Conventional Fluorophores Research Articles

Photolithographic process for the patterning of quantum dots

Recently, quantum dots have been used as molecular probes substituting for conventional organic fluorophores. Quantum dots are stable against photobleaching and have more controllable emission bands, broader absorption spectra, and higher quantum yields. In this study, an array of ZnS-coated CdSe quantum dots on a slide glass has been prepared by photolithographic method. The array pattern was prepared using a positive photoresist (AZ1518) and developer (AZ351). The patterned glass was silanized with 3-aminopropyltriethoxysilane (APTES), and carboxyl-coated quantum dots were selectively attached onto the array pattern. The silanization was examined by measuring contact angle and the surface of the array pattern was analyzed using AFM and fluorescent microscope.

Luminescent Lanthanide Nanoparticles as Labels in DNA Microarrays for Quantification of Methyl Tertiary Butyl Ether Degrading Bacteria

We report application of lanthanide nanoparticles for DNA quantification in a microarray platform as a substitute for conventional organic fluorophores. A non-PCR based DNA microarray assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether (MTBE) was demonstrated. Probe DNA was immobilized on a glass surface, hybridized with biotinylated target DNA and subsequently incubated with Neutravidin-biofunctionalized nanoparticles. The fluorescence spot intensities, measured by a commercial laser scanner, show a linear relationship (R2 = 0.98) with bacterial 16S rDNA over a range of target DNA concentrations, while the background fluorescence remained low. In addition, nanoparticles fluorescence shows a stronger intensity than Quasar570 (Cy3). Present sensitivity of the assay is 10 pM of target DNA. The selectivity of the DNA-nanoparticle-probes to discriminate a non-target DNA with two base pairs mismatch in the 16S rDNA gene sequence was shown. The use of Eu:Gd2O3 nanoparticles as biolabels provides a relatively non-toxic, inexpensive, rapid and sensitive alternative to the materials currently used in DNA microarrays.

Resonance Energy Transfer in Steady-State and Time-Decay Fluoro-Immunoassays for Lanthanide Nanoparticles Based on Biotin and Avidin Affinity

Compared with conventional organic fluorophores, lanthanide-doped nanoparticles as donor and gold nanoparticles as quencher-like acceptor in energy transfer studies offer many technological advantages; in particular, the long-lifetime donor endows the fluorescence resonance energy transfer (FRET) assay with great signal-to-noise ratio, and the quencher acceptor allows monitoring of the donor channel alone. The LaPO4:Ce,Tb nanoparticles with high quantum yield in aqueous solution are readily paired with differently sized gold nanoparticles through the well-established binding affinity of biotin and avidin. The spectra overlap between the emission of lanthanide nanoparticles and absorption of gold nanoparticles meets the prerequisite for energy transfer, and the resonance energy transfer process is characterized by both steady-state and time-decay luminescent measurements. With the emission contributions from Tb3+ ions located at the surface and inside the lanthanide nanoparticles taken into account, the co...

Luminescent lanthanide nanoparticles as labels in DNA microarrays for quantification of methyl tertiary butyl ether degrading bacteria.

We report application of lanthanide nanoparticles for DNA quantification in a microarray platform as a substitute for conventional organic fluorophores. A non-PCR based DNA microarray assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether (MTBE) was demonstrated. Probe DNA was immobilized on a glass surface, hybridized with biotinylated target DNA and subsequently incubated with Neutravidin-biofunctionalized nanoparticles. The fluorescence spot intensities, measured by a commercial laser scanner, show a linear relationship (R2 = 0.98) with bacterial 16S rDNA over a range of target DNA concentrations, while the background fluorescence remained low. In addition, nanoparticles fluorescence shows a stronger intensity than Quasar570 (Cy3). Present sensitivity of the assay is 10 pM of target DNA. The selectivity of the DNA-nanoparticle-probes to discriminate a non-target DNA with two base pairs mismatch in the 16S rDNA gene sequence was shown. The use of Eu:Gd2O3 nanoparticles as biolabels provides a relatively non-toxic, inexpensive, rapid and sensitive alternative to the materials currently used in DNA microarrays.

Quantum dots as donors in fluorescence resonance energy transfer for the bioanalysis of nucleic acids, proteins, and other biological molecules

Quantum dots (QDs) have a number of unique optical properties that are advantageous in the development of bioanalyses based on fluorescence resonance energy transfer (FRET). Researchers have used QDs as energy donors in FRET schemes for the analysis of nucleic acids, proteins, proteases, haptens, and other small molecules. This paper reviews these applications of QDs. Existing FRET technologies can potentially be improved by using QDs as energy donors instead of conventional fluorophores. Superior brightness, resistance to photobleaching, greater optimization of FRET efficiency, and/or simplified multiplexing are possible with QD donors. The applicability of the Förster formalism to QDs and the feasibility of using QDs as energy acceptors are also reviewed.

Fluorescent Imaging Applications of Quantum Dot Probes

Quantum dots (QDs) have rapidly emerged as an attractive alternative to conventional organic fluorophores in a variety of biological imaging applications. Their improved photostability allows for long-term dynamic imaging of cellular processes and their narrow, size-tunable emission permits unprecedented multiplexing capabilities. Additionally, the inherent brightness of core/shell QDs, with quantum yields capable of exceeding 85%, provides increased sensitivity for both diagnostic screening and single molecule tracking applications. To date, the primary focus of research in this field has been directed towards modifying the surface chemistries of the QDs to introduce biological specificity while, at the same time, limiting nonspecific cellular interactions. As such, biomolecules such as antibodies, peptides, streptavidin and biotin have all been conjugated to QDs and been used to demonstrate specific labeling of cellular targets. Additionally, polyethylene glycol (PEG) modification of the QD surface has been shown to limit nonspecific interactions. The use of small molecule QD-conjugates has also been demonstrated as an effective means for targeted labeling of membrane associated receptors. This approach introduces specificity via ligand-receptor interactions, resulting in a highly modular system which is easily modified to interrogate a wide variety of cellular targets. This article provides a comprehensive review of the current status of QD imaging applications in biological systems with a particular emphasis on the design and application of small molecule nanoconjugates.

In Vivo Imaging Using Quantum Dot–Conjugated Probes

This unit describes the use of quantum dots (QDs) for live-cell imaging and the use of QDs in flow cytometry for quantitative analysis of ligand binding constants and receptor density. Conventional fluorophores and visible fluorescent protein (VFP) constructs have allowed visualization of many cellular processes. However, organic and biomolecular fluorophores have limitations in their applications, due to their small Stokes' shift and tendency to photobleach during prolonged imaging. QDs have many advantages over conventional fluorophores, including high brightness and photostability, which make them an exceptional tool for live-cell imaging. There are a large variety of commercially available QDs with different surface reactivities and characteristics. The authors have limited the laboratory protocols presented here to the use of streptavidin-coupled QDs because this gives almost universal applicability to any cell surface receptor by coupling the ligand or antibody that recognizes the receptor to biotin and visualizing the complex by use of QDs.

Surfactant‐Modified Hydrophilic Nanostructured Porous Silicon for the Photosensitized Formation of Singlet Oxygen in Water

Porous silicon (porSi), which contains luminescent Si nanocrystal assemblies, is a promising semiconductor material for the photosensitized formation of singlet oxygen, 1 O2, for biological applications. However, because as-prepared luminescent Si nanocrystals are H-terminated and therefore hydrophobic, the first step to create a prototype of Si nanocrystalbased photosensitizer should be a modification of the Si nanocrystal surface with the aim to make it hydrophilic and able to work in a biological ambient. Such surface modification by surfactants should not, however, result in a decrease of the nanocrystals’ durability that may, in principle, take place because of surfactant-induced weakening of surface tension and the consequent increase of interaction with H2O molecules. Surfactants should not also decrease the ability of the nanocrystals to transfer the excitation energy to acceptors (first of all molecular oxygen) on their surface. These rather contradictory tasks have been largely solved in the present work by use of nonionic surfactants physically adsorbed on the porSi surface. One of the strategic objectives of nanotechnology is the development of new materials of nanometer size that have entirely new physical properties, and, therefore, new functionality. Over the last decade, tailoring of material characteristics by size control has been demonstrated for many types of semiconductors. Optical and electronic properties of semiconductor nanocrystals can be simply engineered by changing their size and composition. When electrons and holes are squeezed into a dimension that approaches a critical size, quantum-confinement effects become apparent. This effect can be seen experimentally as a widening of the semiconductor nanocrystal bandgap. [1] In case of direct-bandgap semiconductors the quantum-confinement effect is now widely used in so-called “quantum dots”, a very promising class of bright fluorescence biological labels that are able to overcome the limitations of conventional organic fluorophores. [2] In case of Si, which is an indirect bandgap semiconductor, radiative transition is forbidden, which is an inherent limitation for light-emitting applications of Si nanocrystals. Nevertheless, the same peculiarity is found to be a great advantage for photosensitizing applications, namely for the energy transfer from long-lived Si nanocrystal excitons to O2 and the production of extremely reactive excited (“singlet”) molecular oxygen, 1 O2. [3] By taking into account the broadband absorption of Si nanocrystals, [4] it be

Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry

Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses.

A novel fluorescent array for mercury (II) ion in aqueous solution with functionalized cadmium selenide nanoclusters

Mono-disperse CdSe nanoclusters have been prepared facilely and functionalized with l-cysteine through two steps by using safe and low cost substances. They are water-soluble and biocompatible. Especially these functionalized quantum dots can be stably soluble in water more than for 30 days, and the intensity of fluorescence and absorbance was decreased less than 15% of fresh prepared CdSe colloids. These functionalized CdSe QDs exhibited strong specific affinity for mercury (II) through QDs interface functional groups. Based on the quenching of fluorescence signals of functionalized CdSe QDs at 530 nm and no obvious wavelength shift or no new emission band in present of Hg (II) at pH 7.75 of phosphate buffer solution, a simple, rapid and specific array for Hg (II) was proposed. In comparison with conventional organic fluorophores, these nanoparticles are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimum conditions, the response of linearly proportional to the concentration of Hg (II) between 0 and 2.0 × 10 −6 mol L −1, and the limit of detection is 6.0 × 10 −9 mol L −1. The relative standard deviation of six replicate measurements is 1.8% for 1.0 × 10 −7 mol L −1 Hg (II). The mechanism of reaction is also discussed. The proposed method was successfully applied for Hg (II) detection in four real samples with a satisfactory result that was obtained by cold vapor atomic fluorescence spectrometry (CV-AFS).

Horseradish Peroxidase-Driven fluorescent Labeling of Nanotubes with Quantum Dots

We describe the first enzyme-driven technique for fluorescent labeling of single-walled carbon nanotubes (SWNTs). The labeling was performed via enzymatic biotinylation of nanotubes in the tyramide-horseradish peroxidase (HRP) reaction. Both direct and indirect fuorescent labeling of SWNTs was achieved using either biotinyl tyramide or fluorescently tagged tyramides. Biotinylated SWNTs later reacted with streptavidin-conjugated fluorophores. Linking semiconductor nanocrystals, quantum dots (Q-dots), to the surface of nanotubes resulted in their fluorescent visualization, whereas conventional fluorophores bound to SWNTs directly or through biotin-streptavidin linkage, were completely quenched. Enzymatic biotinylation permits fluorescent visualization of carbon nanotubes, which could be useful for a number of biomedical applications. In addition, other organic molecules such as proteins, antibodies, or DNA can be conjugated to biotinylated SWNTs using this approach.

Nanoparticles in biomolecular detection

The use of nanoparticles as labels in biomolecular detection in place of conventional molecular fluorophores has led to improvements in sensitivity, selectivity, and multiplexing capacity. Nevertheless, further simplification is needed to take these technologies from the laboratory to point of care. Here, we summarize the latest developments in the use of nanoparticles as labels, especially in bioaffinity sensors for the detection of nucleic acids and proteins.

Liposome biodistribution by time resolved fluorimetry of lipophilic europium complexes

The use of conventional fluorophores suffers from some limitations in biological fluids due to low signal/background ratio. Today, this sensitivity issue might be reasonably improved thanks to lanthanide chelates, by selective detection of long decay fluorescence. Use of pulsed light source time-resolved fluorimetry takes into account the fluorescence decay time of the lanthanide chelates to gain sensitivity in biological media. Lipid-DTPA: Eu compounds have been prepared and incorporated into liposomes to evaluate europium based detection of liposomes in biological media. Fluorescence emission was not modified by this incorporation. Europium labelled liposomes were used for biodistribution studies and showed their use in this context.

High‐Resolution Colocalization of Single Molecules within the Resolution Gap of Far‐Field Microscopy

To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.

Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer.

A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.

Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping.

The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm(2). Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.

A new technique for multiparameter imaging microscopy: use of long decay time photoluminescent labels enables multiple color immunocytochemistry with low channel-to-channel crosstalk.

In this report, we describe luminescence imaging microscopy using five different photoluminescent dyes in a single specimen. We combined the long decay time luminophores, europium(III) chelate, terbium(III) chelate, palladium(II) coproporphyrin, and platinum(II) coproporphyrin, with a green nuclear stain, Syto 25 trade mark, that emits conventional fast decaying fluorescence. The luminescence emissions from the five different luminophores were separated from each other by the differences in spectra and decay times using time-resolved detection. Applicability of this dye-combination for multiparameter analysis of a biological object was verified in a mixed population of peripheral blood leukocytes. Leukocyte cytocentrifugates were incubated in one step with a cocktail of luminophore-conjugated antibodies recognizing neutrophil- and lymphocyte-specific markers, followed by rapid staining with a mixture of nuclear stain and Pt-porphyrin as an eosinophil stain. The results show that multiple luminescent dyes with long decay time can be used together, and in combination with a conventional fluorophore. The separation of the signals of the long decay time labels was distinctive and enabled reliable identification of different leukocyte types, as well as an automated cell count. The long decay time luminophores together with time-resolved luminescence imaging microscopy (TR-LIM) provide a unique tool for studies of simultaneous expression of multiple antigens at the level of a single cell. In comparison with other multiparameter imaging techniques, the described technique offers increased accuracy of results, simplification of preparation procedure, and dramatic shortening of the total processing time. To our knowledge, this is the first time that simultaneous fivefold labeling/staining and analysis in a single specimen has been performed in the field of immunocytochemistry.

Visualization of individual carbon nanotubes with fluorescence microscopy using conventional fluorophores

We demonstrate that individual carbon nanotubes (CNTs) can be visualized with fluorescence microscopy through noncovalent labeling with conventional fluorophores. Reversal of contrast in fluorescence imaging of the CNTs was observed when performing labeling procedure in a nonpolar solvent. Our results are consistent with a CNT-fluorophore affinity mediated by hydrophobic interaction. The reverse-contrast images also provide clear indication of nanotube location.

Novel fluorophores for single-molecule imaging.

Nonlinear optical chromophores based on dicyanodihydrofuran acceptors paired with amine donors have been found to exhibit sufficiently large fluorescence quantum yields and stability to enable single-molecule detection in polymeric hosts. To illustrate the breadth of this class, six fluorophores are presented, spanning the emission range from 505 to 646 nm. In contrast to conventional single-molecule fluorophores, the new molecules feature sensitivity to local rigidity, large ground-state dipole moments, and large polarizability anisotropies, properties that can be used to design new reporter experiments at the single-molecule level.