Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules

We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).     

Aromatic aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live cell imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.     

Capping of CdSe-ZnS quantum dots with DHLA and subsequent conjugation with proteins

We provide a detailed protocol for designing water-soluble CdSe-ZnS quantum dots (QDs) based on cap exchange of the native hydrophobic shell with dihydrolipoic acid (DHLA) ligands, and the preparation of functional QD bioconjugates for use in immunoassays. Our conjugation strategy is based on non-covalent self-assembly between DHLA-capped QDs and protein appended with either an electrostatic attachment domain (namely, the basic leucine zipper) or a polyhistidine tag. These bioconjugates combine the properties of the QD and attached biomolecule to create structures with desirable luminescent and biologically specific properties. This method also allows the preparation of mixed surface conjugates, which results in the conjugates gaining multiple biological activities. Conjugation of DHLA-capped QDs to maltose binding protein (MBP), the immunoglobulin-G-binding beta2 domain of streptococcal protein G (PG) and avidin will be described. MBP and PG were modified by genetic fusion with either a charged leucine zipper or a polyhistidine interaction domain.     

Comprehensive study of interaction between biocompatible PEG‐InP/ZnS QDs and bovine serum albumin

Polyethylene glycol (PEG) surface modified biocompatible InP/ZnS quantum dots (QDs) act as a potential alternative for conventional carcinogenic cadmium‐based quantum dots for in vivo and in vitro studies. Comprehensively, we studied the interaction between a model protein bovine serum albumin (BSA) and PEGylated toxic free InP/ZnS QDs using various spectroscopic tools such as absorption, fluorescence quenching, time resolved and synchronous fluorescence spectroscopic measurements. These studies principally show that tryptophan (Trp) residues of BSA have preferable binding affinity towards PEG‐InP/ZnS QDs surface and a blue shift in Trp fluorescence emission is a signature of conformational changes in its hydrophobic microenvironment. Photoluminescence (PL) intensity of Trp is quenched by ground state complex formation (static quenching) at room temperature. However, InP/ZnS@BSA conjugates become unstable with increasing temperature and PL intensity of Trp is quenched via dynamic quenching by PEG‐InP/ZnS QDs. Experimentally determined thermodynamic parameters for these conjugates have shown spontaneity, entropy driven and exothermic nature of bio‐conjugation. The calculated binding affinity (n ? 1, Hill coefficient) suggest that the affinity of InP/ZnS QDs for a BSA protein is not dependent on whether or not other BSA proteins are already bound to the QD surface. Energy transfer efficiency (E), Trp residue to InP/ZnS QDs distances and energy transfer rate (k_T) were all obtained from FÖrster resonance energy.     

Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria

CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates.     

Aptamer-functionalized quantum dots as theranostic nanotools against cancer and bacterial infections: A comprehensive overview of recent trends

Aptamers (Apts) are synthetic nucleic acid ligands that can be engineered to target various molecules, including amino acids, proteins, and pharmaceuticals. Through a series of adsorption, recovery, and amplification steps, Apts are extracted from combinatorial libraries of synthesized nucleic acids. Using aptasensors in bioanalysis and biomedicine can be improved by combining them with nanomaterials. Moreover, Apt-associated nanomaterials, including liposomes, polymeric, dendrimers, carbon nanomaterials, silica, nanorods, magnetic NPs, and quantum dots (QDs), have been widely used as promising nanotools in biomedicine. Following surface modifications and conjugation with appropriate functional groups, these nanomaterials can be successfully used in aptasensing. Advanced biological assays can use Apts immobilized on QD surfaces through physical interaction and chemical bonding. Accordingly, modern QD aptasensing platforms rely on interactions between QDs, Apts, and targets to detect them. QD-Apt conjugates can be used to directly detect prostate, ovarian, colorectal, and lung cancers or simultaneously detect biomarkers associated with these malignancies. Tenascin-C, mucin 1, prostate-specific antigen, prostate-specific membrane antigen, nucleolin, growth factors, and exosomes are among the cancer biomarkers that can be sensitively detected using such bioconjugates. Furthermore, Apt-conjugated QDs have shown great potential for controlling bacterial infections such as Bacillus thuringiensis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Campylobacter jejuni, Staphylococcus aureus, and Salmonella typhimurium. This comprehensive review discusses recent advancements in the design of QD-Apt bioconjugates and their applications in cancer and bacterial theranostics.     

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

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

Blinking effect and the use of quantum dots in single molecule spectroscopy

Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the “on”/“off” states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein–protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.     

Two-photon fluorescence vascular bioimaging with new bioconjugate probes selective toward the vascular endothelial growth factor receptor 2

We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in "whole" mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 μm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.     

CdSe量子点与蛋白质的作用研究

以油酸为稳定剂,石蜡为还原剂,采用有机相法合成了尺寸均匀的CdSe量子点,并通过巯基乙酸将合成的量子点转移至水相。考查了CdSe量子点与几种结构不同的蛋白质(酶)之间的作用规律。研究发现,经巯基乙酸修饰后的量子点与牛血清白蛋白和胰凝乳蛋白酶作用后,荧光强度明显增大。而铜/锌-超氧化物歧化酶对量子点的荧光有明显的淬灭作用,牛血红蛋白对量子点的影响是随着时间的增加荧光强度先增大后减小,体现出一般蛋白质使荧光增强和部分金属离子使荧光淬灭两者的协同效应。     

Assembly and targeting of liposomal nanoparticles encapsulating quantum dots

Quantum dots (QDs) are attracting intense interest as fluorescence labeling agents for biomedical imaging because biocompatible coatings and relatively nontoxic rare earth metal QDs have emerged as possible options. QD photoemissions are bright, of narrow wavelength range, and very stable. We sought to encapsulate QDs within targeted PEGylated liposomes to reduce their propensity for liver uptake and to amplify the already strong QD emission signal. A novel lipid-QD conjugate initialized a process by which lipids in solution coalesced around the QDs. The liposomal structure was confirmed with size measurements, SEM, and IR spectroscopy. PEGylated QD liposomes injected into a xenograft tumor model largely cleared from the body within 24 h. Residual liver labeling was low. Targeted QD liposomes exhibited robust tumor labeling compared with controls. This study highlights the potential of these near IR emitting QD liposomes for preclinical/clinical applications.     

Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery

We demonstrate the use of self-assembled luminescent semiconductor quantum dot (QD)-peptide bioconjugates for the selective intracellular labeling of several eukaryotic cell lines. A bifunctional oligoarginine cell penetrating peptide (based on the HIV-1 Tat protein motif) bearing a terminal polyhistidine tract was synthesized and used to facilitate the transmembrane delivery of the QD bioconjugates. The polyhistidine sequence allows the peptide to self-assemble onto the QD surface via metal-affinity interactions while the oligoarginine sequence allows specific QD delivery across the cellular membrane and intracellular labeling as compared to nonconjugated QDs. This peptide-driven delivery is concentration-dependent and thus can be titrated. Upon internalization, QDs display a punctate-like staining pattern in which some, but not all, of the QD signal is colocalized within endosomes. The effects of constant versus limited exposure to QD-peptide conjugates on cellular viability are evaluated by a metabolic specific assay, and clear differences in cytotoxicity are observed. The efficacy of using peptides for selective intracellular delivery is highlighted by performing a multicolor QD labeling, where we found that the presence or absence of peptide on the QD surface controls cellular uptake.     

Effects of N‐Acetyl‐L‐Cysteine‐Capped CdTe Quantum Dots on Bovine Serum Albumin and Bovine Hemoglobin: Isothermal Titration Calorimetry and Spectroscopic Investigations

The interactions of N‐acetyl‐L‐cysteine‐capped CdTe quantum dots (QDs) with bovine serum albumin (BSA) and bovine hemoglobin (BHb) were investigated by isothermal titration calorimetry (ITC), fluorescence, synchronous fluorescence, fluorescence lifetime, ultraviolet–visible absorption, and circular dichroism techniques. Fluorescence data of BSA–QDs and BHb–QDs revealed that the quenching was static in every system. While CdTe QDs changed the microenvironment of tryptophan in BHb, the microenvironment of BSA kept unchanged. Adding CdTe QDs affected the skeleton and secondary structure of the protein (BSA and BHb). The ITC results indicated that the interaction between the protein (BSA and BHb) and QDs‐612 was spontaneous and the predominant force was hydrophobic interaction. In addition, the binding constants were determined to be 1.19 × 10⁵ L mol^?1 (BSA–QDs) and 2.19 × 10⁵ L mol^?1 (BHb–QDs) at 298 K. From these results, we conclude that CdTe QDs have a larger impact on the structure of BHb than BSA.     

Lead sulfide near-infrared quantum dot bioconjugates for targeted molecular imaging

In this paper, we report the use of lead sulfide quantum dot (PbS QD) bioconjugates as near infrared (NIR) contrast agents for targeted molecular imaging with expanded emission wavelengths beyond 1000 nm. The red-shifted emission band, coupled with the small particle size, which will facilitate clearance, both afford PbS QDs unique properties for noninvasive, high resolution in vivo NIR imaging applications. We have performed imaging experiments at the molecular level using surface-modified PbS NIR QDs, together with our lab-built NIR imaging system. This novel instrumentation and fluorescent contrast agent have enabled us to study the relatively unexplored NIR biomedical imaging spectral region of 900-1200 nm. Preliminary experimental results indicate that PbS-QD/antibody bioconjugates are promising candidates for targeted NIR molecular imaging and future in vivo NIR tissue imaging applications.     

Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay

Capillary electrophoresis coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates to biological molecules. The CE-LIF was laboratory-built and capable of injection (hydrodynamic and electrokinetic) from sample volumes as low as 4 μL via the use of a modified micro-fluidic chip platform. Commercially available quantum dots were bioconjugated to proteins and immunoglobulins through the use of established techniques (non-selective and selective). Non-selective techniques involved the use of EDCHCl/sulfo-NHS for the conjugation of BSA and myoglobin to carboxylic acid-functionalized quantum dots. Selective techniques involved 1) the use of heterobifunctional crosslinker, sulfo-SMCC, for the conjugation of partially reduced IgG to amine-functionalized quantum dots, and 2) the conjugation of periodate-oxidized IgGs to hydrazide-functionalized quantum dots. The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values. The performance of capillary electrophoresis in characterizing immunoconjugates of quantum dot-labeled IgGs was also evaluated. Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules. This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.     

Multi-Color Single Particle Tracking with Quantum Dots

Quantum dots (QDs) have long promised to revolutionize fluorescence detection to include even applications requiring simultaneous multi-species detection at single molecule sensitivity. Despite the early promise, the unique optical properties of QDs have not yet been fully exploited in e. g. multiplex single molecule sensitivity applications such as single particle tracking (SPT). In order to fully optimize single molecule multiplex application with QDs, we have in this work performed a comprehensive quantitative investigation of the fluorescence intensities, fluorescence intensity fluctuations, and hydrodynamic radii of eight types of commercially available water soluble QDs. In this study, we show that the fluorescence intensity of CdSe core QDs increases as the emission of the QDs shifts towards the red but that hybrid CdSe/CdTe core QDs are less bright than the furthest red-shifted CdSe QDs. We further show that there is only a small size advantage in using blue-shifted QDs in biological applications because of the additional size of the water-stabilizing surface coat. Extending previous work, we finally also show that parallel four color multicolor (MC)-SPT with QDs is possible at an image acquisition rate of at least 25 Hz. We demonstrate the technique by measuring the lateral dynamics of a lipid, biotin-cap-DPPE, in the cellular plasma membrane of live cells using four different colors of QDs; QD565, QD605, QD655, and QD705 as labels.     

Controlling the reactivity of ampiphilic quantum dots in biological assays through hydrophobic assembly of custom PEG derivatives

Modifications of the quantum dot (QD) surface are routinely performed via covalent biomolecule attachment, and poly(ethylene glycol) (PEG) derivatization has previously been shown to limit nonspecific cellular interactions of QD probes. Attempts to functionalize ampiphilic QDs (AMP-QDs) with custom PEG derivatives having a hydrophobic terminus resulted in self-assembly of these PEG ligands to the AMP-QD surface in the absence of covalent coupling reagents. We demonstrate, via electrophoretic characterization techniques, that these self-assembled PEG-QDs exhibit improved passivation in biological environments and are less susceptible to unwanted protein adsorption to the QD surface. We highlight the artifactual fluorescent response protein adsorption can cause in biological assays, and discuss considerations for improved small molecule presentation to facilitate specific QD interactions.     

Synthesis, photophysical effects, and DNA targeting properties of oxazole yellow-peptide bioconjugates

The design of a dsDNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency is described. The bioconjugate has the molecular recognition features of the polypeptide from a DNA-binding protein and the dsDNA-dependent fluorescence of an intercalating dye. The DNA sequence selectivity of the probe was characterized, as were the changes in photophysical properties of the dye upon covalent linkage to the peptide to assess whether such bioconjugates could function as molecular probes of gene sequences. The oxazole yellow-peptide bioconjugate exhibits DNA recognition and binding affinity comparable to the native Hin recombinase protein. Examination of photophysical effects to dye conjugation indicates a negligible affect on the fluorescence quantum yield. Fluorescence studies indicate this molecular probe is useful to determine the presence of a given DNA target sequence and gives negligible fluorescence in the absence of a given target site. Using the synthetic route described here, bioconjugates could be designed using different combinations of DNA recognition polypeptides and cyanine dyes to generate an array of sequence specific and wavelength specific probes.     

Labeling of BSA and imaging of mouse T‐lymphocyte as well as mouse spleen tissue by l‐glutathione capped CdTe quantum dots

l ‐glutathione capped highly fluorescent CdTe quantum dots (QDs) were prepared by an aqueous approach and used as fluorescent labels to link albumin bovine serum (BSA) and rat anti‐mouse CD4, which was expressed on mouse T‐lymphocyte and mouse spleen tissue. The sharp and narrow emission peaks showed that the as‐prepared QDs have desirable dispersibility, uniformity and good fluorescence properties. Both CdTe–BSA and CdTe–CD4 conjugates showed an enhancement of fluorescence intensity over that of bare CdTe QDs. The experimental result of gel electrophoresis confirmed the successful conjugation of CdTe–BSA and CdTe–CD4. The fluorescent microscopic images of CdTe–CD4 labeled mouse T‐lymphocyte cells and mouse spleen tissue were compared with that obtained from fluorescein isothiocyanate labeling. It was demonstrated that the CdTe QDs‐based probe exhibited much better photostability and fluorescence intensity than fluorescein isothiocyanate, showing a good application potential in the immuno‐labeling of cells and tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of ligand density on the spectral, physical, and biological characteristics of CdSe/ZnS quantum dots

Chemical modification of the surface of CdSe/ZnS quantum dots (QDs) with small molecules or functional ligands often alters the characteristics of these particles. For instance, dopamine conjugation quenches the fluorescence of the QDs, which is a property that can be exploited for sensing applications if the conjugates are taken up into living cells. However, different sizes and/or preparations of mercaptocarboxylic acid solubilized QDs show very different properties when incubated with cells. It is unknown what physical parameters determine a QDs ability to interact with a cell surface, be endocytosed, escape from endosomes, and/or enter the nucleus. In this study, we examine the surface chemistry of QD-dopamine conjugates and present an optimized method for tracking the attachment of small biomolecules to the surface. It is found that the fluorescence intensity, surface charge, colloidal stability, and biological interactions of the QDs vary as a function of the density of dopamine on the surface. Successful targeting of QD-dopamine to dopamine receptor positive PC12 cells correlates with greater homogeneity of particle thiol layer, and a minimum number of ligands required for specific association can be estimated. These results will enable users to develop methods for screening QD conjugates for biological activity before proceeding to experiments with cell lines and animals.