The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.     

Biomedical applications of glyconanoparticles based on quantum dots

Background

Quantum dots (QDs) are outstanding nanomaterials of great interest to life sciences. Their conjugation versatility added to unique optical properties, highlight these nanocrystals as very promising fluorescent probes. Among uncountable new nanosystems, in the last years, QDs conjugated to glycans or lectins have aroused a growing attention and their application as a tool to study biological and functional properties has increased.

Development of magnetic separation and quantum dots labeled immunoassay for the detection of mercury in biological samples

A rapid and sensitive immunoassays of mercury (Hg) in biological samples was developed using quantum dots (QDs) and magnetic beads (MBs) as fluorescent and separated probes, respectively. A monoclonal antibody (mAb) that recognizes an Hg detection antigen (BSA-DTPA-Hg) complex was produced by the injection of BALB/c mice with an Hg immunizing antigen (KLH-DTPA-Hg). Then the ascites monoclonal antibodies were purified. The Hg monoclonal antibody (Hg-mAb) is conjugated with MBs to separate Hg from biological samples, and the other antibody, which is associated with QDs, is used to detect the fluorescence. The Hg in biological samples can be quantified using the relationship between the QDs fluorescence intensity and the concentration of Hg in biological samples following magnetic separation. In this method, the detection linear range is 1–1000 ng/mL, and the minimum detection limit is 1 ng/mL. The standard addition recovery rate was 94.70–101.18%. The relative standard deviation values were 2.76–7.56%. Furthermore, the Hg concentration can be detected in less than 30 min, the significant interference of other heavy metals can be avoided, and the simultaneous testing of 96 samples can be performed. These results indicate that the method could be used for rapid monitoring Hg in the body.     

Imaging Escherichia coli using functionalized core/shell CdSe/CdS quantum dots

The internalization of a series of water-soluble CdSe/CdS quantum dots (QDs) stabilized by citrate, isocitrate, succinate, and malate by Escherichia coli is established by epifluorescence and confocal fluorescence scanning microscopy, fluorimetry, and UV–vis spectroscopy on whole and lysed bacterial cells. The organic-acid-stabilized QDs span a range in size from 3.8±1.1 to 6.0±2.4 nm with emission wavelengths from 540 to 630 nm. QDs of different sizes (i.e., 3.8–6 nm) can enter the bacterium and be detected on different fluorescence channels with little interference from other QDs as a result of the distinct emission profiles (i.e., 540–630 nm, respectively). Costaining QD-labeled E. coli with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) demonstrates that the QDs and DAPI are colocalized within E. coli, whereas costaining QD-labeled E. coli with membrane dye FM4-64 shows that the FM4-64 is localized in the outer bacterial membrane and that the QDs are inside.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Application of multiple response optimization design to quantum dot-encoded microsphere bioconjugates hybridization assay

The optimization of DNA hybridization for genotyping assays is a complex experimental problem that depends on multiple factors such as assay formats, fluorescent probes, target sequence, experimental conditions, and data analysis. Quantum dot-doped particle bioconjugates have been previously described as fluorescent probes to identify single nucleotide polymorphisms even though this advanced fluorescent material has shown structural instability in aqueous environments. To achieve the optimization of DNA hybridization to quantum dot-doped particle bioconjugates in suspension while maximizing the stability of the probe materials, a nonsequential optimization approach was evaluated. The design of experiment with response surface methodology and multiple optimization response was used to maximize the recovery of fluorescent probe at the end of the assay simultaneously with the optimization of target–probe binding. Hybridization efficiency was evaluated by the attachment of fluorescent oligonucleotides to the fluorescent probe through continuous flow cytometry detection. Optimal conditions were predicted with the model and tested for the identification of single nucleotide polymorphisms. The design of experiment has been shown to significantly improve biochemistry and biotechnology optimization processes. Here we demonstrate the potential of this statistical approach to facilitate the optimization of experimental protocol that involves material science and molecular biology.     

Facile labeling of lipoglycans with quantum dots

Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of the innate immune system, yet mechanisms of their action and in particular the role of glycans remain elusive. Efficient non-invasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here we report a new method for labeling LPS and other lipoglycans with luminescent quantum dots. The labeling is achieved by partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS-Qdot conjugates is demonstrated by the labeling of mouse monocytes. This simple method should find broad applicability in studies concerned with visualization of LPS biodistribution and identification of LPS binding agents.     

量子点对细菌的标记及抗菌作用

目的量子点是近年来发展起来的一种新型的荧光纳米材料,与传统的材料相比具有独特的性质,所以在生物传感器、实时追踪、多色标记及成像等方面有着广泛的应用。本文主要对量子点在细菌标记和抗菌等方面的应用进行了综述。     

简述量子点与抗体的偶联方法

量子点是一种具有独特光学性质的半导体纳米材料,表面带有功能基团的水溶性量子点可与抗体偶联,作为荧光探针用于多种生物学研究。根据量子点表面所修饰的物质不同,偶联方法可分为共价偶联与非公价偶联两大类。本研究主要对量子点与抗体的偶联方法进行简单介绍。     

Near infrared sensing based on fluorescence resonance energy transfer between Mn:CdTe quantum dots and Au nanorods

A novel sensing system based on the near infrared (NIR) fluorescence resonance energy transfer (FRET) between Mn:CdTe quantum dots (Qdots) and Au nanorods (AuNRs) was established for the detection of human IgG. The NIR-emitting Qdots linked with goat anti-human IgG (Mn:CdTe-Ab1) and AuNRs linked with rabbit anti-human IgG (AuNRs-Ab2) acted as fluorescence donors and acceptors, respectively. FRET occurred by human IgG with the specific antigen–antibody interaction. And human IgG was detected based on the modulation in FRET efficiency. The calibration graph was linear over the range of 0.05–2.5 μM of human IgG under optimal conditions. The proposed sensing system can decrease the interference of biomolecules in NIR region and increase FRET efficiency in optimizing the spectral overlap of AuNRs with Mn:CdTe Qdots. This method has great potential for multiplex assay with different donor–acceptor pairs.     

Rhizopus stolonifer mediated biosynthesis of biocompatible cadmium chalcogenide quantum dots

We report an efficient method to biosynthesize biocompatible cadmium telluride and cadmium sulphide quantum dots from the fungus Rhizopus stolonifer. The suspension of the quantum dots exhibited purple and greenish-blue luminescence respectively upon UV light illumination. Photoluminescence spectroscopy, X-ray diffraction, and transmission electron microscopy confirms the formation of the quantum dots. From the photoluminescence spectrum the emission maxima is found to be 424 and 476 nm respectively. The X-ray diffraction of the quantum dots matches with results reported in literature. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for cell viability evaluation carried out on 3-days transfer, inoculum 3 × 10⁵ cells, embryonic fibroblast cells lines shows that more than 80% of the cells are viable even after 48 h, indicating the biocompatible nature of the quantum dots. A good contrast in imaging has been obtained upon incorporating the quantum dots in human breast adenocarcinoma Michigan Cancer Foundation-7 cell lines.     

CdTe quantum dots (QDs) improve the affinities of baicalein and genistein for human serum albumin in vitro

Baicalein and genistein were studied for the affinities for human serum albumin (HSA) in the presence and absence of three CdTe quantum dots (QDs) with different sizes. Three typical CdTe QDs with maximum emissions of 535 nm (green-emitting, G-QDs), 598 nm (yellow-emitting, Y-QDs), and 654 nm (red-emitting, R-QDs) were tested. The fluorescence intensities of HSA decreased remarkably with increasing concentration of QDs. Baicalein resulted in an obvious blue-shift of the λ_em of HSA from 340 to 334 nm. However, the extents of blue-shifts induced by baicalein and genistein in the presence of QDs were much bigger than that in the absence of QDs. The quenching process of baicalein for HSA was easily affected by the QDs size than that of genistein. QDs increased the quenching constant from 136.97% to 162.24% for baicalein. However, QDs only increased the quenching constants from 20.56% to 32.23% for genistein. G-QDs, Y-QDs, and R-QDs increased the affinities of baicalein for HSA about 3.02%, 6.38% and 9.40%. G-QDs, Y-QDs, and R-QDs increased the affinities of genistein for HSA about 2.56%, 13.46% and 19.44%. The binding affinities of baicalein and genistein for HSA increased with increasing QDs size.     

Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots

Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of “artificial” elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S-XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD-secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and β-tubulin, a protein associated with cytoskeleton microtubules. We set up samples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal specificity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion.     

Detection of quercetin based on Al-amplified phosphorescence signals of manganese-doped ZnS quantum dots

A simple phosphorescence method is proposed for quercetin detection based on Al³⁺-amplified room-temperature phosphorescence (RTP) signals of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs). The sensor was established based on some properties as follows. Al³⁺ can interact with carboxyl groups on the surface of MPA-capped Mn-doped ZnS QDs via chelation, which will lead to the aggregation of QDs and amplification of RTP signals, After the addition of quercetin, it can form more stable complex with Al³⁺ in alkaline aqueous solution and dissociate Al³⁺ from the surface of Mn-doped ZnS QDs, which will result in significant recovery of RTP intensity of the MPA-capped Mn-doped ZnS–Al³⁺ system. Under the optimized conditions, the change of RTP intensity was proportional to the concentration of quercetin in the range from 0.1 to 6.0 mg L⁻¹, with a high correlation coefficient of 0.996 and a detection limit of 0.047 mg L⁻¹. The proposed method is potentially suitable for detection of quercetin in real samples without complicated pretreatment.     

Optical coding of mammalian cells using semiconductor quantum dots

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional fluorescence microscopy or flow cytometry systems. We created unique fluorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create > 100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique fluorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.     

Facile synthesis and photophysical characterization of luminescent CdTe quantum dots for Forster resonance energy transfer based immunosensing of staphylococcal enterotoxin B

Aqueous phase synthesis of CdTe quantum dots (QDs) with surface functionalization for bioconjugation remains the best approach for biosensing and bioimaging applications. We present a facile aqueous phase method to prepare CdTe QDs by adjusting precursor and ligand concentrations. CdTe QDs had photoluminescence quantum yield up to ≈33% with a narrow spectral distribution. The powder X‐ray diffraction profile elucidated characteristic broad peaks of zinc blende cubic CdTe nanoparticles with 2.5–3 nm average crystalline size having regular spherical morphology as revealed by transmission electron microscopy. Infra‐red spectroscopy confirmed disappearance of characteristic absorptions for –SH thiols inferring thiol coordinated CdTe nanoparticles. The effective molar concentration of 1 : 2.5 : 0.5 respectively for Cd²⁺/3‐mercaptopropionic acid/HTe^– at pH 9 ± 0.2 resulted in CdTe quantum dots of 2.2–3.06 nm having band gap in the range 2.74–2.26 eV respectively. Later, QD₅₂₃ and QD₆₀₁ were used for monitoring staphylococcal enterotoxin B (SEB; a bacterial superantigen responsible for food poisoning) using Forster resonance energy transfer based two QD fluorescence. QD₅₂₃ and QD₆₀₁ were bioconjugated to anti‐SEB IgY antibody and SEB respectively according to carbodiimide protocol. The mutual affinity between SEB and anti‐SEB antibody was relied upon to obtain efficient energy transfer between respective QDs resulting in fluorescence quenching of QD₅₂₃ and fluorescence enhancement of QD₆₀₁. Presence of SEB in the range 1–0.05 µg varied the rate of fluorescence quenching of QD₅₂₃, thereby demonstrating efficient use of QDs in the Forster resonance energy transfer based immunosensing method by engineering the QD size. Copyright © 2012 John Wiley & Sons, Ltd.     

Intracellular imaging of targeted proteins labeled with quantum dots

We developed a new method for imaging the movement of targeted proteins in living cancer cells with photostable and bright quantum dots (QDs). QDs were conjugated with various molecules and proteins, such as phalloidin, anti-tubulin antibody and kinesin. These bioconjugated QDs were mixed with a transfection reagent and successfully internalized into living cells. The movements of individual QDs were tracked for long periods of time. Phalloidin conjugated QDs bound to actin filaments and showed almost no movement. In contrast, anti-tubulin antibody conjugated QDs bound to microtubules and revealed dynamic movement of microtubules. Kinesin showed an interesting behavior whereby kinesin came to be almost paused briefly for a few seconds and then moved once again. This is in direct contrast to the smoothly continuous movement of kinesin in an in vitro assay. The maximum velocity of kinesin in cells was faster than that in the in vitro assay. These results suggest that intracellular movement of kinesin is different from that in the in vitro assay. This newly described method will be a powerful tool for investigating the functions of proteins in living cells.     

Facile and sensitive detection of protamine by enhanced room-temperature phosphorescence of Mn-doped ZnS quantum dots

Quantum dot (QD) nanohybrids provide an effective route to explore the new properties of materials and are increasingly used as highly valuable sensitive (bio) chemical probes. Interestingly, the room-temperature phosphorescence (RTP) of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS QDs could be remarkably enhanced by the addition of protamine. Based on the above finding, a simple, sensitive, and selective method for rapid detection of protamine was successfully designed. With this method, protamine as a cationic peptide interacts electrostatically with MPA-capped Mn-doped ZnS QDs to form MPA-capped Mn-doped ZnS QD/protamine complexes, which leads to the aggregation of QDs and enhances the RTP intensity. Under the optimized conditions, the RTP intensity change was linearly proportional to the concentration of protamine in the range 0.2–3.0 μg ml⁻¹, and the limit of detection was 0.14 μg ml⁻¹. The proposed method was successfully applied to detect protamine in protamine sulfate injection and human serum samples with satisfactory results, and the recovery ranged from 96.5 to 105.6%.     

The fluorescence lifetime and quantum yield of chlorophyll a in Triton X-100 solutions

The fluorescence of chlorophyll (Chl) a in 0.007–0.1% Triton X-100 was investigated by a phase-shift technique. The Chl a concentrations varied from 0.7 to 25 μM. Parallel measurements of fluorescence lifetime (τ) and quantum yield (ψ) were made. It was concluded that homogeneous energy transfer takes place at detergent concentrations above 0.025%: (i) the transfer between uniform molecules of the pigment solubilized in Triton X-100 micelles, when τ and ψ are constant; (ii) the transfer towards the quenching centers, resulting in a proportional decrease in τ and ψ. At a Triton X-100 concentration of about 0.025% the Chl a emission becomes heterogeneous. It is evident from the disproportional decrease in τ and ψ (greater in ψ than in τ) and also from the rise of the fluorescence at 730–750 nm. As the Triton X-100 concentration becomes lower than the critical one (0.021%), the number of micelles drops abruptly and Chl a forms colloid particles in the aqueous medium. This manifests itself as a decrease in τ and as a certain stabilization of ψ. Having analyzed the complex pattern of the τ/ψ ratio, we concluded that under these conditions more than 90% of Chl a is in a weakly fluorescent form (τ < 30 ps) and about 1% is in an aggregated state fluorescing at 732 nm with τ about 0.7 ns.     

Development of tyrosinase biosensor based on quantum dots/chitosan nanocomposite for detection of phenolic compounds

A sensitive and simple amperometric biosensor for phenols was developed based on the immobilization of tyrosinase into CdS quantum dots/chitosan nanocomposite matrix. The nanocomposite film with porous nanostructure, excellent hydrophilicity and biocompatibility resulted in high enzyme loading, and the tyrosinase (Tyr) immobilized in this novel matrix retained its activity to a large extent. The CdS quantum dots/chitosan nanocomposite film was characterized by scanning electron microscopy and electrochemical impedance spectroscopy, and the parameters of the various experimental variables for the biosensor were optimized. Under the optimal conditions, the designed biosensor displayed a wide linear response to catechol over a concentration range of 1.0 × 10⁻⁹ to 2.0 × 10⁻⁵ M with a high sensitivity of 561 ± 9.7 mA M⁻¹ and a low detection limit down to 0.3 nM at a signal-to-noise ratio of 3. The CdS quantum dots/chitosan nanocomposites could provide a novel matrix for enzyme immobilization to promote the development of biosensing and biocatalysis.     

Effect of structural modifications on the spectroscopic properties and dynamics of the excited states of peridinin

The spectroscopic properties and dynamics of the lowest excited singlet states of peridinin and two derivatives have been studied by steady-state absorption and fast-transient optical spectroscopic techniques. One derivative denoted PerOlEs, possesses a double bond and a methyl ester group instead of the r-ylidenebutenolide of peridinin. Another derivative denoted PerAcEs, is the biosynthetic precursor of peridinin and possesses a triple bond and a methyl ester group corresponding to the r-ylidenbutenolide function. Ultrafast time-resolved spectroscopic experiments in the visible and near-infrared regions were performed on the molecules and reveal the energies and regarding the structural features and interactions responsible for the unusual solvent-induced changes in the steady-state and transient absorption spectra and dynamics of dynamics of the excited electronic states. The data also provide information peridinin.