Fadi Aldeek

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.

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.

Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms.

Quantum dots (QDs) nanoprobes are emerging as alternatives to small-molecule fluorescent probes in biomedical technology. This paper reports an efficient and rapid method of producing highly dispersed and stable CdSe-core QDs with a hydrophobic gradient. Amphiphilic core/shell CdSe/ZnS QDs were prepared by ligand exchange at the surface of lipophilic CdSe/ZnS QDs using the dihydrolipoic acid (DHLA) dithiol ligand linked to leucine or phenylalanine amino acids. Contact angle relaxations on a hydrophobic surface and surface tension measurements indicated that aqueous dispersions of CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs exhibit increased hydrophobicity compared to CdSe-core QDs capped by the hydrophilic 3-mercaptopropionic acid (MPA) ligand. We found that the surface functional groups and the ligand density at the periphery of these QDs significantly dictated their interactions with a complex biological matrix called biofilm. Using fluorescence confocal microscopy and an autocorrelation function (semi-variogram), we demonstrated that MPA-capped QDs were homogeneously associated to the biopolymers, while amphiphilic CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs were specifically confined allowing identification of hydrophobic microdomains of the biofilms. Results obtained clearly point out that the final destination of QDs in biofilms can properly be controlled by an appropriate design of surface ligands.

UV and Sunlight Driven Photoligation of Quantum Dots: Understanding the Photochemical Transformation of the Ligands

We have recently reported that photoinduced ligation of ZnS-overcoated quantum dots (QDs) offers a promising strategy to promote the phase transfer of these materials to polar and aqueous media using multidentate lipoic acid (LA)-modified ligands. In this study we investigate the importance of the underlying parameters that control this process, in particular, whether or not photoexcited QDs play a direct role in the photoinduced ligation. We find that irradiation of the ligand alone prior to mixing with hydrophobic QDs is sufficient to promote ligand exchange. Furthermore, photoligation onto QDs can also be carried out simply by using sunlight. Combining the use of Ellmans test with matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, we probe the nature of the photochemical transformation of the ligands. We find that irradiation (using either a UV photoreactor or sunlight) alters the nature of the disulfide groups in the lipoic acid, yielding a different product mixture than what is observed for chemically reduced ligands. Irradiation of the ligand in solution generates a mixture of monomeric and oligomeric compounds. Ligation onto the QDs selectively favors oligomers, presumably due to their higher coordination onto the metal-rich QD surfaces. We also show that photoligation using mixed ligands allows the preparation of reactive nanocrystals. The resulting QDs are coupled to proteins and peptides and tested for cellular staining. This optically controlled ligation of QDs combined with the availability of a variety of multidentate and multifunctional LA-modified ligands open new opportunities for developing fluorescent platforms with great promises for use in imaging and sensor design.

Aqueous Growth of Gold Clusters with Tunable Fluorescence Using Photochemically Modified Lipoic Acid-Based Ligands

We report a one-phase aqueous growth of fluorescent gold nanoclusters (AuNCs) with tunable emission in the visible spectrum, using a ligand scaffold that is made of poly(ethylene glycol) segment appended with a metal coordinating lipoic acid at one end and a functional group at the other end. This synthetic scheme exploits the ability of the UV-induced photochemical transformation of LA-based ligands to provide DHLA and other thiol byproducts that exhibit great affinity to metal nanoparticles, obviating the need for chemical reduction of the dithiolane ring using classical reducing agents. The influence of various experimental conditions, including the photoirradiation time, gold precursor-to-ligand molar ratios, time of reaction, temperature, and the medium pH, on the growth of AuNCs has been systematically investigated. The photophysical properties, size, and structural characterization were carried out using UV-vis absorption and fluorescence spectroscopy, TEM, DOSY-NMR, and X-ray photoelectron spectroscopy. The hydrodynamic size (RH) obtained by DOSY-NMR indicates that the size of these clusters follows the trend anticipated from the absorption and PL data, with RH(red) > RH(yellow) > RH(blue). The tunable emission and size of these gold nanoclusters combined with their high biocompatibility would make them greatly promising for potential use in imaging and sensing applications.

Glycosylated quantum dots for the selective labelling of Kluyveromyces bulgaricus and Saccharomyces cerevisiae yeast strains.

Highly fluorescent CdTe quantum dots (QDs) stabilized by thioglycolic acid (TGA) were prepared by an aqueous solution approach and used as fluorescent labels in detecting yeast cells. Sugars (mannose, galactose or glucose) were adsorbed on CdTe@TGA QDs and the interaction of these nanoparticles with yeast cells was studied by fluorescence microscopy. Results obtained demonstrate that galactose and mannose functionalized QDs associate respectively with Kluyveromyces bulgaricus and Saccharomyces cerevisiae yeast strains due to saccharide/lectin specific recognition. Glucose-functionalized CdTe QDs, which are not recognized by cell lectins, preferentially localize in the bud scars of S. cerevisiae.

Patterned Hydrophobic Domains in the Exopolymer Matrix of Shewanella oneidensis MR-1 Biofilms

ABSTRACT Water-dispersible amphiphilic surface-engineered quantum dots (QDs) were found to be strongly accumulated within discrete zones of the exopolymer network of Shewanella oneidensis MR-1 biofilms, but not on the cell surfaces. These microdomains showed a patterned distribution in the exopolymer matrix, which led to a restricted diffusion of the amphiphilic nanoparticles.

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.

Self-Assembled Gold Nanoparticle–Fluorescent Protein Conjugates as Platforms for Sensing Thiolate Compounds via Modulation of Energy Transfer Quenching

The ability of Au and other metal nanostructures to strongly quench the fluorescence of proximal fluorophores (dyes and fluorescent proteins) has made AuNP conjugates attractive for use as platforms for sensor development based on energy transfer interactions. In this study, we first characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-histidine coordination, where parameters such as NP size and number of attached proteins are varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ∼95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further exploited these findings to develop a solution phase sensing platform targeting thiolate compounds. Energy transfer (ET) was employed as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. Our results show that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction, and type of thiol derivatives used. Further analysis of the PL recovery data provides a measure for the equilibrium dissociation constant (Kd-1) for these compounds. These findings combined indicate that the AuNP-fluorescent protein conjugates may offer a potentially useful platform for thiol sensing both in solution and in cell cultures.

Synthesis, Characterization and Biological Applications of Water-Soluble ZnO Quantum Dots

Quantum dots (QDs) or semiconductor nanocrystals are of great interest to fundamental studies but have also potential applications as biological probes (Medintz et al., 2005), fluorescent biosensor (Costa-Fernandez et al., 2006), light-emitting diodes (LEDs) (Lim et al. 2007), and solar cells (Robel et al., 2006). Owing to the effect of quantum confinement, QDs show exceptional physical and chemical properties such as sharp and symmetrical emission spectra, high quantum yield (QY), good photo- and chemical stability, and size-dependent emission-wavelength tunability (Bruchez et al., 1998; Chan et al., 1998). For biological labelling, the most studied QDs are the nanocrystals of CdSe and CdTe (Aldeek et al., 2008) and the corresponding core/shell structured QDs (such as CdSe/ZnS, CdTe/ZnS or CdTe/ZnTe) that are more robust against chemical degradation or photooxidation than the parent cores (Law et al., 2009). Recent findings have highlighted the acute toxicity of II-VI semiconductor QDs without an external layer of a nontoxic material on biological systems (Schneider et al., 2009; Dumas et al. 2010). This toxicity results mainly from the decomposition and release of heavy metal ions and formation of reactive oxygen species. The toxicity of cadmium is a concern that will also limit the use of these visible or near IR emitting nanocrystals, especially for applications directly related to human health. Synthesis of low toxicity QDs and especially Cd-free QDs is the most challenging aspect of working with these materials in biological and medical fields. A promising member of the Cd-free QD is ZnO. However, ZnO nanoparticles are not stable in water. This instability is related to their surface luminescent mechanisms. Water molecules are able to attack the luminescent centers on the ZnO surface and destroy them rapidly. This chapter describes the strategies that have been developed over the last years to transfer ZnO QDs in water.