We describe the effectiveness of a multicoordinating polymer coating to surface functionalize gold nanospheres, nanoshells, and nanorods and promote their steric stabilization in biological media. The polymer ligand synthesized via one-step nucleophilic addition reaction starting with poly(isobutylene-alt-maleic anhydride) precursor presents multiple lipoic acid groups for strong coordination on metal-rich surfaces and several hydrophilic motifs (e.g., zwitterion groups or short poly(ethylene glycol) (PEG) chains) to promote water solubilization. We show that nanocrystals ligated with this polymer are compact in size and exhibit excellent long-term colloidal stability over broad conditions. We compare the ability of zwitterion- and PEG-modified polymer ligands to shield the metal surfaces from sodium cyanide digestion, or resist the competitive removal by dithiothreitol (DTT). We find that polymers appended with either hydrophilic motif essentially eliminate DTT competition for surface binding, while nanocrystals capped with the PEGylated coating exhibits substantially better resistance to sodium cyanide digestion compared with zwitterionic coating. Furthermore, we probe the differences between the two coatings in terms of endowing surface charge to the nanocrystals and affecting their Brownian diffusion properties. Additionally, we show that zwitterionic coating is very effective in preventing the formation of protein corona on such nanostructures, a highly valuable result with direct implications in biotechnology.

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.

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.

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.

We detail the design of hydrophilic metal-coordinating ligands and their use for the effective coating of luminescent quantum dots (QDs). The ligand design exploits the specific, reagent-free nucleophilic addition reaction of amine-modified molecules toward maleic anhydride to introduce several lipoic acid metal anchors, hydrophilic zwitterion moieties, and specific reactive groups along a poly(isobutylene-alt-maleic anhydride) (PIMA) chain. Tunable reactive groups tested in this study include azide, biotin, carboxyl, and amine. Cap exchange with these multilipoic acid ligands via a photochemical ligation strategy yields homogeneous QD dispersions that are colloidally stable over several biologically relevant conditions and for extended periods of time. The zwitterionic coating yields compact nanoparticle size and imparts nonsticky surface properties onto the QDs, preventing protein absorption. The introduction of a controllable number of reactive groups allows conjugation of the QDs to biomolecules via bio-orthogonal coupling chemistries including (1) attachment of the neurotransmitter dopamine to QDs via amine-isothiocyanate reaction to produce a platform capable of probing interactions with cysteine in proteins, based on charge transfer interactions; (2) self-assembly of biotinylated QDs with streptavidin-dye; and (3) ligation of azide-functionalized QDs to cyclooctyne-modified transferrin via copper-free click chemistry, used for intracellular delivery. This ligand design strategy can be used to prepare an array of metal-coordinating ligands adapted for coating other inorganic nanoparticles, including magnetic and plasmonic nanomaterials.

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.

•Unique photophysical properties of luminescent QDs.•External modulation of QD PL via energy transfer (ET) and/or charge transfer (CT) interactions.•Benefits of QD use in CT and ET interactions.•Sensor Design based on ET and CT.•Charge transfer implication for light-emitting and photovoltaic devices.Luminescent quantum dots (QDs) exhibit size- and composition-tunable photophysical properties that are not shared by their bulk parent materials or at the molecular scale. They have attracted considerable interest further motivated by several potential applications. A particular interest has centered on exploiting the ability of QDs to engage in both fluorescence resonance energy transfer (FRET) and charge transfer (CT) interactions with proximal fluorophores and redox active molecules/complexes, respectively. In this review, we highlight how the QD's optical and spectroscopic properties can be controlled via FRET and/or CT interactions. We first show that QDs provide a unique platform for controlling both modes of interactions. We then provide representative examples in biology which include developing sensing assemblies that report on properties such as pH changes, enzymatic activity and ligand–receptor binding. Implications in electronic devices focus on light emitting devices and photovoltaic cells, where we discuss device architecture, control over carrier injection, carrier mobility, and exciton recombination.

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.

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.

We introduce a new set of multicoordinating polymers as ligands that combine two distinct metal-chelating groups, lipoic acid and imidazole, for the surface functionalization of QDs. These ligands combine the benefits of thiol and imidazole coordination to reduce issues of thiol oxidation and weak binding affinity of imidazole. The ligand design relies on the introduction of controllable numbers of lipoic acid and histamine anchors, along with hydrophilic moieties and reactive functionalities, onto a poly(isobutylene-alt-maleic anhydride) chain via a one-step nucleophilic addition reaction. We further demonstrate that this design is fully compatible with a novel and mild photoligation strategy to promote the in situ ligand exchange and phase transfer of hydrophobic QDs to aqueous media under borohydride-free conditions. Ligation with these polymers provides highly fluorescent QDs that exhibit great long-term colloidal stability over a wide range of conditions, including a broad pH range (3–13), storage at nanomolar concentration, under ambient conditions, in 100% growth media, and in the presence of competing agents with strong reducing property. We further show that incorporating reactive groups in the ligands permits covalent conjugation of fluorescent dye and redox-active dopamine to the QDs, producing fluorescent platforms where emission is controlled/tuned by Förster Resonance Energy Transfer (FRET) or pH-dependent charge transfer (CT) interactions. Finally, the polymer-coated QDs have been coupled to cell-penetrating peptides to facilitate intracellular uptake, while subsequent cytotoxicity tests show no apparent decrease in cell viability.

We introduce a set of multicoordinating imidazole- and zwitterion-based ligands suited for surface functionalization of quantum dots (QDs). The polymeric ligands are built using a one-step nucleophilic addition reaction between poly(isobutylene-alt-maleic anhydride) and distinct amine-containing functionalities. This has allowed us to introduce several imidazole anchoring groups along the polymer chain for tight coordination to the QD surface and a controllable number of zwitterion moieties for water solubilization. It has also permitted the introduction of reactive and biomolecular groups for further conjugation and targeting. The QDs capped with these new ligands exhibit excellent long-term colloidal stability over a broad range of pH, toward excess electrolyte, in cell-growth media, and in the presence of natural reducing agents such as glutathione. These QDs are also resistant to the oxidizing agent H2O2. More importantly, by the use of zwitterion moieties as the hydrophilic block, this polymer design provides QDs with a thin coating and compact overall dimensions. These QDs are easily self-assembled with full size proteins expressed with a polyhistidine tag via metal–histidine coordination. Additionally, the incorporation of amine groups allows covalent coupling of the QDs to the neurotransmitter dopamine. This yields redox-active QD platforms that can be used to track pH changes and detect Fe ions and cysteine through charge-transfer interactions. Finally, we found that QDs cap-exchanged with folic acid-functionalized ligands could effectively target cancer cells, where folate-receptor-mediated endocytosis of QDs into living cells was time- and concentration-dependent.

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.

We explored the effects of changing the separation distance on the charge transfer interactions between luminescent QD and proximal dopamine (in QD–dopamine assemblies), and the ensuing photoluminescence (PL) quenching. The separation distance was controlled using a tunable size bridge between the QD and dopamine via a poly(ethylene glycol) (PEG) chain where the average number of monomers was discretely varied. Using steady-state and time-resolved fluorescence measurements, we found that the photoluminescence losses were substantially more pronounced for QD–dopamine complexes prepared with the shortest PEG bridge, but progressively decreased with increasing PEG size. We also found that the charge transfer interactions can be affected by the nature of the capping ligand used. In particular, we found that interactions and PL quenching in these assemblies tracked the effects of separation distance, conjugate valence and the energy mismatch between the dopamine redox levels and QD energy levels, when a compact zwitterion was used to control the conjugate configuration. However, additional effects of shielding the access of reactive dopamine to amine groups on the QD surface, when a longer inert PEG ligand was used, were found to produce heterogeneous conjugates, alter the interactions and produce weaker PL quenching.

We explored the charge transfer interactions between CdSe–ZnS core–shell quantum dots (QDs) and the redox active neurotransmitter dopamine, using covalently assembled QD–dopamine conjugates. We combined steady-state fluorescence, time-resolved fluorescence, and transient absorption bleach measurements to probe the effects of changing the QD size (thus the QD energy levels) and the conjugate valence on the rate of QD photoluminescence quenching when the pH of the medium was adjusted from acidic to alkaline. We measured substantially larger quenching efficiencies, combined with more pronounced shortening of the carrier dynamics of these assemblies for smaller size QDs and in alkaline pH. Moreover, we found that changes in the QD size alter the electron and hole relaxation of photoexcited QDs but with different extents. For instance, a pronounced change in the hole relaxation was measured in alkaline buffers. Moreover, the hole relaxation was faster for conjugates of green-emitting QDs as compared to their red-emitting counterparts. We attribute these results to the more favorable electron transfer rates from the reduced form of the dopamine to the valence band of the QDs, a process that becomes more efficient for green-emitting QDs. The latter benefits from lower oxidation potential and larger energy mismatch with the green QDs in alkaline buffers. In comparison, the effects of pH changes on the rates of electron transfer from excited QDs to dopamine are less affected by the QD size. These findings reflect the importance of the energy mismatch between the QD energy levels and the redox levels of dopamine, and shed light onto the complex interactions involved in these assemblies. Such conjugates also provide promising sensing and imaging tools for use in in vivo experiments.

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.

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.Keywords: biocompatibility; colloidal stability; fluorescence; quantum dots; surface-functionalization;

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.Keywords: fluorescence; functionalization; ligand; metal nanoclusters; reduction

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.Keywords: affinity chromatography; energy transfer; gold nanoparticles; kinetics; luminescent quantum dots; metal-affinity interactions; polyhistidine; self-assembly

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.

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.

We investigated the charge transfer interactions between luminescent quantum dots (QDs) and redox active dopamine. For this, we used pH-insensitive ZnS-overcoated CdSe QDs rendered water-compatible using poly (ethylene glycol)-appended dihydrolipoic acid (DHLA-PEG), where a fraction of the ligands was amine-terminated to allow for controlled coupling of dopamine–isothiocyanate onto the nanocrystal. Using this sample configuration, we probed the effects of changing the density of dopamine and the buffer pH on the fluorescence properties of these conjugates. Using steady-state and time-resolved fluorescence, we measured a pronounced pH-dependent photoluminescence (PL) quenching for all QD-dopamine assemblies. Several parameters affect the PL loss. First, the quenching efficiency strongly depends on the number of dopamines per QD-conjugate. Second, the quenching efficiency is substantially increased in alkaline buffers. Third, this pH-dependent PL loss can be completely eliminated when oxygen-depleted buffers are used, indicating that oxygen plays a crucial role in the redox activity of dopamine. We attribute these findings to charge transfer interactions between QDs and mainly two forms of dopamine: the reduced catechol and oxidized quinone. As the pH of the dispersions is changed from acidic to basic, oxygen-catalyzed transformation progressively reduces the dopamine potential for oxidation and shifts the equilibrium toward increased concentration of quinones. Thus, in a conjugate, a QD can simultaneously interact with quinones (electron acceptors) and catechols (electron donors), producing pH-dependent PL quenching combined with shortening of the exciton lifetime. This also alters the recombination kinetics of the electron and hole of photoexcited QDs. Transient absorption measurements that probed intraband transitions supported those findings where a simultaneous pronounced change in the electron and hole relaxation rates was measured when the pH was changed from acidic to alkaline.

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 N,N-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.

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, via 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.Keywords: ligand; luminescence; nanocluster; nanoparticle; reduction; size focusing

We have designed, prepared, and tested a new set of multidentate catechol- and polyethylene glycol (PEG)-derivatized oligomers, OligoPEG-Dopa, as ligands that exhibit strong affinity to iron oxide nanocrystals. The ligands consist of a short poly(acrylic acid) backbone laterally appended with several catechol anchoring groups and several terminally functionalized PEG moieties to promote affinity to aqueous media and to allow further coupling to target molecules (bio and others). These multicoordinating PEGylated oligomers were prepared using a relatively simple chemical strategy based on N,N′-dicyclohexylcarbodiimide (DCC) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) condensation. The ability of these catechol-functionalized oligomers to impart long-term colloidal stability to the nanoparticles is compared to other control ligands, namely, oligomers presenting several carboxyl groups and monodentate ligands presenting either one catechol or one carboxyl group. We found that the OligoPEG-Dopa ligands provide rapid ligand exchange, and the resulting nanoparticles exhibit greatly enhanced colloidal stability over a broad pH range and in the presence of excess electrolytes; stability is notably improved compared to non-catechol presenting molecular or oligomer ligands. By inserting controllable fractions of azide-terminated PEG moieties, the nanoparticles (NPs) become reactive to complementary functionalities via azide–alkyne cycloaddition (Click), which opens up the possibility of biological targeting of such stable NPs. In particular, we tested the Click coupling of azide-functionalized nanoparticles to an alkyne-modified dye. We also measured the MRI T2 contrast of the OligoPEG-capped Fe3O4 nanoparticles and applied MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to test the potential cytotoxicity of these NPs to live cells; we found no measurable toxicity to live cells.Keywords: biocompatibility; colloidal stability; cytotoxicity; iron oxide nanocrystals; MRI contrast; surface functionalization; synthesis

Water solubilized nanoparticles such as CdSe−ZnS core−shell nanocrystals (quantum dots, QDs) have great potential in bioimaging and sensing applications due to their excellent photophysical properties. However, the efficient modification of QDs with complex biomolecules represents a significant challenge. Here, we describe a straightforward arylhydrazone approach for the chemoselective covalent modification of QDs that is compatible with neutral pH and micromolar concentrations of the peptide target. The kinetics of covalent modification can be monitored spectroscopically at 354 nm in the presence of the QD and average peptide/QD ratios from 2:1 to 11:1 were achieved with excellent control over the desired valency. These results suggest that aniline catalyzed hydrazone ligation has the potencial to provide a general method for the controlled assembly of a variety of nanoparticle-biomolecule hybrids.

We present the design and synthesis of a new set of poly(ethylene glycol) (PEG)-based ligands appended with multidentate anchoring groups and test their ability to provide colloidal stability to semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs) in extreme buffer conditions. The ligands are made of a PEG segment appended with two thioctic acid (TA) or two dihydrolipoic acid (DHLA) anchoring groups, bis(TA)-PEG-OCH3 or bis(DHLA)-PEG-OCH3. The synthesis utilizes Michael addition to create a branch point at the end of a PEG chain combined with carbodiimide-coupling to attach two TA groups per PEG chain. Dispersions of CdSe−ZnS core−shell QDs and AuNPs with remarkable long-term colloidal stability at pHs ranging from 1.1 to 13.9 and in the presence of 2 M NaCl have been prepared and tested using these ligands. AuNPs with strong resistance to competition from dithiothreitol (as high as 1.5 M) have also been prepared. This opens up possibilities for using them as stable probes in a variety of bio-related studies where resistance to degradation at extreme pHs, at high electrolyte concentration, and in thiol-rich environments is highly desirable. The improved colloidal stability of nanocrystals afforded by the tetradentate ligands was further demonstrated via the assembly of stable QD−nuclear localization signal peptide bioconjugates that promoted intracellular uptake.

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.

We characterized the resonance energy-transfer interactions for conjugates consisting of QD donors self-assembled with three distinct fluorescent protein acceptors, two monomeric fluorescent proteins, the dsRed derivative mCherry or yellow fluorescent protein, and the multichromophore b-phycoerythrin light-harvesting complex. Using steady-state and time-resolved fluorescence, we showed that nonradiative transfer of excitation energy in these conjugates can be described within the Förster dipole−dipole formalism, with transfer efficiencies that vary with the degree of spectral overlap, the donor−acceptor separation distance, and the number of acceptors per QD. Comparison between the quenching data and simulation of the conjugate structures indicated that while energy transfer to monomeric proteins was identical to what was measured for QD−dye pairs, interactions with b-phycoerythrin were more complex. For the latter, the overall transfer efficiency results from the cumulative contribution of individual channels between the central QD and the chromophores distributed throughout the protein structure. Due to the biocompatible nature of fluorescent proteins, these QD assemblies may have great potential for use in intracellular imaging and sensing.

In this report we review some of the recent progress made for enhancing the biocompatibility of luminescent quantum dots (QDs) and for developing targeted bio-inspired applications centered on live cell imaging and sensing. We start with a detailed analysis of the surface functionalization strategies developed thus far, and discuss their effectiveness for providing long term stability of the quantum dots in biological media, to changes in pH and to added electrolytes. We then discuss the available conjugation techniques to couple QDs to a variety of biological receptors and compare their effectiveness. In particular, we highlight the implementation of new strategies such as the use of copper-free cyclo-addition reaction (CLICK) chemistry and chemo-selective ligation. We then discuss the advances made for intracellular delivery where ideas such as receptor-driven endocytosis and uptake promoted by cell penetrating peptides are used. We then describe a few representative examples where QDs have been used to investigate specific cell biology processes. Such processes include binding of QDs conjugated to the nerve growth factor to membrane specific receptors and intracellular uptake, tracking of membrane protein at the single molecule level, and recognition of ligand bound QDs by T cell receptors. We conclude by discussing issues of toxicity associated with the use of QDs in biology.Download high-res image (220KB)Download full-size image

We explored the effects of changing the separation distance on the charge transfer interactions between luminescent QD and proximal dopamine (in QD–dopamine assemblies), and the ensuing photoluminescence (PL) quenching. The separation distance was controlled using a tunable size bridge between the QD and dopamine via a poly(ethylene glycol) (PEG) chain where the average number of monomers was discretely varied. Using steady-state and time-resolved fluorescence measurements, we found that the photoluminescence losses were substantially more pronounced for QD–dopamine complexes prepared with the shortest PEG bridge, but progressively decreased with increasing PEG size. We also found that the charge transfer interactions can be affected by the nature of the capping ligand used. In particular, we found that interactions and PL quenching in these assemblies tracked the effects of separation distance, conjugate valence and the energy mismatch between the dopamine redox levels and QD energy levels, when a compact zwitterion was used to control the conjugate configuration. However, additional effects of shielding the access of reactive dopamine to amine groups on the QD surface, when a longer inert PEG ligand was used, were found to produce heterogeneous conjugates, alter the interactions and produce weaker PL quenching.