Quantum dots as strain- and metabolism-specific microbiological labels

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.     

Incorporating fluorescent dyes and quantum dots into magnetic microbeads for immunoassays

Microbeads that are both paramagnetic and fluorescently labeled are commercially available in colors spanning the visible spectrum. Although these commercial beads can be bright, polydispersity in both size and fluorescent intensity limit their use in quantitative assays. Very recently, more monodisperse beads have become available, but their large size and surface properties make them less than ideal for some bioassay applications. Here we describe methods to customize commercial nonfluorescent magnetic microparticles with fluorescent dyes and quantum dots (QDs) without affecting their magnetic or surface chemical properties. Fluorescent dyes and 3.3-nm diameter CdSe/ZnS QDs were sequestered within 0.8-micron diameter magnetic beads by swelling the polystyrene matrix of the bead in organic solvent, letting the chromophores partition, and then collapsing the matrix in polar solvents. Chromophore incorporation has been characterized using both UV-visible absorption spectroscopy and fluorescence microscopy, with an average of 3 x 10(8) rhodamine 6G molecules/bead and 6 x 10(4) QDs/bead. The modified beads are uniform in size and intensity, with optical properties comparable to currently available commercial beads. Immunoassay results obtained with our custom fluorescent magnetic microbeads are consistent with those obtained using conventional magnetic microbeads.     

Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers.

Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei.     

Functionalized fluorescent core-shell nanoparticles used as a fluorescent labels in fluoroimmunoassay for IL-6

Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications. In this paper, a sensitive fluoroimmunoassay for recombinant human interleukin-6 (IL-6) with the functionalized Rubpy-encapsulated fluorescent core-shell silica nanoparticles labeling technique has been proposed. IL-6 was measured based on the specific interaction between captured IL-6 antigen and functionalized fluorescent core-shell nanoparticles-labeled anti-IL-6 monoclonal antibody. The calibration graph for IL-6 was linear over the range 20-1250 pg ml(-1) with a detection limit of 7 pg ml(-1) (3 sigma). The regression equation of the working curve is I(F)=7.665+32.499[IL-6] (ng ml(-1)) (r=0.9980). The relative standard deviation (R.S.D.) for 11 parallel measurements of 78 pg ml(-1) IL-6 was 3.2%. Furthermore, the application of fluorescence microscopy imaging in the study of the antibody labeling and sandwich fluoroimmunoassay with the functionalized fluorescent core-shell silica nanoparticles was also explored. This proposed method has the advantage of showing the specificity of immunoassay and sensitivity of fluorescent nanoparticle labels technology. The results demonstrate that the method offers potential advantages of sensitivity, simplicity and reproducibility for the determination of IL-6, and is applicable to the determination of IL-6 in serum samples and enabling fluorescence microscopy imaging for the determination of IL-6.     

Oligomeric fluorescent labels for DNA

In an effort to find fluorescent labels that have large Stokes shifts and increased emission intensity, a strategy for fluorescence labeling of DNA was explored in which multiple individual fluorophores are incorporated at adjacent positions at the end of a DNA probe. To encourage close interactions, hydrocarbon and heterocycle fluorophores were substituted at C-1 of deoxyribose, replacing the DNA base. The C-glycosides studied contained the well-known fluorophores terphenyl, pyrene, and terthiophene. For comparison, a commercial fluorescein-dU nucleotide was examined. Oligomeric labels containing up to five fluorophores were tested. Interestingly, all four dyes behaved differently on multiple substitution. Fluorescein displayed strong self-quenching properties, with the quantum yield dropping severalfold with each additional substitution and with a constant, small Stokes shift. In contrast, pyrene showed increases in quantum yield on addition of more than one fluorophore and yielded efficient long-wavelength emission on multiple substitution, with Stokes shifts of >130 nm. Oligomeric terphenyl labels gave a small progressive red shift in absorption and a marked red shift in emission wavelength and showed a strong increase in brightness with more monomers. Finally, terthiophene oligomers showed self-quenching combined with increasing Stokes shifts. Overall, the results suggest that some oligomeric fluorescent labels exhibit properties not available in common fluorescein class (or other commercial) labels, such as large Stokes shifts and increasing brightness with increasing substitution.     

Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides

Abstract In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λex=355 nm and λem=440 nm. We concluded that the optimal wavelengths for 3-AQ were λex=355 nm and λem=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λex/em values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent pro\xadperties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.     

DBD dyes as fluorescent probes for sensing lipophilic environments

Small fluorescent organic molecules based on [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) could be used as probes for lipophillic microenvironments in aqueous solutions by indicating the critical micelles concentration of detergents and staining cell organelles. Their fluorescence lifetime decreases drastically by the amount of water in their direct environment. Therefore they are potential probes for fluorescence lifetime imaging microscopy (FLIM).     

Microfluidic bead-based enzymatic primer extension for single-nucleotide discrimination using quantum dots as labels

This study reports the development of an on-chip enzyme-mediated primer extension process based on a microfluidic device with microbeads array for single-nucleotide discrimination using quantum dots as labels. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. The applied allele-specific primer extension method employed a nucleotide-degrading enzyme (apyrase) to achieve specific single-nucleotide detection. Based on the apyrase-mediated allele-specific primer extension with quantum dots as labels, on-chip single-nucleotide discrimination was demonstrated with high discrimination specificity and sensitivity (0.5 pM, signal/noise > 3) using synthesized target DNA. The chip-based signal enhancement for single-nucleotide discrimination resulted in 200 times higher sensitivity than that of an off-chip test. This microfluidic device successfully achieved simultaneous detection of two disease-associated single-nucleotide polymorphism sites using polymerase chain reaction products as target. This apyrase-mediated microfluidic primer extension approach combines the rapid binding kinetics of homogeneous assays of suspended microbeads array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for single-base analysis with small reagent consumption, short assay time, and parallel detection.     

Photostability studies of phycobiliprotein fluorescent labels

Photostability studies of four fluorescent phycobiliproteins were conducted to identify stable chromophores for biological labeling applications. Phycobiliprotein photodestruction was linear with the applied laser power and depended on the total number of photons absorbed per molecule. Photodestruction quantum yields phi of 1.1 X 10(-5) for R-phycoerythrin, 6.6 X 10(-6) for B-phycoerythrin, 4.5 X 10(-6) for allophycocyanin, and 2.5 X 10(-6) for C-phycocyanin were measured. C-Phycocyanin is a factor of 10.8 more photostable than fluorescein. The photostability of R-phycoerythrin was improved by a factor of 1.7 by adding n-propyl gallate. The addition of superoxide dismutase, catalase, sodium dithionite, ascorbate, dithiothreitol, EDTA, or deoxygenation with argon bubbling had no effect on the photostability of R-phycoerythrin.     

Separation-based glycoprofiling approaches using fluorescent labels

Glycoprotein analysis is essential within the biopharmaceutical industry, as the structure of the different glycans present can affect the safety and efficacy of products. However analysis of cleaved glycans presents a major analytical challenge, due to their inherent complexity, lack of chromophore and the existence of various isoforms (both position and linkage). In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants, so the released glycan pool contains a range of structures. Both normal phase chromatography and capillary gel electrophoresis offer excellent selectivity for the analysis of fluorescently labelled glycans. The normal phase (NP) chromatographic approach is sensitive, reliable and well established, with databases available for searching structures assigned relative to retention times. Capillary gel electrophoresis with laser induced fluorescence (CGE-LIF) offers faster analysis times, though currently no databases are available to search mobilities against structures, therefore data has to be cross-correlated with either normal phase chromatography or mass spectrometry approaches when developing and validating methods. The principles of both methods are described and a review is presented that includes evaluation against a set of criteria established through consultation with the biopharmaceutical industry.     

The application of quantum dots as fluorescent label to glycoarray

A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions was demonstrated using quantum dots (QDs) as a fluorescence label coupled with protein. 1,3-Dipolar cycloaddition between azide and alkyne was exploited to attach alpha-d-glucopyranoside to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface, and the product formation was detected by both electrospray ionization-mass spectrometry (ESI-MS) and QD- (or fluorescein isothiocyanate (FITC))-conjugated lectin binding. It indicated that the peak intensity of the fluorescence emission was proportional to the initial concanavalin A (Con A) concentration in the range of 2 x 10(-3) micromol/L to 2 x 10(-2)mmol/L with a detection limit at least 100 times lower than that of the FITC-based method.     

Quantum dots in nanobiotechnology

Research on semiconductor nanocristals (also known as quantum dots of QD) in the field of nanobiotechnology is rapidly evolving thanks to progresses in their synthesis and their surface chemistry. Two types of materials, water soluble and biocompatible single QD and beads containing QDs, are becoming available and exciting applications based on these new materials are developed. We will present the recent progress in the synthesis of these materials and their applications. We will discuss the problems that remain to be solved and the perspectives.     

Simultaneous detection of dual proteins using quantum dots coated silica nanoparticles as labels

Simultaneous detection of multianalytes associated with a particular cancer is beneficial for disease diagnosis. Here, a facile immunosensing strategy was designed to allow simultaneous electrochemical detection of dual proteins, in a single run. CdSe and PbS water-soluble quantum dots (QDs) were prepared and coated on monodisperse silica nanoparticles as labels for proteins detection. Rabbit immunoglobulin G antigen (IgG) and carcinoembryonic antigen (CEA) were chosen as model proteins for analysis. After a typical sandwich immunoassay, CdSe and PbS QDs labels were introduced onto the Au substrates' surface, which were then dissolved and could be simultaneously monitored by square-wave-voltammetric (SWV) stripping measurements. Under selected conditions, IgG and CEA could be assayed in the ranges of 0.05-40 ng mL(-1) and 0.05-25 ng mL(-1), respectively. The proposed method possessed high sensitivity, good precision, and satisfactory reproducibility and regeneration.     

Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix

We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy- requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p- [dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.     

Protein substrates of proteinases with fluorescent labels

Protein substrates for proteinases with double fluorescent fluorophors are synthesized. Pyridoxal-5'-phosphate, dansyl chloride and fluorescein isothiocyanate were used as fluorescent sounds for modification of globin. Phosphoyridoxyl fluorophor was present in the both substrates. The second label was either fluoresceinthiocarbamyl or dansyl fluorophor. Spectral characteristics and ability to hydrolysis of obtained substrates have been studied. The influence of some salts on fluorescent characteristics of those substrates have been analyzed. Differentiation of the hydrolyzed substrate from the initial one by ammonium sulphate is shown to be possible.     

Quantum dots and prion proteins

A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrP^Sc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels.     

Quantum dots brighten biological imaging

Quantum dots (QDs) are novel photostable semiconductor nanocrystals possessing wide excitation spectra and narrow, symmetrical emission spectra and can be conjugated to a wide range of biological targets, including proteins, antibodies and nucleic acid probes. These characteristics have provoked considerable interest in their use for bioimaging. Much investigation has been performed into their use for multiplex immunohistochemistry and in situ hybridisation which, when combined with multispectral imaging, has enabled quantitation and colocalisation of gene expression in clinical tissue. Many advances have recently been made using QDs for live cell and in vivo imaging, in which QD-labelled molecules can be tracked and visualised in 3-D. This review aims to outline the beneficial properties presented by QDs along with important advances in their biological application.     

Natural fruit extracts as non-toxic fluorescent dyes for staining fungal chlamydospores

Currently, most synthetic dyes utilized for fungal fluorescent staining are toxic, carcinogenic, or harmful to animals, humans, and the environment. This study proposes non-toxic extracts of fruits from the genera Rhamnus, Ribes, Sambucus, Viburnum, Sorbus and Beta as simple, safe, and ecological alternatives to chemical fluorescent dye for efficient staining of Fusarium chlamydospore cells using, as test strains, five different pathogenic Fusarium species.     

Pairs of fluorescent dyes as probes of DNA and chromosomes.

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.     

Quantum dots as nano plug-in's for efficient NADH resonance energy routing

The routing of fluorescent signals from NADH to quantum dots (QDs) has been a subject of extensive research for FRET based applications. In the present study, the spectral cross talk of NAD(+)/NADH with QDs was used to monitor the reaction of NAD(+)-dependent dehydrogenase enzyme. CdTe QD may undergo dipolar interaction with NADH as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, non-radiative energy transfer can take place from NADH to CdTe QD enhancing QDs fluorescence. Energy routing assay of NADH-QD was applied for detection of formaldehyde as a model analyte in the range 1000-0.01 ng/mL by the proposed technique. We observed proportionate quenching of CdTe QD fluorescence by NAD(+) and enhancement in the presence of NADH formed by various concentrations of enzyme (0.028-0.4 U). Hence, it was possible to detect formaldehyde in the range 1000-0.01 ng/mL with a limit of detection (LOD) at 0.01 ng/mL and regression coefficient R(2)=0.9982. Therefore, a unique optical sensor was developed for the detection of the formaldehyde in sensitive level based on the above mechanism. This method can be used to follow the activity of NAD(+)-dependent enzymes and detection of dehydrogenases in general.