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

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

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, <i>in situ</i> functionalization of the NCs with reactive groups (<i>e.g</i>., 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

Growth of <i>In Situ</i> Functionalized Luminescent Silver Nanoclusters by Direct Reduction and Size Focusing

We have used one phase growth reaction to prepare a series of silver nanoparticles (NPs) and luminescent nanoclusters (NCs) using sodium borohydride (NaBH₄) reduction of silver nitrate in the presence of molecular scale ligands made of polyethylene glycol (PEG) appended with lipoic acid (LA) groups at one end and reactive (−COOH/–NH₂) or inert (−OCH₃) functional groups at the other end. The PEG segment in the ligand promotes solubility in a variety of solvents including water, while LAs provide multidentate coordinating groups that promote Ag–ligand complex formation and strong anchoring onto the NP/NC surface. The particle size and properties were primarily controlled by varying the Ag-to-ligand (Ag:L) molar ratios and the molar amount of NaBH₄ used. We found that while higher Ag:L ratios produced NPs, luminescent NCs were formed at lower ratios. We also found that nonluminescent NPs can be converted into luminescent clusters, <i>via</i> a process referred to as “size focusing”, in the presence of added excess ligands and reducing agent. The nanoclusters emit in the far red region of the optical spectrum with a quantum yield of ∼12%. They can be redispersed in a number of solvents with varying polarity while maintaining their optical and spectroscopic properties. Our synthetic protocol also allowed control over the number and type of reactive functional groups per nanocluster

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

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 Ellman’s 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

Identification of Penicillin G Metabolites under Various Environmental Conditions Using UHPLC-MS/MS

In this work, we investigate the stability of penicillin G in various conditions including acidic, alkaline, natural acidic matrices and after treatment of citrus trees that are infected with citrus greening disease. The identification, confirmation, and quantitation of penicillin G and its various metabolites were evaluated using two UHPLC-MS/MS systems with variable capabilities (i.e., Thermo Q Exactive Orbitrap and Sciex 6500 QTrap). Our data show that under acidic and alkaline conditions, penicillin G at 100 ng/mL degrades quickly, with a determined half-life time of approximately 2 h. Penillic acid, penicilloic acid, and penilloic acid are found to be the most abundant metabolites of penicillin G. These major metabolites, along with isopenillic acid, are found when penicillin G is used for treatment of citrus greening infected trees. The findings of this study will provide insight regarding penicillin G residues in agricultural and biological applications

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 (<i>R</i>_H) obtained by DOSY-NMR indicates that the size of these clusters follows the trend anticipated from the absorption and PL data, with <i>R</i>_H(red) > <i>R</i>_H(yellow) > <i>R</i>_H(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

Functional-Group-Dependent Formation of Bioactive Fluorescent-Plasmonic Nanohybrids

We detail the assembly, driven by metal-affinity coordination, of fluorescent-plasmonic hybrid constructs that are also biologically active. The hybrid constructs are prepared by first assembling polymer-encapsulated luminescent quantum dots that present amine-, carboxy-, and lipoic acid-terminated groups (QD-FG) and plasmonic gold nanoparticles capped with rather low density of lipoic acid-appended zwitterion ligands (AuNP-LA-ZW). The dual QD-AuNP constructs were then coupled to polyhistidine-appended maltose binding proteins, yielding the final trifunctional assemblies. The coordination of amine-, carboxy-, and lipoic acid-terminated QDs with AuNP-LA-ZW was characterized using steady-state and time-resolved fluorescence quenching measurements. We measured rather different coordination affinities between the functional groups on the QDs and the AuNP surfaces. This assembly mode still allowed the partially exposed AuNPs in the inorganic/polymer hybrid to bind to polyhistidine-appended proteins. This protein assembly was confirmed using amylose affinity chromatography, which also confirmed the structural integrity of the hybrid and biological activity of the bound protein. Owing to the high colloidal stability of the surface-modified QDs and AuNP-LA-ZW, combined with flexible functionalization, we anticipate that this strategy could facilitate the integration of hybrid inorganic/polymer constructs with specific photophysical properties into biological systems

Enhanced Optical Properties of Core/Shell/Shell CdTe/CdS/ZnO Quantum Dots Prepared in Aqueous Solution

New quantum dots (QDs) were fabricated with a core/shell/shell structure consisting of CdTe core/CdS shell/ZnO shell. Despite the high lattice mismatch between CdS and ZnO, a ZnO shell was successfully introduced by basic hydrolysis of Zn(OAc)2 at the surface of core/shell CdTe/CdS QDs stabilized by 3-mercaptopropionic acid (MPA). The core/shell/shell CdTe/CdS/ZnO@MPA QDs exhibited a significant redshift of emission peaks (up to 50 nm for green-emitting CdTe/CdS QDs) when the shell grew. By changing the size of the core or the thickness of the ZnO shell, the emission colors of the obtained nanocrystals can be tuned between the green and red regions of the spectrum following an identical procedure. The influence of ZnO shell growth on photoluminescence (PL) quantum yields was found to be more pronounced for CdTe/CdS samples with green or yellow emission for which quantum yields increased up to three times. The epitaxial growth of the ZnO shell was confirmed by X-ray photoelectron spectroscopy and luminescence decay experiments. Because of the passivation of surface defects, a PL lifetime of 33.6 ns was measured for core/shell/shell CdTe/CdS/ZnO QDs prepared with a Zn/Cd ratio of 0.8, while it was only 17.7 ns for core/shell CdTe/CdS QDs