量子点的近场激发荧光特性研究

近场光学显微镜具有nm量级的空间分辨率,量子点(quantum dots,QDs)荧光探针具有激发谱宽、发射谱线窄、荧光强度高、抗光漂白和稳定性高等优点,两者结合用于生物大分子的成像探测和识别具有广泛的应用前景。用近场光学显微镜对链霉亲和素偶联的QDs进行近场荧光激发,并对其荧光发射特性和光稳定性进行研究,结果表明:近场光学显微镜nm量级的空间分辨率,可以同时观察到了QDs的单体、二聚体和三聚体;QDs的荧光发射强度高,近场荧光像对比度好,单量子点的荧光半高宽达到25nm;对一定入射波长的单色激发光,QDs的近场荧光强度随着激发功率密度的增加线性增加,并很快趋于稳定。与传统的荧光染料如异硫氰酸荧光素相比,QDs的稳定性非常好,在激发功率密度为300W/cm2的近场辐射下,量子点的荧光强度超过6h基本保持不变,其抗光漂白能力远远高于普通荧光染料。     

An antibody-conjugated internalizing quantum dot suitable for long-term live imaging of cells.

Quantum dots (QD) are fluorescent semiconductor nanocrystals that are emerging as superior alternatives to the conventional organic dyes used in biological applications. Although QDs offer several advantages over conventional fluorescent dyes, including greater photostability and a wider range of excitation and (or) emission wavelengths, their toxicity has been an issue in its wider use as an analytic, diagnostic and therapeutic tool. We prepared a conjugate QD with an internalizing antibody and demonstrated that the QD-antibody conjugate is efficiently internalized into cells and is visible even after multiple divisions. We demonstrate that the internalized QD is nontoxic to cells and provides a sensitive tool for long-term molecular imaging.     

A single molecule investigation of the photostability of quantum dots

Quantum dots (QDs) are very attractive probes for multi-color fluorescence imaging in biological applications because of their immense brightness and reported extended photostability. We report here however that single QDs, suitable for biological applications, that are subject to continuous blue excitation from a conventional 100 W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a permanent dark, photobleached state. We further show that β-mercaptoethanol has a dual stabilizing effect on the fluorescence emission of QDs: 1) by increasing the frequency of time that a QD is in its fluorescent state, and 2) by decreasing the photobleaching rate. The observed QD color spectral switching is especially detrimental for multi-color single molecule applications, as we regularly observe spectral blue-shifts of 50 nm, or more even after only ten seconds of illumination. However, of significant importance for biological applications, we find that even small, biologically compatible, concentrations (25 μM) of β-mercaptoethanol has a significant stabilizing effect on the emission color of QDs, but that greater amounts are required to completely abolish the spectral blue shifting or to minimize the emission intermittency of QDs.     

Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy

OBJECTIVE: To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM). STUDY DESIGN: U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei. RESULTS: FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained. CONCLUSION: QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.     

Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.     

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.     

dsDNA-coated quantum dots

Because of their unique spectral properties, quantum dots (QDs) have recently proved useful as fluorescent labels for biosensing probes. We developed a versatile QD label by modifying dsDNA with biotin and thiol groups at opposite ends and attaching it to quantum dots via a metal-thiol bond. These dsDNA-coated QDs fluorescently label their targets through biotin-streptavidin binding and show excellent histological results when used to detect biotin-labeled chromosome probes. The dsDNA coating also circumvented the common problems of aggregation and steric hindrance that occur with other QD probes.     

Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy

Metastasis is an impediment to the development of effective cancer therapies. Our understanding of metastasis is limited by our inability to follow this process in vivo. Fluorescence microscopy offers the potential to follow cells at high resolution in living animals. Semiconductor nanocrystals, quantum dots (QDs), offer considerable advantages over organic fluorophores for this purpose. We used QDs and emission spectrum scanning multiphoton microscopy to develop a means to study extravasation in vivo. Although QD labeling shows no deleterious effects on cultured cells, concern over their potential toxicity in vivo has caused resistance toward their application to such studies. To test if effects of QD labeling emerge in vivo, tumor cells labeled with QDs were intravenously injected into mice and followed as they extravasated into lung tissue. The behavior of QD-labeled tumor cells in vivo was indistinguishable from that of unlabeled cells. QDs and spectral imaging allowed the simultaneous identification of five different populations of cells using multiphoton laser excitation. Besides establishing the safety of QDs for in vivo studies, our approach permits the study of multicellular interactions in vivo.     

Lab-on-a-chip immunoassay for multiple antibodies using microsphere light scattering and quantum dot emission

Double detection of microsphere light scattering and quantum dot emission was demonstrated for lab-on-a-chip immunoassay without using stationary support. We conjugated quantum dots (QDs) onto microspheres to enable multiplex assays as well as to enhance the limit of detection (LOD). We named this configuration "nano-on-micro" or "NOM". Upon radiation with UV light (380nm), a stronger light scattering signal is observed with NOMs than QDs or microspheres alone. Additionally, NOMs are easier to handle than QDs. Since QDs also provide fluorescent emission, we are able to utilize an increase in light scattering for detecting antigen-antibody reaction and a decrease in QD emission to identify which antibody (or antigen) is present. Two types of NOM combinations were used. One batch of microspheres was coated with QDs emitting at 655 nm and mouse IgG (mIgG); the other with QDs emitting at 605 nm and bovine serum albumin (BSA). A mixture of these two NOMs was used to identify either anti-mIgG or anti-BSA. NOM particles and target solutions were mixed in a microfluidic device (using highly carboxylated microspheres as previously demonstrated by our group) and on-chip detection was performed using proximity optical fibers. Forward light scattering at 380 nm was collected. With the positive target, the scattering signal was increased. The LOD was as low as 50 ng ml(-1) (330 pM) with p<0.05. Fluorescent emission (655 or 605 nm) was simultaneously collected. With the positive target, the emission signal was attenuated. Therefore, we were able to detect two different antibodies simultaneously with two different detection protocols. We believe this NOM bioassay has the ability to screen for and detect multiple antibodies with minimal sample processing and handling (one-step lab-on-a-chip immunoassay).     

Near Infrared,Highly Efficient Luminescent Solar Concentrators

The fabrication of a low reabsorption emission loss, high efficient luminescent solar concentrator (LSC) is demonstrated by embedding near infrared (NIR) core/shell quantum dots (QDs) in a polymer matrix. An engineered Stokes shift in NIR core/shell PbS/CdS QDs is achieved via a cation exchange approach by varying the core size and shell thickness through the refined reaction parameters such as reaction time, temperature, precursor molar ratio, etc. The as‐synthesized core/shell QDs with high quantum yield (QY) and excellent chemical/photostability exhibit a large Stokes shift with respect to the bare PbS QDs due to the strong core‐to‐shell electrons leakage. The large‐area planar LSC based on core/shell QDs exhibits the highest value (6.1% with a geometric factor of 10) for optical efficiency compared to the bare NIR QD‐based LSCs and other reported NIR QD‐based LSCs. The suppression of emission loss and the broad absorption of PbS/CdS QDs offer a promising pathway to integrate LSCs and photovoltaic devices with good spectral matching, indicating that the proposed core/shell QDs are strong candidates for fabricating high efficiency semi‐transparent large‐area LSCs.     

Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry

Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD-antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1-3 days depending on the number of biomarkers and QD probes used.     

Enhancement of cell internalization and photostability of red and green emitter quantum dots upon entrapment in novel cationic nanoliposomes

Two quantum dots (QDs), a green emitter, CdSe and a red emitter, CdSe with ZnS shell are encapsulated into novel liposomes in two different formulations including cationic liposomes. Quantum dots have proven themselves as powerful inorganic fluorescent probes, especially for long‐term, multiplexed imaging and detection. Upon delivery into a cell, in endocytic vesicles such as endosomes, their fluorescence is quenched. We have investigated the potential toxic effects, photophysical properties and cell internalization of QDs in new formulation of liposomes as an in vitro vesicle model. Entrapment of QDs into liposomes is brought about with a decrease in their intrinsic fluorescence and toxicities and an increase in their photostability and lifetime. The biomimetic lipid bilayer of liposomes provides high biocompatibility, thereby enhancing the effectiveness of fluorescent nanoparticles for biological recognition in vitro and in vivo. The prepared lipodots could effectively prevent QDs from photo‐oxidation during storage and when exposed to ultraviolet (UV) light. Moreover, the flow cytometry of HEK 293 T cells showed that the cell internalization of encapsulated QDs in (DSPC/CHO/DOPE/DOAB) liposome is enhanced 10 times compared with non‐encapsulated QD (bare QDs).     

Fluorescent quantum dots: An insight on synthesis and potential biological application as drug carrier in cancer

Quantum dots (QDs) are nanocrystals of semiconducting material possessing quantum mechanical characteristics with capability to get conjugated with drug moieties. The particle size of QDs varies from 2 to 10 nm and can radiate a wide range of colours depending upon their size. Their wide and diverse usage of QDs across the world is due to their adaptable properties like large quantum yield, photostability, and adjustable emission spectrum. QDs are nanomaterials with inherent electrical characteristics that can be used as drug carrier vehicle and as a diagnostic in the field of nanomedicine. Scientists from various fields are aggressively working for the development of single platform that can sense, can produce a microscopic image and even be used to deliver a therapeutic agent. QDs are the fluorescent nano dots with which the possibilities of the drug delivery to a targeted site and its biomedical imaging can be explored. This review is mainly focused on the different process of synthesis of QDs, their application especially in the areas of malignancies and as a theranostic tool. The attempt is to consolidate the data available for the use of QDs in the biomedical applications.     

Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria

CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates.     

Cadmium-containing quantum dots: properties,applications, and toxicity

The marriage of biology with nanomaterials has significantly accelerated advancement of biological techniques, profoundly facilitating practical applications in biomedical fields. With unique optical properties (e.g., tunable broad excitation, narrow emission spectra, robust photostability, and high quantum yield), fluorescent quantum dots (QDs) have been reasonably functionalized with controllable interfaces and extensively used as a new class of optical probe in biological researches. In this review, we summarize the recent progress in synthesis and properties of QDs. Moreover, we provide an overview of the outstanding potential of QDs for biomedical research and innovative methods of drug delivery. Specifically, the applications of QDs as novel fluorescent nanomaterials for biomedical sensing and imaging have been detailedly highlighted and discussed. In addition, recent concerns on potential toxicity of QDs are also introduced, ranging from cell researches to animal models.

     

Can earthworms biosynthesize highly luminescent quantum dots?

Band gap tunable cadmium selenide (CdSe) quantum dots (QDs) were synthesized within earthworms that emit in the middle of the visible spectrum. We demonstrated that this luminescence emission is a combination of the earthworm's protein and QD luminescence, such that the contribution of QDs in the luminescence was negligible. Eisenia fetida earthworms were used for QD biosynthesis and were exposed to different concentrations of CdCl₂ and Na₂SeO₃ in standard soil for an adequate exposure time. The size of the CdSe QDs based on the effective mass model was in agreement with the size measured from the transmission electron microscopy analysis, with an average diameter of 7 nm. Ultraviolet–visible and photoluminescence analyses confirmed the synthesis of CdSe QDs with unique absorption and luminescence at 430 nm and 605 nm, respectively.     

Water-soluble quantum dots for biomedical applications

Semiconductor nanocrystals are 1-10nm inorganic particles with unique size-dependent optical and electrical properties due to quantum confinement (so they are also called quantum dots). Quantum dots are new types of fluorescent materials for biological labeling with high quantum efficiency, long-term photostability, narrow emission, and continuous absorption spectra. Here, we discuss the recent development in making water-soluble quantum dots and related cytotoxicity for biomedical applications.     

Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum

Cryptosporidium parvum is a waterborne pathogen that poses potential risk to drinking water consumers. The detection of Cryptosporidium oocysts, its transmissive stage, is used in the latest U.S. Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-Cryptosporidium sp. monoclonal antibody (MAb). However, FITC exhibits low resistance to photodegradation. This property will inevitably limit the detection accuracy after a short period of continuous illumination. In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection. In this study, QD605-streptavidin together with biotinylated MAb was used for C. parvum oocyst detection. The C. parvum oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above. The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 +/- 501 oocysts (mean +/- standard deviation) was determined to be 20 nM. The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (P > 0.126) when various C. parvum oocyst concentrations were used. The QD605 was highly photostable while the FITC intensity decreased to 19.5% +/- 5.6% of its initial intensity after 5 min of continuous illumination. The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water. This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting C. parvum oocysts with the advantage of having a high photobleaching threshold.     

Application of quantum dots as probes for correlative fluorescence, conventional, and energy-filtered transmission electron microscopy.

Luminescent semiconductor quantum dots (QDs) are a new class of fluorescent label with wide-ranging applications for cell imaging. The electron density and elemental composition of these materials permit the extension of their use as probes in conventional electron microscopy (TEM) and energy-filtered TEM (EFTEM). Here we illustrate the feasibility of using streptavidin-conjugated QDs as TEM tags by labeling a nuclear protein on cell sections and obtaining correlative fluorescence and TEM data. We also show that QD probes can be employed in conjunction with immunogold for co-localization of proteins at the ultrastructural level. Furthermore, by obtaining cadmium elemental maps of CdSe/ZnS QDs distributed on a nuclear structure, we demonstrate the potential of QDs for co-localization of multiple proteins when used in combination with EFTEM.