Specificity of fluorophore - streptavidin detection of biotinylated total human DNA probe in metaphase chromosomes and photostability

() Control (no fluorophore – streptavidin conjugate); () streptavidin – Qdot 605 detection of chromosome 1q12 region (vertical and horizontal arrows); () Texas Red – streptavidin detection of biotinylated DNA hybridized to 1q12 (vertical arrows) and () FITC – streptavidin detection of 1q12 sites (vertical arrows). Bar in panel () is 10 μm. () Signal decay upon continuous illumination with fluorescence microscope/mercury illumination in metaphase chromosome band 1q12 during 2 h continuous illumination. Red is Qdot 605, green is Texas Red, and blue is FITC. () Total intensity of whole interphase nuclei during 120 ms illumination (blue bars) and background (red bars). = 3 cells in each. Copyright © 2004 Oxford University Press. Xiao Y, Barker PE. 2004. Semiconductor nanocrystal probes for human metaphase chromosomes. , 32:e28. Reproduced with permission.<p><b>Copyright information:</b></p><p>Taken from "Potential clinical applications of quantum dots"</p><p></p><p>International Journal of Nanomedicine 2008;3(2):151-167.</p><p>Published online Jan 2008</p><p>PMCID:PMC2527673.</p><p>© 2008 Dove Medical Press Limited. All rights reserved</p

Quenching of Quantum Dot Emission by Fluorescent Gold Clusters: What It Does and Does Not Share with the Förster Formalism

Understanding the interactions that control the energy transfer between dyes, or luminescent quantum dots (QDs), and gold nanoparticles still has several unanswered questions. In this study we probed these interactions using a unique model where CdSe-ZnS QDs were coupled to fluorescent gold nanoclusters (AuNCs). Steady-state and time-resolved fluorescence measurements were used to investigate the effects of spectral overlap and separation distance on the quenching of QD photoemission in these assemblies, using three different size QDs with distinct emission spectra and a variable length polyethylene glycol bridge. We found that the QD photoluminescence quenching efficiency depends on the spectral overlap and separation distance, with larger quenching efficiencies than what would be expected for a QD-dye pair with similar overlap. Moreover, despite the large losses in QD PL, we found no resonance enhancement in the cluster emission for any of the sample configurations used. These results indicate that the mechanism driving the quenching by metal clusters shares an important feature (namely dependence on the spectral overlap) with the Förster dipole–dipole coupling at the heart of fluorescence resonance energy transfer (FRET) and widely validated for dye-dye and QD-dye assemblies. They also prove that the energy losses induced by metal nanostructures are governed by a process that is different from the Förster mechanism

Poly(ethylene glycol)-Based Multidentate Oligomers for Biocompatible Semiconductor and Gold Nanocrystals

We have developed a new set of multifunctional multidentate OligoPEG ligands, each containing a central oligomer on which were laterally grafted several short poly­(ethylene glycol) (PEG) moieties appended with either thioctic acid (TA) or terminally reactive groups. Reduction of the TAs (e.g., in the presence of NaBH₄) provides dihydrolipoic acid (DHLA)-appended oligomers. Here the insertion of PEG segments in the ligand structure promotes water solubility and reduces nonspecific interactions, while TA and DHLA groups provide multidentate anchoring onto Au nanoparticles (AuNPs) and ZnS-overcoated semiconductor quantum dots (QDs), respectively. The synthetic route involves simple coupling chemistry using <i>N</i>,<i>N</i>-dicylohexylcarbodiimide (DCC). Water-soluble QDs and AuNPs capped with these ligands were prepared via cap exchange. As prepared, the nanocrystals dispersions were aggregation-free, homogeneous, and stable for extended periods of time over pH ranging from 2 to 14 and in the presence of excess electrolyte (2 M NaCl). The new OligoPEG ligands also allow easy integration of tunable functional and reactive groups within their structures (e.g., azide or amine), which imparts surface functionalities to the nanocrystals and opens up the possibility of bioconjugation with specific biological molecules. The improved colloidal stability combined with reactivity offer the possibility of using the nanocrystals as biological probes in an array of complex and biologically relevant media

Probing Protein Corona Formation around Gold Nanoparticles: Effects of Surface Coating

There has been much interest in integrating various inorganic nanoparticles (nanoscale colloids) in biology and medicine. However, buildup of a protein corona around the nanoparticles in biological media, driven by nonspecific interactions, remains a major hurdle for the translation of nanomedicine into clinical applications. In this study, we investigate the interactions between gold nanoparticles and serum proteins using a series of dihydrolipoic acid (DHLA)-based ligands. We employed gel electrophoresis combined with UV–vis absorption and dynamic light scattering to correlate protein adsorption with the nature and size of the ligand used. For instance, we found that AuNPs capped with DHLA alone promote nonspecific protein adsorption. In comparison, capping AuNPs with polyethylene glycol- or zwitterion-appended DHLA essentially prevents corona formation, regardless of ligand charge and size. Our results highlight the crucial role of surface chemistry and core material in protein corona formation and offer valuable information for the design of colloidal nanomaterials for biological applications

Elucidating the Role of Surface Coating in the Promotion or Prevention of Protein Corona around Quantum Dots

Nonspecific interactions in biological media can lead to the formation of a protein corona around nanocolloids, which tends to alter their behavior and limit their effectiveness when used as probes for imaging or sensing applications. Yet, understanding the corona buildup has been challenging. We hereby investigate these interactions using luminescent quantum dots (QDs) as a model nanocolloid system, where we carefully vary the nature of the hydrophilic block in the surface coating, while maintaining the same dihydrolipoic acid (DHLA) bidentate coordinating motif. We first use agarose gel electrophoresis to track changes in the mobility shift upon exposure of the QDs to protein-rich media. We find that QDs capped with DHLA (which presents a hydrophobic alkyl chain terminated with a carboxyl group) promote corona formation, in a concentration-dependent manner. However, when a polyethylene glycol block or a zwitterion group is appended onto DHLA, it yields a coating that prevents corona buildup. Our results clearly confirm that nonspecific interactions with protein-rich media are strongly dependent on the nature of the hydrophilic motif used. Additional gel experiments using SDS-PAGE have allowed further characterization of the corona protein, and showed that mainly a soft corona forms around the DHLA-capped QDs. These findings will be highly informative when designing nanocolloids that can find potential use in biological applications

Self-Organized Tubular Structures as Platforms for Quantum Dots

The combination of top-down and bottom-up approaches offers great opportunities for the production of complex materials and devices. We demonstrate this approach by incorporating luminescent CdSe-ZnS nanoparticles into macroscopic tube structures that form as the result of externally controlled self-organization. The 1–2 mm wide hollow tubes consist of silica-supported zinc oxide/hydroxide and are formed by controlled injection of aqueous zinc sulfate into a sodium silicate solution. The primary growth region at the top of the tube is pinned to a robotic arm that moves upward at constant speed. Dispersed within the injected zinc solution are 3.4 nm CdSe-ZnS quantum dots (QDs) capped by DHLA-PEG–OCH₃ ligands. Fluorescence measurements of the washed and dried tubes reveal the presence of trapped QDs at an estimated number density of 10¹⁰ QDs per millimeter of tube length. The successful inclusion of the nanoparticles is further supported by electron microscopy and energy dispersive X-ray spectroscopy, with the latter suggesting a nearly homogeneous QD distribution across the tube wall. Exposure of the samples to copper sulfate solution induces quenching of about 90% of the tubes’ fluorescence intensity. This quenching shows that the large majority of the QDs is chemically accessible within the microporous, about 15-μm-wide tube wall. We suggest possible applications of such QD-hosting tube systems as convenient sensors in microfluidic and related applications

One-Phase Synthesis of Water-Soluble Gold Nanoparticles with Control over Size and Surface Functionalities

We report a simple and efficient synthetic method to prepare gold nanoparticles (AuNPs) in aqueous phase using HAuCl4 and poly(ethylene glycol) (PEG) ligands appended with bidentate anchoring groups. Our approach provides narrow size distribution nanocrystals over the size range between 1.5 and 18 nm; this range is much wider than those achieved using other small molecules and polymer ligands. The NP size was simply controlled by varying the molar ratio of Au-to-PEG ligand precursors. Further passivation of the as-prepared AuNPs permitted in situ functionalization of the NP surface with the desired functional groups. The prepared AuNPs exhibit remarkable stability in the presence of high salt concentrations, over a wide range of pHs (2−13), and a strong resistance to competition from dithiothreitol (DTT). These results are a clear manifestation of the advantages offered by our synthetic approach to prepare biocompatible AuNPs, where modular, multifunctional ligands presenting strong anchoring groups and hydrophilic PEG chains are used

Bio-orthogonal Coupling as a Means of Quantifying the Ligand Density on Hydrophilic Quantum Dots

We describe the synthesis of two metal-coordinating ligands that present one or two lipoic acid (LA) anchors, a hydrophilic polyethylene glycol (PEG) segment and a terminal reactive group made of an azide or an aldehyde, two functionalities with great utility in bio-orthogonal coupling techniques. These ligands were introduced onto the QD surfaces using a combination of photochemical ligation and mixed cap exchange strategy, where control over the fraction of azide and aldehyde groups per nanocrystal can be easily achieved: LA-PEG-CHO, LA-PEG-N₃, and bis­(LA)-PEG-CHO. We then demonstrate the application of two novel bio-orthogonal coupling strategies directly on luminescent quantum dot (QD) surfaces that use click chemistry and hydrazone ligation under catalyst-free conditions. We applied the highly efficient hydrazone ligation to couple 2-hydrozinopyridine (2-HP) to aldehyde-functionalized QDs, which produces a stable hydrazone chromophore with a well-defined optical signature. This unique optical feature has enabled us to extract a measure for the ligand density on the QDs for a few distinct sizes and for different ligand architectures, namely mono-LA-PEG and bis­(LA)-PEG. We found that the foot-print-area per ligand was unaffected by the nanocrystal size but strongly depended on the ligand coordination number. Additionally, we showed that when the two bio-orthogonal functionalities (aldehyde and azide) are combined on the same QD platform, the nanocrystal can be specifically reacted with two distinct targets and with great specificity. This design yields QD platforms with distinct chemoselectivities that are greatly promising for use as carriers for in vivo imaging and delivery

Multidentate Zwitterionic Ligands Provide Compact and Highly Biocompatible Quantum Dots

Hydrophilic functional semiconductor nanocrystals that are also compact provide greatly promising platforms for use in bioinspired applications and are thus highly needed. To address this, we designed a set of metal coordinating ligands where we combined two lipoic acid groups, bis­(LA)-ZW, (as a multicoordinating anchor) with a zwitterion group for water compatibility. We further combined this ligand design with a new photoligation strategy, which relies on optical means instead of chemical reduction of the lipoic acid, to promote the transfer of CdSe-ZnS QDs to buffer media. In particular, we found that the QDs photoligated with this zwitterion-terminated bis­(lipoic) acid exhibit great colloidal stability over a wide range of pHs, to an excess of electrolytes, and in the presence of growth media and reducing agents, in addition to preserving their optical and spectroscopic properties. These QDs are also stable at nanomolar concentrations and under ambient conditions (room temperature and white light exposure), a very promising property for fluorescent labeling in biology. In addition, the compact ligands permitted metal–histidine self-assembly between QDs photoligated with bis­(LA)-ZW and two different His-tagged proteins, maltose binding protein and fluorescent mCherry protein. The remarkable stability of QDs capped with these multicoordinating and compact ligands over a broad range of conditions and at very small concentrations, combined with the compatibility with metal–histidine conjugation, could be very useful for a variety of applications, ranging from protein tracking and ligand–receptor binding to intracellular sensing using energy transfer interactions

Understanding the Self-Assembly of Proteins onto Gold Nanoparticles and Quantum Dots Driven by Metal-Histidine Coordination

Coupling of polyhistidine-appended biomolecules to inorganic nanocrystals driven by metal-affinity interactions is a greatly promising strategy to form hybrid bioconjugates. It is simple to implement and can take advantage of the fact that polyhistidine-appended proteins and peptides are routinely prepared using well established molecular engineering techniques. A few groups have shown its effectiveness for coupling proteins onto Zn- or Cd-rich semiconductor quantum dots (QDs). Expanding this conjugation scheme to other metal-rich nanoparticles (NPs) such as AuNPs would be of great interest to researchers actively seeking effective means for interfacing nanostructured materials with biology. In this report, we investigated the metal-affinity driven self-assembly between AuNPs and two engineered proteins, a His₇-appended maltose binding protein (MBP-His) and a fluorescent His₆-terminated mCherry protein. In particular, we investigated the influence of the capping ligand affinity to the nanoparticle surface, its density, and its lateral extension on the AuNP-protein self-assembly. Affinity gel chromatography was used to test the AuNP-MPB-His₇ self-assembly, while NP-to-mCherry-His₆ binding was evaluated using fluorescence measurements. We also assessed the kinetics of the self-assembly between AuNPs and proteins in solution, using time-dependent changes in the energy transfer quenching of mCherry fluorescent proteins as they immobilize onto the AuNP surface. This allowed determination of the dissociation rate constant, <i>K</i>_d^–1 ∼ 1–5 nM. Furthermore, a close comparison of the protein self-assembly onto AuNPs or QDs provided additional insights into which parameters control the interactions between imidazoles and metal ions in these systems