Quantum Dot Probes Research Articles

A Novel Fluorescent Quantum Dot Probe for the Rapid Diagnostic High Contrast Imaging of Tumor in Mice.

A simple probe - antibody conjugated silica over coated cadmium selenide quantum dots (QD-Ab probe) for efficient and rapid diagnostic in vivo imaging of tumors is developed. Compared to unconjugated quantum dots (QD), these probes underwent efficient cellular internalization and tumor targeting behavior, retaining bright emission under in vivo cancer models. Silica over coated cadmium selenide quantum dots were conjugated with Epidermal growth factor receptor (EGFR) monoclonal antibody to detect the over expression of EGFR in cancer models. The in vitro cellular internalization efficiency of QD and QD-Ab probe in cultured stem cells (RADMSCs) and cancer cells (HeLa) were assessed by ICP-OES and cLSM. Results demonstrated a greater internalization efficiency of CdSe-Silica QD-Ab probe than CdSe-Silica QDs. For in vivo imaging solid tumor bearing mice was subjected to tail vein injection of QD and QD-Ab probe. After the specific time interval of injection, mice were anesthetized and subjected into Xenogen IVIS®200 imaging system, followed by ex vivo imaging. Subsequently, ultrathin sections of tumor were imaged by using cLSM. Both in vivo and ex vivo imaging results confirmed the tumor-targeted imaging efficiency of QD-Ab probes compared to unconjugated QDs.

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Bio-compatibility and cytotoxicity studies of water-soluble CuInS2-ZnS-AFP fluorescence probe in liver cancer cells

The oncogenesis of hepatocellular carcinoma (HCC) is not clear. The current methods of the pertinent studies are not precise and sensitive. The present study was to use liver cancer cell line to explore the bio-compatibility and cytotoxicity of ternary quantum dots (QDs) probe and to evaluate the possible application of QDs in HCC. CuInS2-ZnS-AFP fluorescence probe was designed and synthesized to label the liver cancer cell HepG2. The cytotoxicity of CuInS2-ZnS-AFP probe was evaluated by MTT experiments and flow cytometry. The labeling experiments indicated that CuInS2-ZnS QDs conjugated with AFP antibody could enter HepG2 cells effectively and emit intensive yellow fluorescence by ultraviolet excitation without changing cellular morphology. Toxicity tests suggested that the cytotoxicity of CuInS2-ZnS-AFP probe was significantly lower than that of CdTe-ZnS-AFP probe (t test, F=0.8, T=-69.326, P<0.001). For CuInS2-ZnS-AFP probe, time-effect relationship was presented in intermediate concentration (>20%) groups (P<0.05) and dose-effect relationship was presented in almost all of the groups (P<0.05). CuInS2-ZnS-AFP QDs probe had better bio-compatibility and lower cytotoxicity compared with CdTe-ZnS-AFP probe, and could be used for imaging the living cells in vitro.

Ultrasensitive detection of protein biomarker using fluorescent quantum dot probes

Ultrasensitive detection of protein biomarker using fluorescent quantum dot probes

Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots.

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.

Nitrogen and Sulfur Codoped Reduced Graphene Oxide as a General Platform for Rapid and Sensitive Fluorescent Detection of Biological Species.

Nitrogen (N) and sulfur (S) codoped reduced graphene oxide (N,S-rGO) was synthesized through a facile solvothermal process. The introduction of N and S heteroatoms into GO effectively activated the sp(2)-hybridized carbon lattice and made the material an ideal electron/energy acceptor. Such unique properties enable this material to perform as a general platform for rapid and sensitive detection of various biological species through simple fluorescence quenching and recovering. When quantum dot (QD)-labeled HBV (human being disease-related gene hepatitis B virus DNA) and HIV (human being disease-related gene human immunodeficiency virus DNA) molecular beacon probes were mixed with N,S-rGO, QD fluorescence was quenched; when target HBV and HIV DNA were added, QD fluorescence was recovered. By the recovered fluorescence intensity, the target virus DNA detection limits were reduced to 2.4 nM for HBV and 3.0 nM for HIV with detection time of less than 5 min. It must be stressed out that different viruses in the same homogeneous aqueous media could be discriminated and quantified simultaneously through choosing diverse QD probes with different colors. Moreover, even one mismatched target DNA could be distinguished using this method. When altering the molecular beacon loop domain to protein aptamers, this sensing strategy was also able to detect thrombin and IgE in 5 min with detection limits of 0.17 ng mL(-1) and 0.19 ng mL(-1), respectively, which was far more rapid and sensitive than bare GO-based fluorescence detection strategy.

Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels

In this study, we developed a multiplex immunoassay system that combines the suspension and planar microarray formats within a single layer of polydimethylsiloxane (PDMS) using soft lithography technology. The suspension format was based on the target proteins forming a sandwich structure between the magnetic beads and the quantum dot (QD) probes through specific antibody–antigen interactions. The planar microarray format was produced by fabricating an array of micro-wells in PDMS. Each micro-well was designed to trap a single microbead and eventually generated a microbead array within the PDMS chamber. The resultant bead-based on-chip assay could be used for simultaneously detecting three lung cancer biomarkers—carcinoembryonic antigen (CEA), fragments of cytokeratin 19 (CYFRA21-1) and neuron-specific enolase (NSE)—in 10μl of human serum, with a wide linear dynamic range (1.03–111ng/mL for CEA and CYFRA21-1; 9.26–1000ng/ml for NSE) and a low detection limit (CEA: 0.19ng/ml; CYFRA21-1: 0.97ng/ml; NSE: 0.37ng/ml; S/N=3). Our micro-well chip does not require complex e-beam lithography or the reactive ion etching process as with existing micro-well systems, which rely on expensive focused ion beam (FIB) milling or optical fiber bundles. Furthermore, the current approach is easy to operate without extra driving equipment such as pumps, and can make parallel detection for multiplexing with rapid binding kinetics, small reagent consumption and low cost. This work has demonstrated the importance of the successful application of on-chip multiplexing sandwich assays for the detection of biomarker proteins.

Abstract B02: Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging

Abstract The dynamic behavior of proteins in the spatial and temporal context of a cell is critical for dictating downstream signaling and subsequent cellular effects. Human epidermal growth factor receptor 2 (HER2) is a ligand-less receptor tyrosine kinase whose heterodimerization with other HER receptors (EGFR, HER3, HER4) activates potent downstream signaling to modulate cell proliferation and survival. HER2 represents a major therapeutic target as its overexpression is prevalent in a major subset of invasive breast cancers. Yet while current understanding of HER2 signaling entails a broad sequence of signaling events, the spatiotemporal dynamics of how these signaling mechanisms are executed remains undefined. Here, we describe the molecular-scale motions that underlie HER2 signaling behavior in live cells using a bright and photostable anti-HER2 quantum dot (QD) probe designed and validated to track the receptor for long durations with high spatiotemporal resolution. Methods: HER2 was detected using an anti-HER2 affibody that was fused to a fluorogen activating peptide (FAP) which bound with high affinity to a fluorogen (Malachite Green)-QD complex. HER2-QDs were imaged in live unstimulated and stimulated human SKBR3 breast cancer cells. Cells were serum-starved overnight and: 1) received no treatment during the course of imaging (unstimulated) or 2) were chronically treated with the HER3 and HER4 specific ligand, heregulin-beta1 (HRG) during the course of imaging (stimulated). HRG was administered globally across all cells or locally to a specific cellular region. Trajectory information was analyzed using custom single particle tracking MATLAB software to measure key biophysical parameters. Results: The anti-HER2-QD probe showed robust and increased signal-to-noise compared to conventional organic dyes. Validation was measured by sensitive quantitation of single probes in low and high HER2-expressing cell lines with background non-specific binding of less than five probes per cell. HER2 motions were complex and composed of a heterogeneous composite of diffusive, constrained, and stationary motions. These motions were observed in the trajectories of HER2 receptors traveling along cellular protrusions, cell body, and along protrusions between adjacent cells. HRG stimulation produced morphological changes (e.g. membrane ruffling, protrusion growth) as well as changes in HER2 dynamics. HRG-stimulated cells demonstrated an increase in HER2 mobility (as measured by diffusion constant), increased speed (as measured by instantaneous velocity), and wider exploratory area (as measured by maximum trajectory distance) than in unstimulated cells. Interestingly, increased receptor mobility did not correlate strongly with a wider exploratory area, and more mobile receptors showed more constrained motion. Conclusions: In contrast to the simple diffusive motions previously reported for members of major classes of receptors (e.g. GPCRs, tyrosine kinase receptors), our sensitive measurements of HER2 receptor motion demonstrate complex molecular dynamics in both unstimulated and stimulated cells. Ligand stimulation induces changes in cell morphology such as membrane ruffling and protrusion growth that accompany changes in HER2 mobility and range of motion. The diversity of receptor dynamics suggest a heterogeneous environment in which HER2 engages. Future experiments include understanding the dynamic interplay between HER2 with the cytoskeleton and downstream signaling with HER2-targetting therapies using real-time measurements of HER2 molecular motions. Citation Format: Wai Yan Lam, Yi Wang, Mark J. Olah, Keith A. Lidke, Marcel Bruchez, Joe Gray, Tania Q. Vu. Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B02.

Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe.

The large external antigen (LEA) is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC) as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC) combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605) conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05), indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC) combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of the cell lines, which was confirmed by flow cytometry. The results of this study indicate that QD-ICC has the potential to noninvasively detect rare circulating tumor cells in clinical samples in real clinical applications.

Peptide-Conjugated Quantum Dots Act as the Target Marker for Human Pancreatic Carcinoma Cells

Background/Aims: In the present study, we describe a novel and straightforward approach to produce a cyclic- arginine-glycine-aspartic (RGD)-peptide-conjugated quantum dot (QD) probe as an ideal target tumor biomarker. Due to its specific structure, the probe can be used for targeted imaging of pancreatic carcinoma cells. Methods: Pancreatic carcinoma cells were routinely cultured and marked with QD-RGD probe. The QD-RGD probe on the fluorescence-labeled cancer cell was observed by fluorescence microscopy and laser confocal microscopy. Cancer cell viability was detected by MTT assay after culturing with QD-RGD probe. Results: Fluorescence microscopy and laser confocal microscopy displayed that 10nmol/L QD-RGD probe was able to effectively mark pancreatic carcinoma cells. In comparison with organic dyes and fluorescent proteins, the quantum dot-RGD probe had unique optical and electronic properties. Conclusion: QD-RGD probe has a low cytotoxicity with an excellent optical property and biocompatibility. These findings support further evaluation of QD-RGD probes for the early detection of pancreatic cancer.

Nanosized Fluorescent Diagnostic Probes Consisting of Single-domain Antibodies Conjugated with Quantum Dots

In state-of-the-art nanoprobes, monoclonal antibodies (mAbs) used as capture molecules are often too large for nanoparticle targeting and are randomly orientated relative to the nanoparticle surface due to the inherent characteristics of the procedure for their fabrication. Here, we are reviewing our recent publications on the smallest known nanoprobes consisting of quantum dots (QDs) and single-domain antibodies (sdAbs), low-molecular-weight fragments of llama immunoglobulins produced in E. coli and attached to the QDs in a strictly specified orientation. The nanoprobe developed has a hydrodynamic diameter smaller than 12nm and consists of four sdAbs and a QD. They are sensitive and specific enough to accurately estimate even a small number of cells with target biomarkers by means of flow cytometry. The small size of the probes enables immunohistochemical staining much deeper areas of tissue samples as compared to standard probes. Tests with clinical biopsies have shown that sdAb–QD probes provide the same or higher quality of biomarker detection compared to the gold-standard histochemical approach. The nanoprobes developed have many implications for quick multiplexed diagnosis and FRET-based detection platforms.

A novel aptamer–quantum dot fluorescence probe for specific detection of antibiotic residues in milk

Herein, a facile, signal-on and homogenous fluorescence assay using novel aptamer-dsDNA antibody–quantum dot probes was designed for detecting antibiotic residues.

Detection of Leishmania-specific DNA and surface antigens using a combination of functionalized magnetic beads and cadmium selenite quantum dots

Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125ng/μl and 103cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings.

Nanoimaging: photophysical and pharmaceutical characterization of poly-lactide-co-glycolide nanoparticles engineered with quantum dots

Quantum dots (QDs) and polymeric nanoparticles (NPs) are considered good binomials for the development of multifunctional nanomedicines for multimodal imaging. Fluorescent imaging of QDs can monitor the behavior of QD-labeled NPs in both cells and animals with high temporal and spatial resolutions. The comprehension of polymer interaction with the metallic QD surface must be considered to achieve a complete chemicophysical characterization of these systems and to describe the QD optical properties to be used for their unequivocal identification in the tissue. In this study, by comparing two different synthetic procedures to obtain polymeric nanoparticles labeled with QDs, we investigated whether their optical properties may change according to the formulation methods, as a consequence of the different polymeric environments. Atomic force microscopy, transmission electron microscopy, confocal and fluorescence lifetime imaging microscopy characterization demonstrated that NPs modified with QDs after the formulation process (post-NPs-QDs) conserved the photophysical features of the QD probe. In contrast, by using a polymer modified with QDs to formulate NPs (pre-NPs-QDs), a significant quenching of QD fluorescence and a blueshift in its emission spectra were observed. Our results suggest that the packaging of QDs into the polymeric matrix causes a modification of the QD optical properties: these effects must be characterized in depth and carefully considered when developing nanosystems for imaging and biological applications.

CdTe/ZnS quantum dots as fluorescent probes for ammonium determination.

Novel CdTe/ZnS quantum dot (QD) probes based on the quenching effect were proposed for the simple, rapid, and specific determination of ammonium in aqueous solutions. The QDs were modified using 3-mercaptopropionic acid, and the fluorescence responses of the CdTe/ZnS QD probes to ammonium were detected through regularity quenching. The quenching levels of the CdTe/ZnS QDs and ammonium concentration showed a good linear relationship between 4.0 × 10(-6) and 5.0 × 10(-4) mol/L; the detection limit was 3.0 × 10(-7) mol/L. Ammonium contents in synthetic explosion soil samples were measured to determine the practical applications of the QD probes and a probable quenching mechanism was described. Copyright © 2015 John Wiley & Sons, Ltd.

Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH.

Design and preparation of quantum dots fluorescent probes for in situ identification of Microthrix parvicella in bulking sludge.

A series of quantum dots (QDs) fluorescent probes for the in situ identification of Microthrix parvicella (M. parvicella) in bulking sludge were designed and prepared. In the preparation of CdTe/CdS QDs, the 11-mercaptoundecanoic acid (11-acid) and 16-mercaptohexadecanoic acid (16-acid) were used as the stabilizer. The prepared QDs probes were characterized by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and transmission electron microscopy (TEM), and the results showed that the CdTe/CdS QDs formed a core-shell structure and the long carbon chain was successfully grafted onto its surface. And the three QDs probes had different crystallinity and particle size, which was due to the inhibition effect of long carbon chain. The optical properties test results showed that although the formed core-shell structure and long carbon chain affected the fluorescent intensity, adsorption, and emission spectra of the QDs probes, the probes B and C had a large stokes-shift of 82 and 101 nm, which was a benefit for their fluorescent labeling property. In the fluorescent identification of M. parvicella, the probes B and C effectively adsorbed onto the surface of M. parvicella through a hydrophobic bond, and then identified M. parvicella by their unique fluorescence. In addition, it was found that a better hydrophobic property resulted in better identification efficiency.

Facile Synthesis of Glutathione-capped CdS Quantum Dots as a Fluorescence Sensor for Rapid Detection and Quantification of Paraquat.

This paper describes a convenient and rapid fluorescence sensor for determination of paraquat (PA) based on glutathione-capped CdS quantum dots (QDs). The methodology enabled the use of a simple synthesis procedure for water solubilization of CdS QDs via a fast route using glutathione as a capping agent within 15 min. The resulting water-soluble QDs exhibit a strong fluorescence emission at 536 nm with high and reproducible photostability. PA is an important class of electron acceptors for QDs. Thus, the fluorescence intensity of the glutathione-capped CdS QDs probe could be dramatically quenched by PA due to the electron transfer mechanism. The fluorescence intensity of the CdS QDs system was proportional to PA concentration in the range of 0.025 to 1.5 μg mL(-1), with a detection limit of 0.01 μg mL(-1). The time of analysis sample, including preparation of QDs and fluorescent measurement for PA, was only 20 min. Most of the potentially coexisting substances did not interfere with the PA-induced quenching effect except diquat. Furthermore, the analytical applicability of the proposed method was demonstrated by analyzing PA in water, rice and cabbage samples, and the recoveries were between 86 and 105% which satisfied the requirement of detection for PA. These results showed that the proposed method was simple in design and fast in operation, and could be used as a sensitive tool for detecting PA in environmental and agricultural samples.