatively consistent gap size dependence. However, the average fluorescence enhancement factor was rather repressed even in the nanostructure with the smallest gap size compared to the large growth of photoluminescence. The variation of the position of the dye molecule attached to the nanostructure may influence the wide distribution of the fluorescence enhancement factor and cause the rather small average value of the fluorescence enhancement factor.     

Homogeneous assay technology based on upconverting phosphors

Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening.     

Frequency upconversion and two-photon absorption of salicylaldehyde azine 1

Two-photon absorption and two-photon excitation fluorescence of salicylaldehyde azine 1 crystals were investigated. It was observed an intense visible fluorescence when this material was excited with a laser tuned at the near infrared region. Varying the laser intensity we characterized this phenomenon as a simultaneous two-photon laser absorption process. Using open aperture Z-scan measurements we characterized this two-photon absorption phenomenon and measured the value of the two-photon absorption cross-section of this molecule to be equal to 87 GM. Our results indicate that this is a promising organic material aiming nonlinear photonics applications.     

Verification of antiparallel G-quadruplex structure in human telomeres by using two-photon excitation fluorescence lifetime imaging microscopy of the 3,6-Bis(1-methyl-4-vinylpyridinium)carbazole diiodide molecule

Different G-quadruplex structures for the human telomeric sequence d(T2AG3)4 in vitro have been documented in the presence of sodium and potassium. Verification of the G-quadruplex structures in human telomeres in vivo is the main issue in establishing the biological function of the G-quadruplex structures in telomeres as well as the development of anticancer agents. Here we have applied two-photon excitation fluorescence lifetime imaging microscopy to measure the fluorescence lifetime of the BMVC molecule upon interaction with various DNA structures. The distinction in lifetime measured with submicrometer spatial resolution in two-photon excitation fluorescence lifetime imaging microscopy provides a powerful approach not only to verify the existence of the antiparallel G-quadruplex structure in human telomeres but also to map its localizations in metaphase chromosomes.     

Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium

Image contrast enhancement is investigated for two-photon excitation fluorescence images of a microscopic sample that is buried underneath a turbid medium. The image contrast, which deteriorates rapidly with sample depth because of scattering loss, is enhanced by an increase in the average excitation power of the focused Gaussian (the TEM(00) mode) beam according to a compensation relation that has been derived by use of a Monte Carlo analysis of the scattering problem. A correct increase in the excitation power results in a detected fluorescence signal that remains invariant with sample depth. The scheme is demonstrated on images of DAPI-stained nuclei cells viewed underneath a suspension of 0.105-mum-diameter polystyrene spheres.     

Homogeneous noncompetitive immunoassay for 17beta-estradiol based on fluorescence resonance energy transfer

We previously presented a homogeneous noncompetitive assay principle based on quenching of the fluorescence of europium(III) chelate. This assay principle has now been applied to the measurement of 17beta-estradiol (E2) using europium(III) chelate labeled estradiol specific antibody Fab fragment (Eu(III)-Fab) as a donor and E2 conjugated with nonfluorescent QSY21 dye as an acceptor. Fluorescence could be measured only from those Eu(III)-Fab that were bound to E2 of the sample, while the fluorescence of the Eu(III)-Fab that were not occupied by E2 were quenched by E2-QSY21 conjugates. The performance of the assay was tested both in buffer and in the presence of serum. Because approximately half of the Fabs were incapable of binding to E2, the maximum quenching efficiency was only 55%. The lowest limits of detection were 18 and 64 pM in buffer and serum-based calibrators, respectively. The highest measurable concentrations were 0.4 nM in buffer and 1 nM in serum. The presented noncompetitive assay principle requires only one binder, and it can be applied to other haptens as well, providing that a suitable antibody is available.     

Compact detector for proteins based on two-photon excitation of native fluorescence

A compact, inexpensive detector for proteins has been constructed based on two-photon excitation of fluorescence from phenylalanine, tyrosine, and tryptophan. The fluorescence was excited by a solid-state microchip laser operating at 532 nm. Detection limits for phenylalanine, tyrosine, and tryptophan were 62 microM, 2.0 microM, and 470 nM, respectively, in a volume of 3 fL. The detection limit for a test protein, bovine serum albumin, was 130 nM.     

Efficient single-molecule fluorescence resonance energy transfer analysis by site-specific dual-labeling of protein using an unnatural amino acid

Single-molecule fluorescence resonance energy transfer (smFRET) measurement provides a unique and powerful approach to understand complex biological processes including conformational and structural dynamics of individual biomolecules. For effective smFRET analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. Here we demonstrate that site-specific dual-labeling of protein via incorporation of unnatural amino acid provides a clearer picture for the folded and unfolded states of the protein in smFRET analysis than conventional labeling using double cysteines. As a model study, maltose-binding protein (MBP) was dually labeled via incorporation of ρ-azido-l-phenylalanine and cysteine at specific positions, immobilized on a surface, and subjected to smFRET analysis under native and denaturing conditions. The resulting histograms show that site-specific dual-labeling results in a more homogeneous distribution in protein populations, enabling a precise smFRET analysis of protein.     

CdTe量子点的微波合成及其在双光子显微成像中的应用

采用微波辐射加热的方法,以亚碲酸钠(Na2TeO3)作碲源,以谷胱甘肽(GSH)作稳定剂,在水相中合成出高质量的CdTe量子点。所合成量子点的发射波长从515～630nm可调,荧光量子产率(PLQYs)最高达95%。利用X射线粉末衍射(XRD)、高分辨透射电镜(HRTEM)、紫外-可见吸收光谱(UV-Vis)和荧光发射光谱(PL)等技术表征产物的物相结构和光学性质。用双光子激发荧光法研究CdTe量子点的双光子吸收性质。用双光子激发荧光成像技术,以发红光的CdTe量子点作为双光子荧光探针成功标记了人肺腺癌细胞(A549)。     

Two-photon excited coherence gratings in inhomogeneously broadened organic solid

For the first time a frequency-domain grating is observed in the visible region as a result of coherence excited by two-photon absorption of time-delayed near-IR femtosecond pulses in an inhomogeneously broadened organic solid-state system. The grating appears in chlorin (2,3-dihydroporphyrin)-doped polymer film at low temperature and is detected in two different ways. First, modulation is observed in the pure electronic fluorescence band upon 'resonant' two-photon excitation with laser carrier frequency equal to one-half of the fluorescence band maximum. Second, upon long exposure time at half of the transition frequency, a persistent spectral hole with periodic structure is observed. The two-photon absorption spectrum of the chlorin molecule in the spectral range 1130–1310nm is also presented for the first time.     

Nucleic acid-based fluorescence sensors for detecting proteins

We report here development of a rapid, homogeneous, aptamer-based fluorescence assay ("molecular beacons") for detecting proteins. The assay involves protein-induced coassociation of two aptamers recognizing two distinct epitopes of the protein. The aptamers contain short fluorophore-labeled complementary "signaling" oligonucleotides attached to the aptamer by non-DNA linker. Coassociation of the two aptamers with the protein results in bringing the two "signaling" oligonucleotides into proximity, producing a large change of fluorescence resonance energy transfer between the fluorophores. We used thrombin as a model system to provide proof-of-principle evidence validating this molecular beacon design. Thrombin beacon was capable of detecting the protein with high selectivity (also in complex biological mixtures), picomolar sensitivity, and high signal-to-background ratio. This is a homogeneous assay requiring no sample manipulation. Since the design of molecular beacons described here is not limited to any specific protein, it will be possible to develop these beacons to detect a variety of target proteins of biomedical importance.     

Influence of optical properties on two-photon fluorescence imaging in turbid samples

A numerical model was developed to simulate the effects of tissue optical properties, objective numerical aperture (N.A.), and instrument performance on two-photon-excited fluorescence imaging of turbid samples. Model data are compared with measurements of fluorescent microspheres in a tissuelike scattering phantom. Our results show that the measured two-photon-excited signal decays exponentially with increasing focal depth. The overall decay constant is a function of absorption and scattering parameters at both excitation and emission wavelengths. The generation of two-photon fluorescence is shown to be independent of the scattering anisotropy, g, except for g > 0.95. The N.A. for which the maximum signal is collected varies with depth, although this effect is not seen until the focal plane is greater than two scattering mean free paths into the sample. Overall, measurements and model results indicate that resolution in two-photon microscopy is dependent solely on the ability to deliver sufficient ballistic photon density to the focal volume. As a result we show that lateral resolution in two-photon microscopy is largely unaffected by tissue optical properties in the range typically encountered in soft tissues, although the maximum imaging depth is strongly dependent on absorption and scattering coefficients, scattering anisotropy, and objective N.A..     

Two-photon nitric oxide laser-induced fluorescence measurements in a diesel engine

A two-photon nitric oxide (NO) laser-induced fluorescence (LIF) technique was developed and applied to study in-cylinder diesel combustion. The technique prevents many problems associated with in-cylinder, single-photon NO planar-laser-induced fluorescence measurements, including fluorescence interference from the Schumann-Runge bands of hot O2, absorption of a UV excitation beam by in-cylinder gases, and difficulty in rejecting scattered laser light while simultaneously attempting to maximize fluorescence signal collection. Verification that the signal resulted from NO was provided by tuning of the laser to a vibrational off-resonance wavelength that showed near-zero signal levels, which resulted from either fluorescence or interference at in-cylinder pressures of as much as 20 bar. The two-photon NO LIF signal showed good qualitative agreement with NO exhaust-gas measurements obtained over a wide range of engine loads.     

Two-photon fluorescence imaging super-enhanced by multishell nanophotonic particles, with application to subcellular pH

A novel nanophotonic method for enhancing the two-photon fluorescence signal of a fluorophore is presented. It utilizes the second harmonic (SH) of the exciting light generated by noble metal nanospheres in whose near-field the dye molecules are placed, to further enhance the dye's fluorescence signal in addition to the usual metal-enhanced fluorescence phenomenon. This method enables demonstration, for the first time, of two-photon fluorescence enhancement inside a biological system, namely live cells. A multishell hydrogel nanoparticle containing a silver core, a protective citrate capping, which serves also as an excitation quenching inhibitor spacer, a pH indicator dye shell, and a polyacrylamide cladding are employed. Utilizing this technique, an enhancement of up to 20 times in the two-photon fluorescence of the indicator dye is observed. Although a significant portion of the enhanced fluorescence signal is due to one-photon processes accompanying the SH generation of the exciting light, this method preserves all the advantages of infrared-excited, two-photon microscopy: enhanced penetration depth, localized excitation, low photobleaching, low autofluorescence, and low cellular damage.     

Monte carlo analysis of two-photon fluorescence imaging through a scattering medium

The behavior of two-photon fluorescence imaging through a scattering medium is analyzed by use of the Monte Carlo technique. The axial and transverse distributions of the excitation photons in the focused Gaussian beam are derived for both isotropic and anisotropic scatterers at different numerical apertures and at various ratios of the scattering depth with the mean free path. The two-photon fluorescence profiles of the sample are determined from the square of the normalized excitation intensity distributions. For the same lens aperture and scattering medium, two-photon fluorescence imaging offers a sharper and less aberrated axial response than that of single-photon confocal fluorescence imaging. The contrast in the corresponding transverse fluorescence profile is also significantly higher. Also presented are results comparing the effects of isotropic and anisotropic scattering media in confocal reflection imaging. The convergence properties of the Monte Carlo simulation are also discussed.