Conjugated Quantum Dots Research Articles

Advanced procedure for oriented conjugation of full-size antibodies with quantum dots.

Ideal nanoparticle-based nanoprobes should contain on their surface homogeneously oriented highly active affinity molecules, e.g., antibodies (Abs), and should not exceed 15 nm in diameter. Direct conjugation of quantum dots (QDs) with Abs through cross-linking of QD amines with the sulfhydryl groups resulting from the reduction of the Ab disulfide bonds is a generally accepted technique. However, this procedure yields conjugates where Abs are oriented irregularly. This decreases the number of functionally active Abs on the nanoparticle surface, because some Ab recognition sites face inwards and cannot interact with the target moieties. Here, we describe an advanced procedure of Ab reduction, affinity purification, and QD-Ab conjugation with optimized critical steps. We have developed a method for partially reducing the Abs yielding highly functional 75 kDa heavy-light chain Ab fragments. Affinity purification of these Ab fragments followed by their tagging with QDs results in QD-Ab conjugates with largely improved functionality compared to those obtained according to the standard procedures. The new approach can be extended to conjugation of any type of Abs with different semiconductor, noble metal, or magnetic nanocrystals.

Photocatalytic Carbon Disulfide Production via Charge Transfer Quenching of Quantum Dots

Carbon disulfide, a potentially therapeutic small molecule, is generated via oxidative cleavage of 1,1-dithiooxalate (DTO) photosensitized by CdSe quantum dots (QDs). Irradiation of DTO-QD conjugates leads to λ(irr) independent photooxidation with a quantum yield of ~4% in aerated pH 9 buffer solution that drops sharply in deaerated solution. Excess DTO is similarly decomposed, indicating labile exchange at the QD surfaces and a photocatalytic cycle. Analogous photoreaction occurs with the O-tert-butyl ester (t)BuDTO in nonaqueous media. We propose that oxidation is initiated by hole transfer from photoexcited QD to surface DTO and that these substrates are a promising class of photocleavable ligands for modifying QD surface coordination.

Signal Amplification via Biological Self-Assembly of Surface-Engineered Quantum Dots for Multiplexed Subattomolar Immunoassays and Apoptosis Imaging

The parallel and highly sensitive detection of biomolecules is of paramount importance to understand biological functions at the single cell level and for various medical diagnoses. Surface-engineered semiconductor quantum dots (QDs) have been demonstrated to act as a signal amplifiable reporter in immunoassays. This takes advantage of the QDs' robustness against self-quenching in proximity and the tunability of their surface properties. A streptavidin (SA) and biotin QD conjugate pair containing a zwitterionic surface modification was designed for QD self-assembly with minimal nonspecific adsorption. Typical sandwich-type immunoassay procedures were adopted, and the targeted protein binding events were effectively transduced and amplified by the fluorescence of the SA-biotin QD conjugates. The detection limit of myoglobin in 100% serum was determined to be at the subattomolar (tens of copies per milliliter) level, which was achieved by using 100 cycles of the layer-by-layer QD assembly. Adsorption kinetics studies and Monte Carlo simulations revealed that this highly sensitive signal amplification was accomplished by the zwitterionic surface, which gave equilibrium constants 5 orders of magnitude larger for specific binding than for nonspecific binding. The QD conjugates showed an effective multivalency of two, which resulted in a broad linear dynamic range spanning 9 orders of magnitude of target protein concentrations. The assay can be highly miniaturized and multiplexed, and as a proof-of-concept, parallel and rapid detection of four different cancer markers has been successfully demonstrated. To demonstrate that this QD signal amplification can be a universal platform, sensitive imaging and early detection of apoptotic cells were also showcased.

Synthesis of the conjugates of DNA polymerase β with cadmium sulfide nanoparticles and their activity in reverse micelles

Abstract Quantum dots are semiconductor nanocrystals, having very useful luminescent properties. We synthesized cadmium sulfide nanocrystals with infinite solubility and long-time stability in water by using the thioglycolic acid as capping agent and Tris-base as counter ion. The obtained nanoparticles in the concentration range up to 200 nM did not influence on the activity of DNA polymerase β in reverse micelles. The primed M13mp19 DNA upon the irradiation of nanoparticles with UV-light at 312 nm can absorb them. We obtained and characterized by photon correlation spectroscopy the covalent conjugates of DNA polymerase and CdS quantum dots. The conjugates sustained enzymatic activity; however, two-fold decrease in activity of the enzyme was observed. Using DNA template as a ruler and the activity of DNA polymerase, we estimated the Forster distance (2.3 nm) for CdS nanoparticles (donor) and 5(6)-carboxytetramethylrhodamine (acceptor) in the Forster resonance energy transfer (FRET). The conjugate we obtained is functional enzyme, opening the possibility of using this nanohybrid in DNA repair study, for example, to track DNA polymerase β on DNA within DNA repair machine. The proposed experimental protocols are optimized for a convenient, economic and practical use by many researchers.

Using Graphene Quantum Dots as Photoluminescent Probes for Protein Kinase Sensing

A simple and sensitive photoluminescence (PL) assay for the activity of a protein kinase based on the selective aggregation of phosphorylated peptide-graphene quantum dot (GQD) conjugates triggered by Zr(4+) ion coordination has been established. With more sophisticated design of the peptide substrate sequences, detecting other enzymes could also be possible. Under optimal conditions, a linear relationship between the decreased PL intensity of peptide-GQD conjugates and the concentration of casein kinase II (CK2) in the range from 0.1 to 1.0 unit mL(-1) with a detection limit of 0.03 unit mL(-1) (3σ) was obtained. The EC50 value (i.e., the enzyme concentration producing 50% substrate conversion) for CK2 was evaluated to be 0.34 unit mL(-1). The proposed method showed potential applications in kinase inhibitor screening. To demonstrate the potential of this GQD-based platform for screening of kinase inhibitors in real biological systems, the inhibition of CK2 phosphorylation activity by four different inhibitors (ellagic acid, 5,6-dichlorobenzimidazole-l-β-d-ribofuranoside, emodin, and quercetin) was tested in human serum by comparing signals from samples incubated with the inhibitors against that without any inhibitor. As expected, in the presence of inhibitors, the PL intensity increased with increasing inhibitor efficiency. The IC50 value (inhibitor concentration producing 50% inhibition) for ellagic acid was estimated to be 0.041 μM. The developed protocol provides a new and promising tool for the analysis of both the enzyme and its inhibitors with low cost and excellent performance.

Allergen immobilisation and signal amplification by quantum dots for use in a biosensor assay of IgE in serum

The production of biosensors for point of care diagnostics usually requires the immobilisation and storage of protein (for example, antigen or antibody) on a sensor surface, in a manner that retains a high degree of activity and low levels of non-specific binding. These characteristics have been assessed for polymer immobilised antigens (allergens) using an IgG binding assay and demonstrated further by assay with serum containing reactive IgEs.The activity of allergens immobilised on sensor chips using copoly(DMA–NAS–MAPS) and a spotting technique, as well as the specificity of their binding interactions with cognate immunoglobulins was assessed using Dual Polarisation Interferometry (DPI). The data obtained indicate that the allergens studied remain stable over long periods of time (at least 114 days). This performance compared favourably with other immobilisation methods. Allergen coated chips were tested in an anti-casein IgE assay using human serum from allergic and non-allergic donors. Detection of both total Ig and specific IgE was demonstrated using a secondary anti-IgE antibody. Furthermore, optical signal enhancement with streptavidin conjugated quantum dots was shown to yield responses for samples below 0.84ng/mL (0.35KU/L) of IgE, which overlap with the industrial quasi-standard ImmunoCAP® and is the clinically relevant threshold used to classify serum samples from allergic individuals.

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

Sequential injection immunoassay for human bone morphogenic protein-7 using an immunoreactor immobilized with anti-human bone morphogenic protein-7 antibody–CdSe/ZnS quantum dot conjugates

The detection of human bone morphogenic protein-7 (BMP-7) was achieved using a sequential injection immunoassay (SIIA) system. The SIIA system is based on the binding between BMP-7 and anti-human BMP-7 (AbBMP7)–CdSe/ZnS quantum dot (QD) conjugates immobilized onto a glass disk or an optical fiber, using fluorescence detection at excitation and emission wavelengths of 470nm and 580nm, respectively. The AbBMP7–QD conjugates were prepared by conjugating anti-human BMP-7 antibody (AbBMP7) to hydrophilic CdSe/ZnS core/shell quantum dots (QDs). The SIIA system was fully automated using software written in the LabVIEW™ development environment. The analytical performance of the SIIA system was characterized with a number of variables such as carrier flow rate and elution buffer. Under partially optimized operating conditions, the SIIA system had a linear calibration graph at up to 10.0ngmL−1 BMP-7 (R2≥0.975) and a sample frequency of two samples per hour. The SIIA system with an optical fiber immunosensor was used to detect and quantify BMP-7 in spiked real samples obtained from a biological process with recoveries in the range of 95–102%.

Single domain antibody–quantum dot conjugates for ricin detection by both fluoroimmunoassay and surface plasmon resonance

The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe–ZnS core–shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb–QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb–QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD–sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ∼10-fold over sdAb reporters and 2–4 fold over full sized antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD–sdAb conjugates in this format.

Inorganic Nanoparticles and Nanomaterials Based on Titanium (Ti): Applications in Medicine

Nanomedicine is a relatively new field of science and technology. By interacting with biomolecules, therefore at nanoscale, nanotechnology opens up a vast field of research and application. Current and potential applications of nanotechnology in medicine range from research involving diagnostic devices, drug delivery vehicles to enhanced gene therapy and tissue engineering procedures. Its advantage over conventional medicine lies on its size. Operating at nanoscale allows to exploit physical properties different from those observed at microscale such as the volume/surface ratio. This allows drugs of nanosize be used in lower concentration and has an earlier onset of therapeutic action. It also provides materials for controlled drug delivery by directing carriers to a specific location. Inorganic nanomedicine is likely to remain one of the most prolific fields of nanomedicine, which refers to the use of inorganic or hybrid (inorganic-inorganic or inorganic-organic) nanomaterials (INMs) and nanoparticles (INPs) to achieve innovative medical advances for body parts implantation, drug and gene discovery and delivery, discovery of biomarkers, and molecular diagnostics. Among the most promising INMs being developed are metal, silica, dendrimers, organic-inorganic hybrids, ceramics (e.g. ZrO2, TiO2, Al2O3, etc.) and bioinorganic hybrids. Metal NP contrast agents enhance magnetic resonance imaging and ultrasound results in biomedical applications of in vivo imaging. Hollow and porous INMs have been exploited for drug and gene delivery, diagnostic imaging, and photothermal therapy. Biomolecular inorganic nanohybrids and nanostructured biomaterials have been exploited for targeted imaging and therapy, drug and gene delivery, and regenerative medicine. Potential uses for fluorescent quantum dots (QDs) include cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. Biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastasis cell tracking. This article outlines present developments and future prospects for the use of Ti-based NPs and NMs in experimental in vivo and in vitro studies and in engineering nanodevices and biosensors for clinical and investigative use in diagnosis and therapy in diverse fields of medical sciences, such as oncology, infection control, orthopedics, dentistry, dermatology, genetics, cardiology, ophthalmology, etc. Toxicological considerations of these INPs and INMs are also discussed.

Detecting cell adhesion molecules in intact lung using quantum dot conjugates targeted to endothelial cells

Quantum dots (QD) coupled with fluorescence imaging techniques are a powerful tool for detecting proteins in vivo. Here we used dual QD labeling to detect cellular adhesion molecules, CAM (endothelial marker, PECAM and vascular cell adhesion molecule, VCAM) expression in a LPS model of lung inflammation. QDs were functionalized via an adaptor protein and linked to anti‐PECAM and anti‐VCAM Abs to create QD655‐PECAM (QDP) and QD525‐VCAM (QDV), respectively. We established probe specificity in vitro in a PECAM expressing cell line and in LPS treated endothelial cells (for VCAM). For in situ experiments, mice lungs were removed at 24h after LPS instillation, and perfused with medium containing QDV and QDP. For in vivo lung studies, QDs were injected into the jugular vein at 24h after LPS treatment and lungs were removed and processed after overnight fixation. Imaged sections from both studies showed that LPS treated mice had 8–10 fold higher QDV signal, compared to naïve lungs, which significantly colocalized with QDP, indicating increased VCAM expression in pulmonary endothelium. Thus, QD labeling for intravital microscopy will be useful for detection and localization of proteins in pulmonary tissue.

Conjugation of nano and quantum materials with bovine serum albumin (BSA) to study their biological potential

Conjugates of gold nanoparticles (AuNPs) and semiconductor quantum dots (CdS/T) have been synthesized with bovine serum albumin (BSA) using wet chemistry. The optical properties of nano and quantum materials and their BSA conjugate have been studied using UV–Visible and Fluorescence spectroscopy. UV–Visible spectrum of pure BSA showed an absorption maximum at 278nm, which showed blue shift after its conjugation with nano and quantum materials. Increased concentration of AuNPs during conjugation resulted in broadening of BSA peak (278nm), which can be related to the formation of ground state complex formation, caused by the partial adsorption of BSA on the surface of NPs. However, increased concentrations of BSA resulted in decrease in SPR intensity of gold nanoparticles (528nm) and absorbance peak of BSA started diminishing. AuNPs acted as quencher for BSA fluorescence intensity, when excited at 280nm. The binding constant (K) and the number of binding sites (n) between AuNPs and BSA have been found to be 1.97×102 LM−1 and 0.6 respectively. With quantum dots, conjugation resulted in enhancement of fluorescence emission of quantum dots when excited at 300nm, which might be due to the stabilizing effect of BSA on QDs or due to energy transfer from tryptophan moieties of albumin to quantum dots.

Insulin and insulin like growth factor II endocytosis and signaling via insulin receptor B

BackgroundInsulin and insulin-like growth factors (IGFs) act on tetrameric tyrosine kinase receptors controlling essential functions including growth, metabolism, reproduction and longevity. The insulin receptor (IR) binds insulin and IGFs with different affinities triggering different cell responses.ResultsWe showed that IGF-II induces cell proliferation and gene transcription when IR-B is over-expressed. We combined biotinylated ligands with streptavidin conjugated quantum dots and visible fluorescent proteins to visualize the binding of IGF-II and insulin to IR-B and their ensuing internalization. By confocal microscopy and flow cytometry in living cells, we studied the internalization kinetic through the IR-B of both IGF-II, known to elicit proliferative responses, and insulin, a regulator of metabolism.ConclusionsIGF-II promotes a faster internalization of IR-B than insulin. We propose that IGF-II differentially activates mitogenic responses through endosomes, while insulin-activated IR-B remains at the plasma membrane. This fact could facilitate the interaction with key effector molecules involved in metabolism regulation.

A highly sensitive differential pulse anodic stripping voltammetry for determination of 17β-estradiol (E2) using CdSe quantum dots based on indirect competitive immunoassay

In this study a new and fast procedure was developed to determine trace 17β-estradiol (E2) concentrations using CdSe quantum dots (QDs) conjugation with bovine serum albumin (BSA)-E2. To increase the high efficiency of the method, the immunoassay design was restricted to an indirect competitive format. The E2 antigen and bioconjugate were incubated in a microtiter plate with an anti-E2 antibody and competition for antibody binding sites was established. The in situ bismuth-coated carbon electrodes were used for detecting the cadmium ions (Cd2+) released during the acid dissolution step. After optimization, the well-defined sharp anodic stripping voltammograms curves of the E2 concentration ranging from 50 to 1000pg/mL was recorded, and the lowest detection limit was 50pg/mL with 6% reproducibility and 7% repeatability. Finally, the assay was applied to tap water and wastewater samples. The detection limits were 52.56±0.125pg/mL for tap water and 51.42±0.453pg/mL for wastewater. These results show that the assay exhibited sensitive analytical performance in E2 detection with high sensitivity and accuracy with satisfactory results.

Lab-on-a-Chip for Anticancer Drug Screening Using Quantum Dots Probe Based Apoptosis Assay

Quantitative data acquisition of the drug induced cell apoptosis is one of the most important aspects of anticancer drug assessment. Traditional methods using photo-bleachable organic dyes to detect the apoptotic cells, may lead to some difficulties in long time observation of the cells. Moreover, the culture plates inevitably require large number of cells resulting in inefficiency in analysis of fewer cell samples such as a patient's own cells. To address these problems, we developed a microfluidic platform to assess the adherent cell cytotoxicity for anticancer drug screening using Annexin V conjugated quantum dots and Calcein AM as dual apoptotic probes. The results showed that camptothecin possessed more potent cytotoxic effect on these adherent tumor cells. Interestingly, the MCF-7 exhibited a certain resistance to these two anti-tumor drugs. For the first time, we achieved the evaluation of the impacts of model anti-cancer drugs on different adherent tumor cell lines using bio-functional quantum dots probe based cytotoxicity assays in a microfluidic device.

Homing peptide conjugated quantum dots for colorectal cancer imaging

Peptide-labeled quantum dots (QDs) have found wide applications in tumor imaging. Especially, homing peptide labeled QDs can target specific tumor tissues and thus serve as an indicator of the location of the tumor. However, most tumor homing agents target general markers instead of a biomarker specific to a certain type of cancers; for example, no specific imaging agents have been reported for colorectal cancer yet. Herein, we report the conjugation of a colorectal tumor homing peptide TCP-1-H6 with QDs by metal affinity-driven assembly, and also the characterization of the details of the assembly process by capillary electrophoresis. TCP-1-H6 conjugated QDs (TCP-1-H6-QD) were then applied to the imaging of colorectal cancer cells as well as tumor tissues from a mouse model of orthotopic colorectal cancers. TCP-1-H6-QD efficiently targeted colon 26 cells, and were internalized into the intracellular space. On tumor tissue sections, TCP-1-H6-QD specifically labeled the tumor tissues but not health tissues. These results indicate that TCP-1-H6-QD retains the unique homing property of the peptide and thus can be used for specific imaging of colorectal cancer. This method thereby widens the road for specific identification and diagnosis of cancer.

Facile synthesis of QD-anchored composite particles with magnetite cluster cores (nFe3O4@SiO2@QDs)

Magnetism-controlled fluorescent composite particles (nFe3O4@SiO2@QDs) were prepared as QD-anchored silica nanoparticles with magnetite cluster cores. First, citrate-capped magnetites (C-Fe3O4) were prepared by the co-precipitation method and subsequently complexed with 3-aminopropyl-trimethoxysilane (APTMS) at room temperature, consequently leading to the formation of magnetite clusters (A-nFe3O4) with alkoxy terminated interfaces. The sol–gel reaction between A-nFe3O4 and tetraethoxysilane (TEOS) produced the core-shell nFe3O4@SiO2. The resulting core-shell particles exhibited superparamagnetic properties and controllable magnetism simply by adjusting the thickness of nonmagnetic silica layer. After then, amine-terminated nFe3O4@SiO2 (NH2–nFe3O4@SiO2) was conjugated with carboxy quantum dots (QDs) using an EDC coupling agent. The QD conjugation with NH2–nFe3O4@SiO2 of lower pH exhibited the higher photoluminescence (PL) intensity, and the resulting composite particles (nFe3O4@SiO2@QDs) can be a useful biomedical agent. This chemical strategy can be further applied to prepare the core-shell magnetic nanostructure with various oxide layers for specific applications.

Combining Ligand Design with Photoligation to Provide Compact, Colloidally Stable, and Easy to Conjugate Quantum Dots

We describe the design and synthesis of two compact multicoordinating (lipoic acid-appended) zwitterion ligands for the capping of luminescent quantum dots, QDs. This design is combined with a novel and easy to implement photoligation strategy to promote the in situ ligand exchange and transfer of the QDs to buffer media. This method involves the irradiation of the native hydrophobic nanocrystals in the presence of the ligands, which promotes in situ cap exchange and phase transfer of the QDs, eliminating the need for a chemical reduction of the dithiolane groups. Applied to the present LA-zwitterion ligands, this route has provided QDs with high photoluminescence yields and excellent colloidal stability over a broad range of conditions, including acidic and basic pH, in the presence of growth media and excess salt conditions. The small lateral extension of the capping layer allowed easy conjugation of the QDs to globular proteins expressing a terminal polyhistidine tag, where binding is promoted by metal-affinity interactions between the accessible Zn-rich surface and imidazoles in the terminal tag of the proteins. The ability to carry out conjugation in acidic as well as basic conditions opens up the possibility to use such self-assembled QD-protein conjugates in various biological applications.

Study of Interaction between Metallothionein and CdTe Quantum Dots

Quantum dots (QDs) belong to a new class of fluorescent agent for biochemical, medicinal or other purposes. However, QDs based on cadmium or other metals can be risky for an organism. As one of the mechanism how to detoxify cadmium-based QDs expression of metallothioneins (MT) can be considered. Due to high affinity of metallothionein to cadmium(II) ions, we attempted to develop an approach for studying of possible interaction with QDs. We prepared QDs with CdTe core and studied the interaction with MT, which we isolated from livers of Cd-administered rabbits. To study the interaction, we used the mixture of both components MT (3.6 μM): CdTe QDs (0, 0.34, 0.68, 1.02, 1.36, 1.7, 2.04 and 2.47 μM). The mixtures were studied by spectrophotometry within the range from 200 to 750 nm with detected maxima at 260 and 505 nm. Same mixtures were also analysed by differential pulse voltammetry Brdicka reaction, which supported data from spectrophotometry. Subsequently, we used fast protein liquid chromatography for purification of protein–quantum dot conjugates. We obtained the different chromatograms for (1) Apo MT, (2) CdTe QDs and (3) MT–QD complex. We also collected the fractions and subsequently analysed them on the content of Cd and MT, which confirmed the formation of CdTe QDs–MT complex.

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