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1.
Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications. 相似文献
2.
Five two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF2 labels) with fluorescence emission maximum between 530 and 590 nm, and a frequently used rhodamine label, TAMRA, were conjugated to aminomodified oligonucleotides. The performance of the labeled oligonucleotides was studied in a separation-free nucleic acid hybridization assay using ArcDia™ TPX bioaffinity assay technology. The results show that oligonucleotide conjugates of dipyrrylmethene-BF2 labels provide higher two-photon excited fluorescence yield and better assay sensitivity than corresponding TAMRA conjugate. The effect of conjugation on photophysical properties of the labels and performance of the labeled oligonucleotides in separation-free hybridization assay is discussed. 相似文献
3.
Seven different two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF2 labels) and a frequently used TAMRA label were conjugated to mouse IgG against α-fetoprotein in variable substitution degrees. Altogether 40 IgG conjugates were prepared, and studied with respect to one-photon absorption and emission properties, and two-photon fluorescence efficiency using 1064 nm laser as illumination source. Performance of the IgG conjugates as tracers in a separation-free immunometric assay of α-fetoprotein was evaluated using two-photon excitation assay technology, ArcDiaTM TPX. The results show that the dipyrrylmethene-BF2 labels provide subpicomolar sensitivity, which is an order of magnitude better than that of TAMRA label. The effect of chromophore structure and substitution degree of IgG-label conjugates on the assay performance is discussed. 相似文献
4.
A. V. Burenin 《Optics and Spectroscopy》2017,122(1):1-7
Molecules recognizing biomarkers of diseases (monoclonal antibodies (monoABs)) are often too large for biomedical applications, and the conditions that are used to bind them with nanolabels lead to disordered orientation of monoABs with respect to the nanoparticle surface. Extremely small nanoprobes, designed via oriented conjugation of quantum dots (QDs) with single-domain antibodies (sdABs) derived from the immunoglobulin of llama and produced in the E. coli culture, have a hydrodynamic diameter less than 12 nm and contain equally oriented sdAB molecules on the surface of each QD. These nanoprobes exhibit excellent specificity and sensitivity in quantitative determination of a small number of cells expressing biomarkers. In addition, the higher diffusion coefficient of sdABs makes it possible to perform immunohistochemical analysis in bulk tissue, inaccessible for conventional monoABs. The necessary conditions for implementing high-quality immunofluorescence diagnostics are a high specificity of labeling and clear differences between the fluorescence of nanoprobes and the autofluorescence of tissues. Multiphoton micros-copy with excitation in the near-IR spectral range, which is remote from the range of tissue autofluorescence excitation, makes it possible to solve this problem and image deep layers in biological tissues. The two-photon absorption cross sections of CdSe/ZnS QDs conjugated with sdABs exceed the corresponding values for organic fluorophores by several orders of magnitude. These nanoprobes provide clear discrimination between the regions of tumor and normal tissues with a ratio of the sdAB fluorescence to the tissue autofluorescence upon two-photon excitation exceeding that in the case of single-photon excitation by a factor of more than 40. The data obtained indicate that the sdAB-QD conjugates used as labels provide the same, or even better, quality as the “gold standard” of immunohistochemical diagnostics. The developed nanoprobes are expected to find wide application in high-efficiency imaging of tumor and multiparameter diagnostics. 相似文献
5.
Two asymmetrical molecules with substituted acetylene as central rigid elongated conjugation are reported as potential chromophores
for two-photon microscopic imaging. These molecules consist of a typical D–π–A structure, have different donors (D), the same
π-conjugated center (π) and the same acceptor (A). Structural characterization and spectroscopic properties, including single-photon
(linear) absorption, quantum yields, single-photon fluorescence, and two-photon absorption spectra, were studied in solvents
with different polarity. These acetylene-substituted molecules were found to have high two-photon absorption cross-sections
(for example, 690 GM for molecule 1 in toluene), which were determined by a two-photon induced fluorescence method using a
femtosecond Ti: sapphire laser as excitation source. Single- and two-photon cellular imaging experiments demonstrate that
the substituted acetylene derivatives could be one kind of promising two-photon fluorescence probes for cellular imaging. 相似文献
6.
Molecular assays such as immunoassays are often performed using solid carriers and fluorescent labels. In such an assay format
a question can be raised on how much the fluorescence of the label is influenced by the bio-affinity binding events and the
solid carrier surface. Since changes in fluorescence intensity as labels bind to surfaces are notoriously difficult to quantify
other approaches are preferred. A good indicator, independent of the fluorescence intensity of the label, is the fluorescence
lifetime of the marker fluorophore. Changes in fluorescence lifetime reliably indicate the presence of dynamic quenching,
energy transfer or other de-excitation processes. A microsphere based assay system is studied under two-photon excitation.
Changes in fluorescence lifetime are studied as labeled protein conjugates bind on microsphere surfaces – both direct on the
surface and with a few nanometer distance from the surface. Fluorescence signal is measured from individual polystyrene microspheres
and the fluorescence lifetime histogram is simultaneously recorded. The results indicate that self-quenching and quenching
by the polystyrene surface are both present in such a system. However, the effect of the surface can be avoided by increasing
the distance between the surface and the label. Typical distances achieved by a standard sandwich type of assay, are already
sufficient to overcome the surface induced quenching in fluorescence detection. 相似文献
7.
In this work we demonstrate that non-symmetrically substituted phthalo-naphthalocyanine molecule can have extremely large 2PA cross section in near-resonance conditions of excitation. We also show the possibility to switch between its two tautomeric forms upon two-photon excitation at 77 K. By measuring 2PA cross section, quantum efficiency of transformation in these two forms, as well as quantum yield of fluorescence, we evaluate the figure of merit for fast re-writable 3D information storage and show that this figure meets the criteria of reliable and fast (one bit per one femtosecond pulse) 2PA writing or reading. 相似文献
8.
The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules. 相似文献
9.
The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label
free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these
fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths,
fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application
of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism
which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H
and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function
(IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H,
are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside
the spores, is revealed. 相似文献
10.
双光子荧光染料分子在生物医学成像中具有广阔的应用前景,但取代效应对分子结构以及光物理性质影响的探求相对匮乏. 本文设计并研究了一系列脂滴检测染料分子,分析了分子的光学性质以及无辐射跃迁等. 通过分子内弱相互作用和电子- 空穴布居分析,阐述了其内在机理. 结果表明,所研究的分子均具有优良的光物理性能、高效荧光量子产量、大的斯托克斯位移以及显著的双光子吸收截面等. 本工作合理地解释了实验现象并阐述了取代效应对脂滴检测NAPBr染料分子的双光子吸收和激发态性质的影响,这为设计新型的高效有机分子提供了理论指导. 相似文献
11.
Different from organic fluorescence dyes, fluorescent lanthanide complexes have the fluorescence properties of long fluorescence lifetime, large Stokes shift and sharp emission profile, which makes them favorable be used as the fluorescent labeling reagents for microsecond time-resolved fluorescence bioassay. Lanthanide complex-based fluorescence labels have been successfully used for highly sensitive time-resolved fluorescence immunoassay, DNA hybridization assay, cell activity assay, and bioimaging microscopy assay. Since the technique allows easy distinction of the specific fluorescence signal of the long-lived label from short-lived background noises associated with biological samples, scattering lights (Tyndall, Rayleigh and Raman scatterings) and the optical components (cuvettes, filters and lenses), the sensitivity of fluorescence bioassay has been remarkably improved. This paper summarized the recent developments of lanthanide complex-based fluorescence labels and their applications in time-resolved fluorescence bioassays mainly based on the authors’ researches and relative publications. 相似文献
12.
The subject of phosphorescent metalloporphyrins is reviewed, focusing mainly on the development and application of Pt- and Pd-porphyrins. A summary of their general chemical and photophysical properties, and guidelines for rational design of the phosphorescent labels, bioconjugates and probes is given. Examples of different detection formats and particular bioanalytical applications developed in recent years are presented. The potential of phosphorescent porphyrin label methodology is discussed and compared to that of the long-decay fluorescent lanthanide chelates and other common fluorophores. 相似文献
13.
It is shown that colloidal semiconductor nanocrystals (quantum dots), which are promising fluorophores for multiphoton fluorescence
microscopy, exhibit a two-photon absorption saturation effect at moderate powers (not exceeding 10 mW) of femtosecond pumping
radiation. An analytical expression for the power of two-photon fluorescence of quantum dots as a function of the average
pumping power is obtained. With this expression, the deviation of the found dependence from the quadratic law is explained
by two factors, i.e., a large two-photon absorption cross section of quantum dots and their slow (compared to the typical
pumping pulse-repetition period) relaxation to an unexcited state. Using an LSM 510 Carl Zeiss laser scanning microscope equipped
with a Ti:Sa tunable femtosecond laser, a series of model experiments is performed to reveal the saturation effect in a solution
of commercially available quantum dots. Good agreement is obtained between the measured dependence of the power of two-photon
fluorescence on the average pump power and the theoretical calculation results. It is also experimentally demonstrated that,
under fluorescence saturation conditions, the spatial resolution of the method of multiphoton fluorescence microscopy is lost;
this effect is analyzed numerically. 相似文献
14.
Yu X Chen L Deng Y Li K Wang Q Li Y Xiao S Zhou L Luo X Liu J Pang D 《Journal of fluorescence》2007,17(2):243-247
A new class of fluorescent probe produced by conjugating semiconductor quantum dots (QDs) with protein molecule is proposed as an alternative to conventional organic labels. However the fluorescence characteristics of the QD bioconjugates are not clear while they are excitied with one- or two-photon laser pulse. We synthesized specific immunofluorescent probes by linking QDs to alpha fetoprotein (AFP) antibody for specific binding alpha-fetoprotein -an important marker for hepatocellular carcinoma cell lines, and archived specific fluorescence detection with the QDs-Anti-AFP in nude mice. Then, we have analyzed the fluorescence characteristics of QDs-Anti-AFP and original QDs both under one- and two-photon excitations. The results demonstrated that QDs-Anti-AFP's fluorescent spectral and lifetime haven't varied much from that of original QDs. Moreover, QDs-Anti-AFP have exhibited higher fluorescence efficiency than QDs under two-photon examination. 相似文献
15.
In this paper, we present the optical properties and the platforms on fluorescent quantum dots for biological labeling, biomedical
engineering and biosensor in molecular imaging. Quantum dots possess several properties that make them very attractive for
fluorescent tagging: broad excitation spectrum, narrow emission spectrum, precise tunability of their emission peak, longer
fluorescence lifetime than organic fluorophores and negligible photobleaching. We describe how to take such advantages of
quantum dots to develop the technology and employ it to build assay platforms. Finally, ultrasensitivity, multicolor, and
multiplexing of the technology of semiconductor quantum dots open up promising and interesting possibilities for bioassay
platform. 相似文献
16.
Belfield KD Corredor CC Morales AR Dessources MA Hernandez FE 《Journal of fluorescence》2006,16(1):105-110
The synthesis, photophysical characterization, and determination of singlet oxygen quantum yields (ΦΔ) for a class of fluorene derivatives with potential application in two-photon photodynamic therapy (PDT) is reported. It
has been demonstrated that these compounds possess the ability to generate singlet oxygen (1O2) upon excitation. A photochemical method, using 1,3-diphenylisobenzofuran (DPBF) as 1O2 chemical quencher, was employed to determine the singlet oxygen quantum yields (ΦΔ) of the fluorene-based photosensitizers in ethanol. ΦΔ values ranged from 0.35 to 0.75. These derivatives may have potential application as two-photon photosensitizers when pumped
via two-photon excitation in the near-IR spectral region. 相似文献
17.
S. N. Smetanin 《Optics and Spectroscopy》2016,120(3):395-402
The spectral luminescent, photophysical, photochemical, lasing, nonlinear optical, and sensor characteristics of a series of new synthesized complexes of zinc and difluoroborate with dipyrrines of different structure have been studied. It is found that many of these compounds exhibit stimulated emission in different solvents when excited by the second (532 nm) and third (355 nm) harmonics of a Nd:YAG laser in the range of 548–692 nm. It is shown that not only efficient fluorophores belonging to dipyrrine difluoroborates (with a quantum fluorescence yield close to 1), but also compounds with a fluorescence yield equal to 0.3, generate laser radiation with a high resource; phosphorescence is also observed along with fluorescence. Transmission of UV radiation (355 nm) is shown to decrease with an increase in the pulsed excitation power density; this is a manifestation of nonlinear optical properties. The change of phosphorescence signal in dependence of the composition of the gas mixture around a solid-state sample colored by dipyrrinate complexes indicates that a number of the compounds under consideration exhibit sensor abilities. 相似文献
18.
Computational design of ratiometric two-photon fluorescent Zn2+ probes based on quinoline and di-2-picolylamine moieties
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To improve two-photon absorption (TPA) response of a newly synthesized probe, a series of ratiometric two-photon fluorescent Zn2+ sensors based on quinoline and DPA moieties have been designed. The one-photon absorption, TPA, and emission properties of the experimental and designed probes before and after coordination with Zn2+ are investigated employing the density functional theory in combination with response functions. The design consists of two levels. In the first level of design, five probes are constructed through using several electron acceptors or donors to increase accepting or donating ability of the fluorophores. It shows that all the designed probes have stronger TPA intensities at longer wavelengths with respect to the experimental probe because of the increased intra-molecular charge transfer. Moreover, it is found that the probe 4 built by adding an acyl unit has the largest TPA cross section among the designed structures due to the form of longer conjugated length and more linear backbone. One dimethylamino terminal attached along the skeleton can improve TPA intensity more efficiently than two side amino groups. Therefore, in the second level of design, a new probe 7 is formed by both an acyl unit and a dimethylamino terminal. It exhibits that the TPA cross sections of probe 7 and its zinc complex increase dramatically. Furthermore, the fluorescence quantum yields of the designed probes 4 and 7 are calculated in a new way, which makes use of the relation between the computed difference of dipole moment and the measured fluorescence quantum yield. The result shows that our design also improves the fluorescence quantum yield considerably. All in all, the designed probes 4 and 7 not only possess enhanced TPA intensities but also have large differences of emission wavelength upon Zn2+ coordination and strong fluorescence intensity, which demonstrates that they are potential ratiometric two-photon fluorescent probes. 相似文献
19.
20.
首次用谷胱甘肽(GSH)作为稳定剂,在水溶液中制备了稳定地发射绿色荧光和橙色荧光的两种 CdSe/CdS核/壳结构的纳米量子点。用紫外-可见分光光度和荧光光谱方法研究了CdSe/CdS量子点的发光特性。透射电镜(TEM)结果表明CdSe/CdS量子点近似球形,在水中分散性良好,比CdSe量子点具有更优异的发光特性,发射光谱和吸收光谱都有红移现象。将CdSe/CdS量子点与鼠抗人CD3抗体连接,制备了水溶性CdSe/CdS-CD3复合物探针,对人血淋巴细胞进行标记和成像。结果表明用该探针对人血淋巴细胞成像清晰,其荧光在30 min的连续蓝光激发下无明显衰退,而FITC荧光在20 min内基本完全猝灭。 相似文献