Study of fluorescence quenching and dialysis process of CdTe quantum dots, using ensemble techniques and fluorescence correlation spectroscopy

Luminescence properties of quantum dots (QDs) are closely related to their surface structure and chemical properties. In this work some ensemble techniques and fluorescence correlation spectroscopy (FCS) were used to study the fluorescence quenching and dialysis process of CdTe QDs. It is found that when some heavy metal ions, such as silver ions (Ag+), quench QDs, the free Ag+ ions bind with bare Te atoms and form the AgTe structure on the surface. The FCS experimental results show that the quenching process is not the gradual reduction of fluorescence intensity of single QDs, but the decrease in the number of bright QDs with the addition of Ag+ ions. In other words, the bright QDs turn into dark directly in the quenching process. It is observed that some dark QDs converse into the bright QDs in the dialysis experiments and the dialysis process can improve the brightness per QDs. Furthermore, the results of FCS and fluorescence spectroscopy illustrate that the increase of the fluorescence quantum yield (QY) is mainly attributed to the removal of excess unreacted Cd-MPA complex and the possible chemical change of the QDs surface in the dialysis process. These new results can help us to further understand the complex surface structure of water-soluble QDs, improve their surface chemical features, and expand their applications in some fields.     

Magnetic beads-based electrochemiluminescence immunosensor for determination of cancer markers using quantum dot functionalized PtRu alloys as labels

A novel electrochemiluminescence (ECL) immunosensor for sensitive detection of human chorionic gonadotrophin antigen (HCG-Ag) was constructed using CdTe quantum dot functionalized nanoporous PtRu alloys (QDs@PtRu) as labels for signal amplification. In this paper, nanoporous PtRu alloy was employed as the carrier for immobilization of CdTe QDs and antibodies. Primary monoclonal antibody to alfa-HCG antigen (McAb(1)) was immobilized onto the surface of chitosan coated Fe(3)O(4) magnetic nanoparticles (Fe(3)O(4)/CS MNPs) by glutaraldehyde (GA) as coupling agent. Then McAb(1) could be easily separated and assembled on the surface of indium tin oxide glass (ITO) owing to their excellent magnetic properties with external magnetic forces holding the MNPs. Due to signal amplification from the high loading of CdTe QDs, 4.67-fold enhancements in ECL signal for HCG-Ag detection was achieved compared to the unamplified method (single QDs as labels). Under optimal conditions, a wide detection range (0.005~50 ng mL(-1)) and low detection limit (0.8 pg mL(-1)) were achieved through the sandwich-type immunosensor. The novel immunosensor showed high sensitivity and selectivity, excellent stability, and good reproducibility, and thus has great potential for clinical detection of HCG-Ag. In particular, this approach presents a novel class of combining bifunctional nanomaterials with preferable ECL properties and excellent magnetism, which suggests considerable potential in a wide range of applications for bioassays.     

Size dependence of protein diffusion very close to membrane surfaces: measurement by total internal reflection with fluorescence correlation spectroscopy

The diffusion coefficients of nine fluorescently labeled antibodies, antibody fragments, and antibody complexes have been measured in solution very close to supported planar membranes by using total internal reflection with fluorescence correlation spectroscopy (TIR-FCS). The hydrodynamic radii (3-24 nm) of the nine antibody types were determined by comparing literature values with bulk diffusion coefficients measured by spot FCS. The diffusion coefficients very near membranes decreased significantly with molecular size, and the size dependence was greater than that predicted to occur in bulk solution. The observation that membrane surfaces slow the local diffusion coefficient of proteins in a size-dependent manner suggests that the primary effect is hydrodynamic as predicted for simple spheres diffusing close to planar walls. The TIR-FCS data are consistent with predictions derived from hydrodynamic theory. This work illustrates one factor that could contribute to previously observed nonideal ligand-receptor kinetics at model and natural cell membranes.     

Reversible modulation of quantum dot photoluminescence using a protein- bound photochromic fluorescence resonance energy transfer acceptor

Multiple copies ( approximately 20) of Escherichia coli maltose binding protein (MBP) were coordinated to luminescent semiconductor quantum dots (QDs) via a C-terminal oligohistidine segment. The MBP was labeled with a sulfo-N-hydroxysuccinimide-activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]) at two different dye-to-MBP ratios; D/P = 1 and 5. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using white light (>500 nm) or UV light ( approximately 365 nm), respectively. QDs surrounded by MBP-BIPS with D/P = 1 were quenched on average approximately 25% with consecutive repeated switches, while QDs surrounded by MBP-BIPS with D/P = 5 were quenched approximately 60%. This result suggests a possible use of BIPS-labeled proteins in QD-based nanostructures as part of a threshold switch or other biosensing device.     

Studies on bioconjugation of quantum dots using capillary electrophoresis and fluorescence correlation spectroscopy

In this paper, we systematically investigated the conjugation of quantum dots (QDs) with certain biomolecules using capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) methods. Commercial QDs and aqueous-synthesized QDs in our lab were used as labeling probes, certain bio-macromolecules, such as proteins, antibodies, and enzymes, were used as mode samples, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfo-succinimide (Sulfo-NHS) were used as linking reagents. We studied the effects of certain factors such as the isoelectric points (pIs) of bio-macromolecules and buffer pH on the bioconjugation of QDs, and found that the pIs of bio-macromolecules played an important role in the conjugation reaction. By the optimization of the buffer pH some proteins with different pIs were efficiently conjugated with QDs using EDC and Sulfo-NHS as linking agents. Furthermore, we on-line investigated the kinetic process of QDs-bioconjugation by FCS and found that the conjugation reaction of QDs with protein was rapid and the reaction process almost completed within 10 min. We also observed that QDs conjugated with proteins were stable for at least 5 days in phosphate buffer. Our work described here will be very helpful for the improvement of the QDs conjugation efficiency in bioapplications.     

Detection of analyte binding to microarrays using gold nanoparticle labels and a desktop scanner

Microarray hybridization or antibody binding can be detected by many techniques, however, only a few are suitable for widespread use since many of these detection techniques rely on bulky and expensive instruments. Here, we describe the usefulness of a simple and inexpensive detection method based on gold nanoparticle labeled antibodies visualized by a commercial, office desktop flatbed scanner. Scanning electron microscopy studies showed that the signal from the flatbed scanner was proportional to the surface density of the bound antibody-gold conjugates, and that the flatbed scanner could detect six attomoles of antibody-gold conjugates. This detection system was used in a competitive immunoassay to measure the concentration of the pesticide metabolite 2,6-dichlorobenzamide (BAM) in water samples. The results showed that the gold labeled antibodies functioned comparably with a fluorescent based immunoassay for detecting BAM in water. A qualitative immunoassay based on gold-labeled antibodies could determine if a water sample contained BAM above and below 60-70 ng L(-1), which is below the maximum allowed BAM concentration for drinking water (100 ng L(-1)) according to European Union legislation.     

Diffusion current at microdisk electrodes—application to accurate measurement of diffusion coefficients

Stationary electrodes (platinum and glassy carbon) were used for accurate measurement of diffusion coefficients. The theoretical diffusion current-time profile was calculated by digital simulation, by means of which the diffusion coefficient and the product of the charge number of electrode reaction n and the reactant concentration c^o were determined simultaneously from a single chronoamperogram. The feasibility of the method was demonstrated by measuring the diffusion coefficients of Tl(I) ions. When the nc^o value agreed with the corresponding known value, the diffusion coefficients were in good agreement with the standard values determined by the thin-walled hanging mercury drop electrode method. The diffusion coefficient of hexacyanoferrate(III) ions in 1 mol dm^?3 KCl at, 25°C was also determined.     

Quantitative measurement of quantum dot uptake at the cell population level using microfluidic evanescent-wave-based flow cytometry

The intracellular uptake of nanoparticles (NPs) is an important process for molecular and cellular labeling, drug/gene delivery and medical imaging. The vast majority of investigations into NP uptake have been conducted using confocal imaging that is limited to observation of a small number of cells. Such data may not yield quantitative information about the cell population due to the tiny sample size and the potential heterogeneity. Flow cytometry is the technique of choice for studying cell populations with single cell resolution. Unfortunately, classic flow cytometry detects fluorescence from whole cells and does not shed light on subcellular dynamics. In this report, we demonstrate the use of microfluidics-based total internal reflection fluorescence flow cytometry (TIRF-FC) for examining initial quantum dot (QD) entry into cells and the associated subcellular movement at the single cell level with a rate of ～200 cells s(-1). Our cytometric tool allows extraction of quantitative data from a large cell population and reveals details about the QD transport in the periphery of the cell membrane (～100 nm deep into the cytosol). Our data indicate that the fluorescence density at the membrane vicinity decreases after initial QD dosage due to the decline in the density of QDs in the evanescent field and the transport into the cytosol is very rapid.     

量子点荧光共振能量转移检测赭曲霉毒素A

基于量子点与荧光猝灭基团之间构成的荧光共振能量转移体系,以量子点标记赭曲霉毒素A适配体与荧光猝灭基团标记的补体杂交构成荧光传感探针,当有赭曲霉毒素A存在时,由于其适配体与赭曲霉毒素A的高度亲和作用,使传感探针上结合的荧光猝灭剂减少,荧光增强,从而建立了一种检测赭曲霉毒素A的荧光分析方法.该方法简单、快速、特异性强,在适...     

A study of two-electron quantum dot spectrum using discrete variable representation method

A variational method called discrete variable representation is applied to study the energy spectra of two interacting electrons in a quantum dot with a three-dimensional anisotropic harmonic confinement potential. This method, applied originally to problems in molecular physics and theoretical chemistry, is here used to solve the eigenvalue equation to relative motion between the electrons. The two-electron quantum dot spectrum is determined then with a precision of at least six digits. Moreover, the electron correlation energies for various potential confinement parameters are investigated for singlet and triplet states. When possible, the present results are compared with the available theoretical values.     

A theoretical method to analyze diffusion of probe molecules in nanostructured fluids by fluorescence correlation spectroscopy

In this paper, we study the fluorescence fluctuation correlation function in structured fluids where the diffusion coefficients of probe molecules have different values depending on the distance from initial position, and we derive two simple expressions. Both of them reproduce the exact numerical results rather accurately. One of the expressions contains a time-dependent diffusion coefficient and has a clear physical meaning. We show a procedure to analyze experimental data using the time-dependent diffusion coefficient which results from crossover from free diffusion inside a mesh to hindered diffusion through mesh structures.     

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.     

离子液体中硝基苯的扩散系数与量子化学参数的相关研究

采用量子化学半经验AM1方法,分别对[CMIM]+(1,3-二甲基咪唑离子)、[C2MIM]+(1-甲基-3-乙基咪唑离子)、[C4MIM]+(1-甲基-3-丁基咪唑离子)、[C6MIM]+(1-甲基-3-己基咪唑离子)和硝基苯,以及它们1∶1复合物构象进行优化,在此基础上以DFT(B3LYP/6-31+G)计算它们的...     

Efficient assembly of multi-walled carbon nanotube-CdSe/ZnS quantum dot hybrids with high biocompatibility and fluorescence property

CdSe/ZnS core-shell quantum dots (QDs) were efficiently tethered onto polyamidoamine dendrimer-modified multi-walled carbon nanotubes (MWCNTs) by covalent linkage and mercapto-mediated assembly. The obtained MWCNT-QD hybrids were both photophysically and morphologically characterized. The QDs are well-distributed on single nanotube surface in high density and the assembly of QDs onto MWCNTs does not change the fluorescence emission wavelength of QDs but significantly decreases the emission density. Cytotoxicity of MWCNT-QD hybrids to HeLa cells and their fluorescence property in living cell system were evaluated in detail. The hybrids show a little effect on cell viability even at very high concentration (100 μg mL(-1)). Moreover, they possess intense red fluorescence signal under optical fluorescence microscopy and good fluorescence stability over 72-h exposure in living cell system.     

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3 ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.     

Construction of a molecular beacon based on two-photon excited fluorescence resonance energy transfer with quantum dot as donor

A new molecular beacon (MB) driven by two-photon excitation (TPE) using quantum dots as energy donor is constructed, which provides reduced direct excitation of acceptor and is free of interferences from autofluorescence or scattering light in a complicated biological matrix.     

Quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling

We presented a sensitive method to quantify antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling. In this method, the biotinylated monoclonal anti-human IgG molecules were immobilized on the silanized glass substrate surface. By the strong biotin-streptavidin affinity, streptavidin-coated quantum dots were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots in the evanescent wave field were obtained by a high-sensitivity electron multiplying charge-coupled device. Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted, one by one. The linear range of 8.0 × 10⁻¹⁴ to 5.0 × 10⁻¹² mol L⁻¹ was obtained between the number of single molecules and the sample concentration.     

Determination of nicotinyl pesticide residues in vegetables by micellar electrokinetic capillary chromatography with quantum dot indirect laser-induced fluorescence

A new assay was developed by use of micellar electrokinetic capillary chromatography with indirect LIF fluorescence for the determination of thiamethoxam, acetamiprid, and imidacloprid residues in vegetables, in which the cadmium telluride quantum dots (QDs) synthesized in aqueous phase were used as fluorescent background substance and their excitation and emission wavelengths matched with LIF detector by engineering their size. The factors that affected the peak height and the resolution were optimized. The running buffer was composed of 4.4 μM cadmium telluride QDs as fluorescent background substance, 40 mM borate and 60 mM SDS, and its pH was adjusted to 8.0. The separation voltage was 25 kV. Under the optimum conditions, the detection limits were 0.05, 0.01, and 0.009 mg/kg; the linear dynamic ranges were 0.5-30, 0.1-30, and 0.1-30 mg/L; and the average recoveries of spiked samples were 72.0-101.2, 74.0-106.7, and 77.8-105.1% for thiamethoxam, acetamiprid, and imidacloprid, respectively. The assay can meet the requirement of maximum residue limits to these three pesticides in the regulations of European Union and Japan, and has been applied for determining their residues in vegetables.     

Determination of five quinolone antibiotic residues in foods by micellar electrokinetic capillary chromatography with quantum dot indirect laser-induced fluorescence

A new assay was developed for the determination of five quinolone antibiotic residues in foods, loxacin, enrofloxacin, ciprofloxacin, lomefloxacin, and norfloxacin, by micellar electrokinetic capillary chromatography with indirect laser-induced fluorescence, in which cadmium telluride quantum dots were used as a fluorescent background substance. Some factors that affected the peak height and the resolution were examined. The optimized running buffer was composed of 20 mM SDS, 7.2 mg/L quantum dots, and 10 mM borate at pH 8.8. The separation voltage was 20 kV. Under these conditions, five quinolone antibiotic residues were separated successfully within 8 min. The detection limits ranged from 0.003 to 0.008 mg/kg; the linear dynamic ranges were all 0.01?～?10 mg/kg; and the average recoveries of the spiked samples were 81.4?～?94.6 %. The assay can meet the requirement of maximum residue limits to these five quinolone antibiotics in the regulations of the European Union and Japan and has been applied for determining their residues in animal-derived food.