Efficient Assembly of Quantum Dots with Homogenous Glycans Derived from Natural N-Linked Glycoproteins.

Coating inorganic nanoparticles with polyethylene glycol (PEG)-appended ligands, as means to preserve their physical characteristics and promote steric interactions with biological systems, including enhanced aqueous solubility and reduced immunogenicity, has been explored by several groups. Conversely, macromolecules present in the human serum and on the surface of cells are densely coated with hydrophilic glycans that act to reduce nonspecific interactions, while facilitating specific binding and interactions. In particular, N-linked glycans are abundant on the surface of most serum proteins and are composed of a branched architecture that is typically characterized by a significant level of molecular heterogeneity. Here we provide two distinct methodologies, covalent bioconjugation and self-assembly, to functionalize two types of Quantum Dots with a homogeneous, complex-type N-linked glycan terminated with a sialic acid moiety. A detailed physical and functional characterization of these glycan-coated nanoparticles has been performed. Our findings support the potential use of such fluorescent platforms to sense glycan-involved biological processes, such as lectin recognition and sialidase-mediated hydrolysis.

Photochemical transformation of lipoic acid-based ligands: probing the effects of solvent, ligand structure, oxygen and pH.

We have combined optical absorption with the Ellman's test to identify the parameters that affect the transformation of the 5-membered dithiolanes to thiols in lipoic acid (LA) and its derivatives during UV-irradiation. We found that the nature and polarity of the solvent, the structure of the ligands, acidity of the medium and oxygen can drastically affect the amount of photogenerated thiols. These findings are highly relevant to the understanding of the photochemical transformation of this biologically relevant compound, and would benefit the increasing use of LA-based ligands for the surface functionalization of various nanomaterials.

Intracellular Delivery of Luminescent Quantum Dots Mediated by a Virus-Derived Lytic Peptide.

We describe a new quantum dot (QD)-conjugate prepared with a lytic peptide, derived from a nonenveloped virus capsid protein, capable of bypassing the endocytotic pathways and delivering large amounts of QDs to living cells. The polypeptide, derived from the Nudaurelia capensis Omega virus, was fused onto the C-terminus of maltose binding protein that contained a hexa-HIS tag at its N-terminus, allowing spontaneous self-assembly of controlled numbers of the fusion protein per QD via metal-HIS interactions. We found that the efficacy of uptake by several mammalian cell lines was substantial even for small concentrations (10-100 nM). Upon internalization the QDs were primarily distributed outside the endosomes/lysosomes. Moreover, when cells were incubated with the conjugates at 4 °C, or in the presence of chemical endocytic inhibitors, significant intracellular uptake continued to occur. These findings indicate an entry mechanism that does not involve endocytosis, but rather the perforation of the cell membrane by the lytic peptide on the QD surfaces.

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.

Controlling the Architecture, Coordination, and Reactivity of Nanoparticle Coating Utilizing an Amino Acid Central Scaffold.

We have developed a versatile strategy to prepare a series of multicoordinating and multifunctional ligands optimized for the surface-functionalization of luminescent quantum dots (QDs) and gold nanoparticles (AuNPs) alike. Our chemical design relies on the modification of l-aspartic acid precursor to controllably combine, through simple peptide coupling chemistry, one or two lipoic acid (LA) groups and poly(ethylene glycol) (PEG) moieties in the same ligand. This route has provided two sets of modular ligands: (i) bis(LA)-PEG, which presents two lipoic acids (higher coordination) appended onto a single end-functionalized PEG, and (ii) LA-(PEG)2 made of two PEG moieties (higher branching, with various end reactive groups) appended onto a single lipoic acid. These ligands are combined with a new photoligation strategy to yield hydrophilic and reactive QDs that are colloidally stable over a broad range of conditions, including storage at nanomolar concentration and under ambient conditions. AuNPs capped with these ligands exhibit excellent stability in various biological conditions and improved resistance against NaCN digestion. This route also provides compact nanocrystals with tunable surface reactivity. As such, we have covalently coupled QDs capped with bis(LA)-PEG-COOH to transferrin to facilitate intracellular uptake. We have also characterized and quantified the coupling of dye-labeled peptides to QD surfaces using fluorescence resonance energy transfer interactions in QD-peptide-dye assemblies.

Preparation of compact biocompatible quantum dots using multicoordinating molecular-scale ligands based on a zwitterionic hydrophilic motif and lipoic acid anchors.

Luminescent quantum dots (QDs) can potentially be used for many biological experiments, provided that they are constructed in such a way as to be stable in biological matrices. Furthermore, QDs that are compact in size and easy to couple to biomolecules can be readily used for applications ranging from protein tracking to vasculature imaging. In this protocol, we describe the preparation of ligands comprising either one or two lipoic acid (LA) groups chemically linked to a zwitterion moiety. These ligands are then used to functionalize luminescent QDs via a photochemical transformation of LA. This route produces nanocrystals that are compact in size and stable over a broad range of conditions. In addition, the resulting QDs are readily self-assembled with polyhistidine-appended proteins. This mode of conjugation maintains the protein biological activity and its orientation, yielding highly promising fluorescent conjugates that can be used for imaging and sensing. The protocol in its entirety can be completed in 3 weeks.

Ylidenemalononitrile enamines as fluorescent "turn-on" indicators for primary amines.

Ylidenemalononitrile enamines undergo rapid amine exchange followed by a cyclization with primary amines to yield fluorescent products with emission intensities as high as 900 times greater than the starting materials. After identifying the fluorescent species by X-ray crystallography, we demonstrate that the rate of amine exchange is substrate dependent and that by simple structural variation the fluorescence can be tuned over the entire visible spectrum. We further demonstrate their potential application in biomolecule labeling.

Photoligation combined with zwitterion-modified lipoic acid ligands provides compact and biocompatible quantum dots.

We describe the design and synthesis of a series of compact ligands made of lipoic acid (LA)-based coordinating anchors and hydrophilic zwitterion groups. This ligand design is combined with a novel photoligation strategy to promote the transfer of QDs to polar and buffer media. This approach has provided hydrophilic QDs that exhibit great colloidal stability over a broad range of pHs and in the presence of cell culture media. Our photoligation strategy drastically improves previous phase transfer methods by eliminating the need for chemical reduction of the dithiolane ring using NaBH4 prior to the cap exchange, and it is adapted to several LA-based ligands. We also found that QDs stabilized with these compact zwitterionic ligands are fully compatible with metal-histidine-driven self-assembly where the protein activity is maintained after forming conjugation with the QDs.

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, Kd(-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.

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.

Growth of highly fluorescent polyethylene glycol- and zwitterion-functionalized gold nanoclusters.

We have prepared and characterized a new set of highly fluorescent gold nanoclusters (AuNCs) using one-step aqueous reduction of a gold precursor in the presence of bidentate ligands made of lipoic acid anchoring groups, appended with either a poly(ethylene glycol) short chain or a zwitterion group. The AuNCs fluoresce in the red to near-infrared region of the optical spectrum with emission centered at ∼750 nm and a quantum yield of ∼10-14%, and they exhibit long fluorescence lifetimes (up to ∼300 ns). Dispersions of these AuNCs exhibit great long-term colloidal stability, over a wide range of pHs (2-13) and in the presence of high electrolyte concentrations, and a strong resistance to reducing agents such as glutathione. The growth strategy further permitted the controlled, in situ functionalization of the NCs with reactive groups (e.g., carboxylic acid or amine), making these nanoclusters compatible with common and simple-to-implement coupling strategies, such as carbodiimide chemistry. These properties combined make these fluorescent NCs greatly promising for use in various imaging and sensing applications where NIR and long-lived excitations are desired.

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.

Photoinduced phase transfer of luminescent quantum dots to polar and aqueous media.

We report a new strategy for the photomediated phase transfer of luminescent quantum dots, QDs, and potentially other inorganic nanocrystals, from hydrophobic to polar and hydrophilic media. In particular, we demonstrate that UV-irradiation (λ < 400 nm) promotes the in situ ligand exchange on hydrophobic CdSe QDs with lipoic acid (LA)-based ligands and their facile QD transfer to polar solvents and to buffer media. This convenient method obviates the need to use highly reactive agents for chemical reduction of the dithiolane groups on the ligands. It maintains the optical and spectroscopic properties of the QDs, while providing high photoluminescence yield and robust colloidal stability in various biologically relevant conditions. Furthermore, development of this technique significantly simplifies the preparation and purification of QDs with sensitive functionalities. Application of these QDs to imaging the brain of live mice provides detailed information about the brain vasculature over the period of a few hours. This straightforward approach offers exciting possibilities for expanded functional compatibilities and reaction orthogonality on the surface of inorganic nanocrystals.

A novel multinozzle electrospinning process for preparing superhydrophobic PS films with controllable bead-on-string/microfiber morphology.

Superhydrophobic polystyrene (PS) surfaces with mechanical integrity were manufactured by electrospinning in this work. We first report a novel strategy here to combine bead-on-string fibers from 4% PS solution and micro-sized fibers from 20% PS solution homogeneously in one electrospinning step by multinozzle electrospinning. The superhydrophobicity of electrospun sheet can be achieved by the presence of bead-on-string fibers, while micro-sized PS fibers are responsible for the improvement of mechanical property of electrospun mat due to their elastic and flexible behavior. The distinctive design of our multinozzle electrospinning setup places two nozzles in separate electrical fields which guarantee that fibers with different structures are mixed homogeneously. We investigate the relationship between the mass ratio of fibers from two types of solutions and the CA of electrospun mat, the effect of mass ratio to the mechanical property of electrospun mat can also be observed. The result shows that CA value of PS surface merely comprised of bead-on-string fibers could reach up to 154.65°. As the content of microfibers increased, the value of CA decreased, ranging from 153.66° to 145.94°, but the tensile strength of composite mat was enhanced from 0.50 MPa to 1.22 MPa correspondingly which is beneficial to put the mats into practice.