Evaluating the Catalytic Efficiency of the Human Membrane-type 1 Matrix Metalloproteinase (MMP-14) Using AuNP-Peptide Conjugates.

Interactions of plasmonic nanocolloids such as gold nanoparticles and nanorods with proximal dye emitters result in efficient quenching of the dye photoluminescence (PL). This has become a popular strategy for developing analytical biosensors relying on this quenching process for signal transduction. Here, we report on the use of stable PEGylated gold nanoparticles, covalently coupled to dye-labeled peptides, as sensitive optically addressable sensors for determining the catalytic efficiency of the human matrix metalloproteinase-14 (MMP-14), a cancer biomarker. We exploit real-time dye PL recovery triggered by MMP-14 hydrolysis of the AuNP-peptide-dye to extract quantitative analysis of the proteolysis kinetics. Sub-nanomolar limit of detections for MMP-14 has been achieved using our hybrid bioconjugates. In addition, we have used theoretical considerations within a diffusion-collision framework to derive enzyme substrate hydrolysis and inhibition kinetics equations, which allowed us to describe the complexity and irregularity of enzymatic proteolysis of nanosurface-immobilized peptide substrates. Our findings offer a great strategy for the development of highly sensitive and stable biosensors for cancer detection and imaging.

Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14.

We detail the assembly and characterization of quantum dot (QD)-dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarker─matrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides. The analytical capability of the conjugates is enabled by correlating changes in the FRET efficiency with the conjugate valence and/or QD-to-dye separation distance, triggered and modulated by enzymatic proteolysis of surface-tethered peptides. The FRET probe exhibits great sensitivity to enzyme digestion with sub-nanomolar limit of detection. We further analyze the proteolysis data within the framework of the Michaelis-Menten model, which considers the fact that surface-attached peptides have a slower diffusion coefficient than free peptides. This results in reduced collision frequency and lower catalytic efficiency, kcat/KM. Our results suggest that our conjugate design is promising, effective, and potentially useful for in vivo analysis.

Inorganic Nanoparticles Embedded in Polydimethylsiloxane Nanodroplets.

To stabilize and transport them through complex systems, nanoparticles are often encapsulated in polymeric nanocarriers, which are tailored to specific environments. For example, a hydrophilic polymer capsule maintains the circulation and stability of nanoparticles in aqueous environments. A more highly designed nanocarrier might have a hydrophobic core and a hydrophilic shell to allow the transport of hydrophobic nanoparticles and pharmaceuticals through physiological media. Polydimethylsiloxane, PDMS, is a hydrophobic material in a liquid-like state at room temperature. The preparation of stable, aqueous dispersions of PDMS droplets in water is problematic due to the intense mismatch in surface energies between PDMS and water. The present work describes the encapsulation of hydrophobic metal and metal oxide nanoparticles within PDMS nanodroplets using flash nanoprecipitation. The PDMS is terminated by amino groups, and the nanodroplet is capped with a layer of poly(styrenesulfonate), forming a glassy outer shell. The hydrophobic nanoparticles nucleate PDMS droplet formation, decreasing the droplet size. The resulting nanocomposite nanodroplets are stable in aqueous salt solutions without the use of surfactants. The hierarchical structuring, elucidated with small-angle X-ray scattering, offers a new platform for the isolation and transport of hydrophobic molecules and nanoparticles through aqueous systems.

A Multifunctional Contrast Agent for 19F-Based Magnetic Resonance Imaging.

Magnetic resonance imaging, MRI, relying on 19F nuclei has attracted much attention, because the isotopes exhibit a high gyromagnetic ratio (comparable to that of protons) and have 100% natural abundance. Furthermore, due to the very low traces of intrinsic fluorine in biological tissues, fluorine labeling allows easy visualization in vivo using 19F-based MRI. However, one of the drawbacks of the available fluorine tracers is their very limited solubility in water. Here, we detail the design and preparation of a set of water-compatible fluorine-rich polymers as contrast agents that can enhance the effectiveness of 19F-based MRI. The agents are synthesized using the nucleophilic addition reaction between poly(isobutylene-alt-maleic anhydride) copolymer and a mixture of amine-appended fluorine groups and polyethylene glycol (PEG) blocks. This allows control over the polymer architecture and stoichiometry, resulting in good affinity to water solutions. We further investigate the effects of introducing additional segmental mobility to the fluorine moieties in the polymer, by inserting a PEG linker between the moieties and the polymer backbone. We find that controlling the polymer stoichiometry and introducing additional segmental mobility enhance the NMR signals and narrow the peak profile. In particular, we assess the impact of the PEG linker on T2* and T1 relaxation times, using a series of gradient-recalled echo images with varying echo times, TE, or recovery time, TR, respectively. We find that for equivalent concentrations, the PEG linker greatly increases T2*, while maintaining high T1 values, as compared to polymers without this linker. Phantom images collected from these compounds show bright signals over a background with high intensities.

Luminescent Quantum Dots Stabilized by N-Heterocyclic Carbene Polymer Ligands.

We have tested the ability of N-heterocyclic carbene (NHC)-modified ligands to coordinate and stabilize luminescent CdSe-ZnS core-shell quantum dot (QD) dispersions in hydrophilic media. In particular, we probed the effects of ligand structure and coordination number on the coating affinity to the nanocrystals. We find that such NHC-based ligands rapidly coordinate onto the QDs (requiring ∼5-10 min of reaction time), which reflects the soft Lewis base nature of the NHC groups, with its two electrons sharing capacity. Removal of the hydrophobic cap and promotion of carbene-driven coordination on the nanocrystals have been verified by 1H NMR spectroscopy, while 13C NMR was used to identify the formation of carbene-Zn complexes. The newly coated QD dispersions exhibit great long-term colloidal stability over a wide range of conditions. Additionally, we find that coordination onto the QD surfaces affects the optical and spectroscopic properties of the nanocrystals. These include a size-dependent red-shift of the absorption and fluorescence spectra and a pronounced increase in the measured fluorescence intensity when the samples are stored under white light exposure compared to those stored in the dark.

Olfactory bulb-targeted quantum dot (QD) bioconjugate and Kv1.3 blocking peptide improve metabolic health in obese male mice.

The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by the metabolic state of the individual. The excitability of the major output neurons of the olfactory bulb (OB) can be modulated through targeting a voltage-dependent potassium channel, Kv1.3, which responds to changes in metabolic factors such as insulin, glucose, and glucagon-like peptide-1. Because gene-targeted deletion or inhibition of Kv1.3 in the periphery has been found to increase energy metabolism and decrease body weight, we hypothesized that inhibition of Kv1.3 selectively in the OB could enhance excitability of the output neurons to evoke changes in energy homeostasis. We thereby employed metal-histidine coordination to self-assemble the Kv1.3 inhibitor margatoxin (MgTx) to fluorescent quantum dots (QDMgTx) as a means to label cells in vivo and test changes in neuronal excitability and metabolism when delivered to the OB. Using patch-clamp electrophysiology to measure Kv1.3 properties in heterologously expressed cells and native mitral cells in OB slices, we found that QDMgTx had a fast rate of inhibition, but with a reduced IC50, and increased action potential firing frequency. QDMgTx was capable of labeling cloned Kv1.3 channels but was not visible when delivered to native Kv1.3 in the OB. Diet-induced obese mice were observed to reduce body weight and clear glucose more quickly following osmotic mini-pump delivery of QDMgTx/MgTx to the OB, and following MgTx delivery, they increased the use of fats as fuels (reduced respiratory exchange ratio). These results suggest that enhanced excitability of bulbar output neurons can drive metabolic responses.

Engineering the Bio-Nano Interface Using a Multifunctional Coordinating Polymer Coating.

In the past three decades, interest in using nanoparticles as diagnostic tools to interrogate various biosystems has witnessed remarkable growth. For instance, it has been shown that nanoparticle probes enable the study of cellular processes at the single molecule level. These advances provide new opportunities for understanding fundamental problems in biology, innovation in medicine, and the treatment of diseases. A multitude of nanoparticles have been designed to facilitate in vitro or in vivo sensing, imaging, and diagnostics. Some of those nanoparticle platforms are currently in clinical trials or have been approved by the U.S. Food and Drug Administration. Nonetheless, using nanoparticles in biology is still facing several obstacles, such as poor colloidal stability under physiological conditions, nonspecific interactions with serum proteins, and low targeting efficiency in biological fluids, in addition to issues of uncontrolled biodistribution and cytotoxicity. All these problems are primarily controlled by the surface stabilizing coating used.In this Account, we summarize recent progress made in our laboratory focused on the development of multifunctional polymers as coordinating ligands, to tailor the surface properties of nanoparticles and facilitate their application in biology. We first detail the advantageous features of the coating strategy, followed by a discussion of the key parameters in the ligand design. We then describe the synthesis and use of a series of multicoordinating polymers as ligands optimized for coating quantum dots (QDs), gold nanoparticles (AuNPs), and magnetic nanoparticles (MNPs), with a focus on (i) how to improve the colloidal stability and antifouling performance of materials in biological conditions; (ii) how to design highly compact coating, without compromising colloidal stability; and (iii) how to tailor the surface functionalities to achieve conjugation to target biomolecules. We also highlight the ability of a phase transfer strategy, mediated by UV irradiation, to promote rapid ligand exchange while preserving the integrity of key functional groups. We then summarize the bioconjugation approaches applied to polymer-coated nanoparticles, with emphasis on the ability of metal-histidine self-assembly and click chemistry, to control the final nanoparticle bioconjugates. Finally, we demonstrate the use of polymer-coated nanoparticles for sensor design based on redox-active interactions and peptide-mediated intracellular delivery. We anticipate that the coating design presented in this Account would advance the integration of nanoparticles into biology and medicine.

Enhanced Stabilization and Easy Phase Transfer of CsPbBr3 Perovskite Quantum Dots Promoted by High-Affinity Polyzwitterionic Ligands.

The successful growth of colloidal lead halide perovskite quantum dots (PQDs) has generated tremendous interest in the community, due to the unique properties and the promise PQDs offer for use in applications involving light-emitting devices and solar cell technology. However, tangible progress in probing their fundamental properties and/or their integration into optoelectronic devices has been hampered by issues of colloidal and photophysical instability. Here, we introduce a promising surface coating strategy relying on a polyzwitterion polymer, where high-affinity binding onto the QDs is driven by multicoordinating electrostatic interactions with the ion-rich surfaces of CsPbBr3 PQDs. The polymer ligands were synthesized by installing a stoichiometric mixture of amine-modified sulfobetaine anchors and solubilizing motifs on poly(isobutylene-alt-maleic anhydride), PIMA, via nucleophilic addition reaction. We find that this coating approach imparts enhanced colloidal and photophysical stability to the nanocrystals over a broad range of solvent conditions and in powder form. This approach also allows easy phase transfer of the PQDs from nonpolar media to an array of solutions with varying polarities and properties. Additionally, the stabilization strategy preserves the photophysical and structural characteristics of the nanocrystals over a period extending to 1.5 years under certain conditions.

Compact, "Clickable" Quantum Dots Photoligated with Multifunctional Zwitterionic Polymers for Immunofluorescence and In Vivo Imaging.

We detail the preparation of highly fluorescent quantum dots (QDs), surface-engineered with multifunctional polymer ligands that are compact and readily compatible with strain-promoted click conjugation, and the use of these nanocrystals in immunofluorescence and in vivo imaging. The ligand design combines the benefits of mixed coordination (i.e., thiol and imidazole) with zwitterion motifs, yielding sterically-stabilized QDs that present a controllable number of azide groups, for easy conjugation to biomolecules via the selective click chemistry. The polymer coating was characterized using NMR spectroscopy to extract estimates of the diffusion coefficient, hydrodynamic size, and ligand density. The azide-functionalized QDs were conjugated to anti-tropomyosin receptor kinase B antibody (α-TrkB) or to the brain-derived neurotrophic factor (BDNF). These conjugates were highly effective for labeling the tropomyosin receptor kinase B (TrkB) in pyramidal neurons within cortical tissue and for monitoring the BDNF induced activation of TrkB signaling in live neuronal cells. Finally, the polymer-coated QDs were applied for in vivo imaging of Drosophila melanogaster embryos, where the QDs remained highly fluorescent and colloidally stable, with no measurable cytotoxicity. These materials would be of great use in various imaging applications, where a small size, ease of conjugation, and great colloidal stability for in vivo studies are needed.

Delayed Photoluminescence in Metal-Conjugated Fluorophores.

Assemblies of metal nanostructures and fluorescent molecules represent a promising platform for the development of biosensing and near-field imaging applications. Typically, the interaction of molecular fluorophores with surface plasmons in metals results in either quenching or enhancement of the dye excitation energy. Here, we demonstrate that fluorescent molecules can also engage in a reversible energy transfer (ET) with proximal metal surfaces, during which quenching of the dye emission via the energy transfer to localized surface plasmons can trigger delayed ET from metal back to the fluorescent molecule. The resulting two-step process leads to the sustained delayed photoluminescence (PL) in metal-conjugated fluorophores, as was demonstrated here through the observation of increased PL lifetime in assemblies of Au nanoparticles and organic dyes (Alexa 488, Cy3.5, and Cy5). The observed enhancement of the PL lifetime in metal-conjugated fluorophores was corroborated by theoretical calculations based on the reverse ET model, suggesting that these processes could be ubiquitous in many other dye-metal assemblies.

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

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

Modification of Poly(maleic anhydride)-Based Polymers with H2N-R Nucleophiles: Addition or Substitution Reaction?

Reacting poly(maleic anhydride)-based polymers with H2N-R nucleophiles is a flexible and highly effective approach for preparing a variety of multifunctional, multicoordinating, and multireactive polymers. The exact transformation of the anhydride ring during this addition reaction is still an open question. In this report, we characterize the transformation of a representative block copolymer, poly(isobutylene- alt-maleic anhydride), with a few H2N-R nucleophiles. In particular, we test the effects of varying a few reaction parameters/conditions (e.g., temperature, solvent, reaction time, and addition of thionyl chloride) on the nature of the anhydride transformation and bond formed between the polymer and the lateral R groups. The resulting polymers are characterized using a combination of analytical techniques including FT-IR, one- and two-dimensional NMR, and gel electrophoresis. We find that the ring opening transformation occurs under mild conditions. Conversely, cyclic imide transformation can take place for reactions carried out at high temperature (e.g., in DMF under refluxing conditions). We also find that use of a protic solvent, such as methanol, or addition of thionyl chloride (SOCl2) to the reaction mixture under refluxing conditions can promote cyclic imide transformation. The cyclic imide transformation is nonetheless partial, as carboxyl groups could still be accounted for in the resulting compounds. Depending on the type of transformation, the resulting polymer can exhibit a few distinct properties, such as net charge buildup along the chain, or the appearance of weak UV-vis absorption and fluorescence properties. These findings are useful for understanding the properties exhibited by polymer materials prepared via this flexible and highly effective route using anhydride containing polymers and oligomers.

Highly fluorescent hybrid Au/Ag nanoclusters stabilized with poly(ethylene glycol)- and zwitterion-modified thiolate ligands.

We report a simple strategy to grow highly fluorescing, near-infrared-emitting nanoclusters (NCs) made of bimetallic Au/Ag cores, surface capped with a mixture of triphenylphosphine and various monothiol ligands. The ligands include short chain aliphatic monothiols, which yields hydrophobic NCs, and poly(ethylene glycol)- or zwitterion-appended monothiols, which yield NCs that are readily dispersible in buffer media. The reaction uses well-defined triphenylphosphine-protected Au11 clusters (as precursors) that are reacted with Ag(i)-thiolate complexes. The prepared materials are small (diameter <2 nm, as characterized by TEM) with emission peak at 730-760 nm and long lifetime (∼8-12 µs). The quantum yield measured for these materials in both hydrophobic and hydrophilic dispersions is ∼40%. High-magnification dark field STEM and X-ray photoelectron spectroscopy measurements show the presence of both metal atoms in the core, with measured binding energies that agree with reported values for nanocluster materials. The NIR emission combined with high quantum yield, small size, colloidal stability in buffer media and ease of surface functionalization afforded by the coating, make these materials suitable for investigating fundamental questions and potentially useful for biological sensing and imaging applications.

The dual-function of lipoic acid groups as surface anchors and sulfhydryl reactive sites on polymer-stabilized QDs and Au nanocolloids.

Coating inorganic nanocrystals [e.g., quantum dots (QDs) and gold nanoparticles] with polymer ligands presenting multiple lipoic acid anchoring groups provides nanocolloids with remarkable long-term colloidal and photophysical stability. Here, we show that the natural swelling of macromolecules leaves a fraction of the lipoic acid groups in the surface coating free, which are targeted for activation and conjugation to target molecules, using the reliable sulfhydryl-to-maleimide reaction. This implies that simple and efficient functionalization of the nanocrystals can be achieved without introducing additional reactive groups in the coating. We apply a photomediated ligand exchange strategy to luminescent QDs and AuNPs and react the resulting nanocrystals with maleimide Cy3 dye. We then use optical absorption and resonance energy transfer measurements applied to QD-Cy3 and AuNP-Cy3 conjugates to extract estimates for the fraction of accessible lipoic acid groups per QD or AuNP. In addition, we demonstrate the potential utility of this approach by constructing a ratiometric pH sensor made of QD-SNARF conjugates. Our ligand design combined with the photoligation strategy yield colloidally stable dispersions of QDs and AuNPs that present accessible reactive thiols, without introducing new functionalities or requiring disulfide reducing reagents, making them useful for potential use in applications such as biological sensing and imaging.

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.

Gold-doped silver nanoclusters with enhanced photophysical properties.

We detail the characterization of atomically precise, luminescent silver and gold bimetallic nanoclusters (Ag and AgAuNCs) grown in the presence of bidentate lipoic acid (LA, the oxidized form) and dihydrolipoic acid (DHLA, the reduced form) ligands. We found that while doping AuNCs with Ag or Cu precursors using up to a 50% molar fraction (during growth) did not lead to any photoluminescence enhancement, doping of AgNCs with Au resulted in a six-fold enhancement of the PL emission compared to undoped AgNCs. The effect of doping is also reflected in the optical absorption and PL excitation spectra of the gold-doped NCs (AgAuNCs), where a clear blue shift in the absorbance features with respect to the pure AgNCs has been measured. Mass spectrometry measurements using ESI-MS showed that the AgNCs and Au-doped AgNCs had the compositions Ag29(DHLA)12 and Ag28Au(DHLA)12, respectively. The bimetallic nature of the AgAuNC cores was further supported by X-ray Photoelectron Spectroscopy (XPS) measurements. Data showed that the binding energies of the Ag and Au atoms measured from the nanoclusters were shifted with respect to those of the Ag and Au metals. Furthermore, the change in the Ag binding energy was affected by the presence of Au atoms. DOSY-NMR measurements performed on both sets of nanoclusters yielded no change in the hydrodynamic radius measured for either set of NCs when capped with the same ligands.

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.

Margatoxin-bound quantum dots as a novel inhibitor of the voltage-gated ion channel Kv1.3.

Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9 ± 1.1 min, MgTx; vs. 15.3 ± 1.2 min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1 nM concentration, whereas it does not inhibit other screened Shaker channels. We tested the ability of QD-MgTx to inhibit native Kv1.3 expressed in the mouse olfactory bulb (OB). In brain slices of the OB, the conjugate acted similarly to MgTx to inhibit Kv1.3, causing an increased action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues in vivo.

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.

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.