We are developing new and useful ways to prepare bio-inorganic conjugates of highly luminescent s... more We are developing new and useful ways to prepare bio-inorganic conjugates of highly luminescent semiconductor quantum nanocrystals (Quantum Dots; QDs) and proteins for use in biosensing applications. Conjugate assembly isdriven by electrostatic interaction of negatively charged QD surfaces with positively charged proteins or protein subdomains. Conjugates retain the properties of both starting materials, i.e., biological activity of the proteins and optical characteristics of the QDs. We have used these hybrid bio-inorganic conjugates as tracking reagents in fluoroimmunoassays.
The unique photophysical properties of gold nanomaterials combined with progress in developing ef... more The unique photophysical properties of gold nanomaterials combined with progress in developing effective surfacefunctionalization strategies has motivated researchers to employ them as tools for use in biomedical imaging, biosensing, diagnostics, photothermal therapy, and as drug and gene delivery vehicles. However, a major challenge limiting these advancements has been the unavailability of effective strategies to deliver these and other nanocrystals into the cytoplasm of live cells. In this study, we demonstrate that the use of a chemically-synthesized anti-microbial peptide, SVS-1, can promote non-endocytic uptake of both small size gold nanoparticles (AuNPs) and larger size gold nanorods (AuNRs) into mammalian cells. For this, colloidally stable AuNP and AuNRs, surface ligated with an amine-functionalized polymer, His-PIMA-PEG-OCH3/NH2 were prepared. The amine groups allow dual, covalent attachment of cysteine terminated SVS-1 (via a thioether linkage) and NHS-ester-Texas-Red dy...
The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by th... more 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 voltagedependent 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 IC 50, 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.
Effective and easy to implement ligand exchange strategy is paramount to the design of stable and... more Effective and easy to implement ligand exchange strategy is paramount to the design of stable and multifunctional gold and other inorganic nanocolloids. This is also crucial for their use in biology and medicine. In this contribution, we demonstrate that photo-mediated ligand substitution of gold nanocrystals with a series of lipoic acid-modified ligands yields several advantages, including rapid phase transfer and great long-term colloidal stability. This strategy combines photochemical reduction of the dithiolane group with energetically favorable in situ ligand chemisorption, yielding rapid modification of the surfaces. It requires substantially smaller amounts of excess ligands, compared conventional incubation starting with the oxidized form of the ligands. Complete substitution of the ligands is confirmed using 1 H NMR and FT-IR spectroscopy. The colloidal properties of the resulting materials have been tested using a combination of long-term stability in ion-rich media, sodium cyanide digestion and dithiothreitolcompetition tests. They show that photoligation preserves the structure and photophysical properties of the various colloids. Mechanistic arguments have been discussed to explain the effectiveness of this ligation strategy. These findings prove the practical benefits of this approach for designing biocompatible gold colloids, and bode well for using such materials in a variety of biological assays and photo-thermal therapy.
In this letter, we highlight that a low fraction of functional fusion proteins can significantly ... more In this letter, we highlight that a low fraction of functional fusion proteins can significantly change the interaction of complex nanohybrids with cells and induce cellular uptake.
We describe the growth and characterization of highly fluorescing, near-infrared-emitting nanoclu... more We describe the growth and characterization of highly fluorescing, near-infrared-emitting nanoclusters made of bimetallic Au25-xAgx cores, prepared using various monothiol-appended hydrophobic and hydrophilic ligands. The reaction uses well-defined triphenylphosphine-protected Au11 clusters (as precursors), which are reacted with Ag(I)-thiolate complexes. The prepared nanoclusters are small (diameter < 2nm, as characterized by TEM) with emission peak at 760 nm and long lifetime (~12 µs). The quantum yield measured for these materials was 0.3 - 0.4 depending on the ligand. XPS 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 and ease of surface functionalization afforded by the coating, make these materials suitable to implement investigations that address fundamental questions and potentially useful for biological s...
Forster resonance energy transfer (FRET) using colloidal semiconductor quantum dots (QDs) and dye... more Forster resonance energy transfer (FRET) using colloidal semiconductor quantum dots (QDs) and dyes is of importance in a wide range of biological and biophysical studies. Here, we report a study on FRET between CuInS 2 /ZnS QDs and dark quencher dye molecules (IRDye QC-1). Oleate-capped QDs with photoluminescence quantum yields (PLQYs) of 55 ± 4% are transferred into water by using two types of multifunctional polymer ligands combining imidazole groups and specific moieties with amine or methoxy groups as the terminal sites. The resulting water-dispersible QDs show PLQYs as high as 44 ± 4% and exhibit long-term colloidal stability (at least 10 months at 4°C in the dark) with a hydrodynamic diameter of less than 20 nm. A side-by-side comparison experiment was performed using the amine or methoxy-functionalized QDs for coupling to dark quencher dye molecules. The aminefunctionalized QDs bind to the dye molecules via covalent bonds, while methoxy-functionalized ones bind only weakly and nonspecifically. The progressive quenching of the QD emission and shortening of its photoluminescence decay time upon increasing the number of conjugated dye molecules demonstrate that the QD acts as the energy donor and the dark quencher dye as the energy acceptor in a donor−acceptor FRET pair. The FRET dynamics of the QD−dye conjugates are simulated using two different models based on the possible origin of the multiexponential PL decay of the QDs (i.e., variations in nonradiative or radiative decay rates). The model based on the radiative decay rates provides a better fit of our experimental data and estimates a donor−acceptor distance (6.5 nm) that matches well the hydrodynamic radius of the amine-functionalized QDs.
An experimental approach is presented for identifying the scaling laws for polymer chains grafted... more An experimental approach is presented for identifying the scaling laws for polymer chains grafted onto gold nanoparticles. Poly(ethylene oxide) of various molecular weights are grafted onto gold nanoparticles via thiol end‐functional groups. The polymer‐grafted nanoparticles are self‐assembled into monolayers from solvents of different quality. Over a significant range of graft densities, nanoparticle monolayers deposited from good (athermal) solvent exhibit particle spacing that scales according to theoretical predictions for chains in dilute solution. This unexpected result for ordered nanoparticle monolayers is discussed in the context of the deposition process. In monolayers deposited from theta solvent, molecular weight scaling of particle spacing breaks down, possibly due to chain length dependence of solvent quality. In poor solvent, the structure of nanoparticle assemblies is not sufficiently ordered to obtain reliable measurements, possibly due to loss of nanoparticle dispe...
Colloidal quantum dots, made of semiconductor cores and surface coated with an organic shell, hav... more Colloidal quantum dots, made of semiconductor cores and surface coated with an organic shell, have generated much interest in areas ranging from spectroscopy, charge and energy transfer interactions, device design and as probes in biology. Despite the remarkable progress in the growth of these materials, rather limited information about the molecular arrangements of the organic coating is available. Here, several nuclear magnetic resonance (NMR) spectroscopic techniques have been combined to characterize the surface ligand structure(s) on biocompatible CdSe-ZnS quantum dots (QDs). These materials have been prepared via a photo-induced ligand exchange where the native hydrophobic coating is substituted, in-situ, with a series of polyethylene glycolmodified lipoic acid-based (LA-PEG) ligands. We first combined diffusion ordered spectroscopy (DOSY) with heteronuclear single quantum coherence (HSQC) measurements to outline the conditions where the detected proton signals emanate only from surface-bound ligands and identify changes in the proton shifts between free and QD-bound ligands in the sample. Quantification of the ligand density on different size QDs was implemented by comparing the sharp 1 H-signature(s) of lateral groups in the ligands (e.g., the OCH3 group) to an external standard. We found that both molecular architecture of the ligand and surface curvature of the QDs combined play important roles in the surface coverage. Given the non-invasive nature of NMR as an analytical technique, the extracted information on the ligand arrangements on the QD surfaces in hydrophilic media will be highly valuable; it has great implications for the use of QDs in targeting and bioconjugation, cellular imaging as well as energy and charge transfer interactions.
We have designed a new set of coordinating ligands made of a lipoic acid (LA) anchor and poly(eth... more We have designed a new set of coordinating ligands made of a lipoic acid (LA) anchor and poly(ethylene glycol) (PEG) hydrophilic moiety appended with a terminal aldehyde for the surface functionalization of QDs. This ligand design was combined with a recently developed photoligation strategy to prepare hydrophilic CdSe-ZnS QDs with good control over the fraction of intact aldehyde (-CHO) groups per nanocrystal. We further applied the efficient hydrazone ligation to react aldehyde-QDs with 2-hydrazinopyridine (2-HP). This covalent modification produces QD-conjugates with a well-defined absorption feature at 350 nm ascribed to the hydrazone chromophore. We exploited this unique optical signature to accurately measure the number of aldehyde groups per QD when the fraction of LA-PEG-CHO per nanocrystal was varied. This allowed us to extract an estimate for the number of LA-PEG ligands per QD. These results suggest that hydrazone ligation has the potential to provide a simple and general analytical method to estimate the number of surface ligands for a variety of nanocrystals such as metal, metal oxide and semiconductor nanocrystals.
Publisher Summary This chapter reviews the progress made in bio-related applications of luminesce... more Publisher Summary This chapter reviews the progress made in bio-related applications of luminescent colloidal quantum dots (QDs). The material reviewed represents only the prologue of an unfolding story, as quantum dots are a relatively recent discovery and their biological applications are newer still. Nonetheless, a significant body of research literature exists pointing the way toward future advances. We begin with a basic introduction to quantum dots, including their synthesis and some characteristic physical properties, followed by a review of bio-related work involving semiconductor nanocrystals published to date. Work involving the preparation and use of QD-protein conjugates in cellular imaging, quantitative immunoassays, and in early-stage energy transfer applications is reviewed, in addition to the uses of QD-DNA conjugates as nanoscale building blocks. A listing of early patents in this area is also included for those who contemplate utilizing these materials in the commercial arena. Advantages and limitations in bio-related applications are discussed based on the current state-of-the-art in QD technology.
Abstract A method for the preparation and characterization of bioinorganic conjugates made with h... more Abstract A method for the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe–ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays is presented. The conjugation strategy employs ...
We describe a new quantum dot (QD)-conjugate prepared with a lytic peptide, derived from a nonenv... more 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 perf...
We are developing new and useful ways to prepare bio-inorganic conjugates of highly luminescent s... more We are developing new and useful ways to prepare bio-inorganic conjugates of highly luminescent semiconductor quantum nanocrystals (Quantum Dots; QDs) and proteins for use in biosensing applications. Conjugate assembly isdriven by electrostatic interaction of negatively charged QD surfaces with positively charged proteins or protein subdomains. Conjugates retain the properties of both starting materials, i.e., biological activity of the proteins and optical characteristics of the QDs. We have used these hybrid bio-inorganic conjugates as tracking reagents in fluoroimmunoassays.
The unique photophysical properties of gold nanomaterials combined with progress in developing ef... more The unique photophysical properties of gold nanomaterials combined with progress in developing effective surfacefunctionalization strategies has motivated researchers to employ them as tools for use in biomedical imaging, biosensing, diagnostics, photothermal therapy, and as drug and gene delivery vehicles. However, a major challenge limiting these advancements has been the unavailability of effective strategies to deliver these and other nanocrystals into the cytoplasm of live cells. In this study, we demonstrate that the use of a chemically-synthesized anti-microbial peptide, SVS-1, can promote non-endocytic uptake of both small size gold nanoparticles (AuNPs) and larger size gold nanorods (AuNRs) into mammalian cells. For this, colloidally stable AuNP and AuNRs, surface ligated with an amine-functionalized polymer, His-PIMA-PEG-OCH3/NH2 were prepared. The amine groups allow dual, covalent attachment of cysteine terminated SVS-1 (via a thioether linkage) and NHS-ester-Texas-Red dy...
The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by th... more 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 voltagedependent 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 IC 50, 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.
Effective and easy to implement ligand exchange strategy is paramount to the design of stable and... more Effective and easy to implement ligand exchange strategy is paramount to the design of stable and multifunctional gold and other inorganic nanocolloids. This is also crucial for their use in biology and medicine. In this contribution, we demonstrate that photo-mediated ligand substitution of gold nanocrystals with a series of lipoic acid-modified ligands yields several advantages, including rapid phase transfer and great long-term colloidal stability. This strategy combines photochemical reduction of the dithiolane group with energetically favorable in situ ligand chemisorption, yielding rapid modification of the surfaces. It requires substantially smaller amounts of excess ligands, compared conventional incubation starting with the oxidized form of the ligands. Complete substitution of the ligands is confirmed using 1 H NMR and FT-IR spectroscopy. The colloidal properties of the resulting materials have been tested using a combination of long-term stability in ion-rich media, sodium cyanide digestion and dithiothreitolcompetition tests. They show that photoligation preserves the structure and photophysical properties of the various colloids. Mechanistic arguments have been discussed to explain the effectiveness of this ligation strategy. These findings prove the practical benefits of this approach for designing biocompatible gold colloids, and bode well for using such materials in a variety of biological assays and photo-thermal therapy.
In this letter, we highlight that a low fraction of functional fusion proteins can significantly ... more In this letter, we highlight that a low fraction of functional fusion proteins can significantly change the interaction of complex nanohybrids with cells and induce cellular uptake.
We describe the growth and characterization of highly fluorescing, near-infrared-emitting nanoclu... more We describe the growth and characterization of highly fluorescing, near-infrared-emitting nanoclusters made of bimetallic Au25-xAgx cores, prepared using various monothiol-appended hydrophobic and hydrophilic ligands. The reaction uses well-defined triphenylphosphine-protected Au11 clusters (as precursors), which are reacted with Ag(I)-thiolate complexes. The prepared nanoclusters are small (diameter < 2nm, as characterized by TEM) with emission peak at 760 nm and long lifetime (~12 µs). The quantum yield measured for these materials was 0.3 - 0.4 depending on the ligand. XPS 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 and ease of surface functionalization afforded by the coating, make these materials suitable to implement investigations that address fundamental questions and potentially useful for biological s...
Forster resonance energy transfer (FRET) using colloidal semiconductor quantum dots (QDs) and dye... more Forster resonance energy transfer (FRET) using colloidal semiconductor quantum dots (QDs) and dyes is of importance in a wide range of biological and biophysical studies. Here, we report a study on FRET between CuInS 2 /ZnS QDs and dark quencher dye molecules (IRDye QC-1). Oleate-capped QDs with photoluminescence quantum yields (PLQYs) of 55 ± 4% are transferred into water by using two types of multifunctional polymer ligands combining imidazole groups and specific moieties with amine or methoxy groups as the terminal sites. The resulting water-dispersible QDs show PLQYs as high as 44 ± 4% and exhibit long-term colloidal stability (at least 10 months at 4°C in the dark) with a hydrodynamic diameter of less than 20 nm. A side-by-side comparison experiment was performed using the amine or methoxy-functionalized QDs for coupling to dark quencher dye molecules. The aminefunctionalized QDs bind to the dye molecules via covalent bonds, while methoxy-functionalized ones bind only weakly and nonspecifically. The progressive quenching of the QD emission and shortening of its photoluminescence decay time upon increasing the number of conjugated dye molecules demonstrate that the QD acts as the energy donor and the dark quencher dye as the energy acceptor in a donor−acceptor FRET pair. The FRET dynamics of the QD−dye conjugates are simulated using two different models based on the possible origin of the multiexponential PL decay of the QDs (i.e., variations in nonradiative or radiative decay rates). The model based on the radiative decay rates provides a better fit of our experimental data and estimates a donor−acceptor distance (6.5 nm) that matches well the hydrodynamic radius of the amine-functionalized QDs.
An experimental approach is presented for identifying the scaling laws for polymer chains grafted... more An experimental approach is presented for identifying the scaling laws for polymer chains grafted onto gold nanoparticles. Poly(ethylene oxide) of various molecular weights are grafted onto gold nanoparticles via thiol end‐functional groups. The polymer‐grafted nanoparticles are self‐assembled into monolayers from solvents of different quality. Over a significant range of graft densities, nanoparticle monolayers deposited from good (athermal) solvent exhibit particle spacing that scales according to theoretical predictions for chains in dilute solution. This unexpected result for ordered nanoparticle monolayers is discussed in the context of the deposition process. In monolayers deposited from theta solvent, molecular weight scaling of particle spacing breaks down, possibly due to chain length dependence of solvent quality. In poor solvent, the structure of nanoparticle assemblies is not sufficiently ordered to obtain reliable measurements, possibly due to loss of nanoparticle dispe...
Colloidal quantum dots, made of semiconductor cores and surface coated with an organic shell, hav... more Colloidal quantum dots, made of semiconductor cores and surface coated with an organic shell, have generated much interest in areas ranging from spectroscopy, charge and energy transfer interactions, device design and as probes in biology. Despite the remarkable progress in the growth of these materials, rather limited information about the molecular arrangements of the organic coating is available. Here, several nuclear magnetic resonance (NMR) spectroscopic techniques have been combined to characterize the surface ligand structure(s) on biocompatible CdSe-ZnS quantum dots (QDs). These materials have been prepared via a photo-induced ligand exchange where the native hydrophobic coating is substituted, in-situ, with a series of polyethylene glycolmodified lipoic acid-based (LA-PEG) ligands. We first combined diffusion ordered spectroscopy (DOSY) with heteronuclear single quantum coherence (HSQC) measurements to outline the conditions where the detected proton signals emanate only from surface-bound ligands and identify changes in the proton shifts between free and QD-bound ligands in the sample. Quantification of the ligand density on different size QDs was implemented by comparing the sharp 1 H-signature(s) of lateral groups in the ligands (e.g., the OCH3 group) to an external standard. We found that both molecular architecture of the ligand and surface curvature of the QDs combined play important roles in the surface coverage. Given the non-invasive nature of NMR as an analytical technique, the extracted information on the ligand arrangements on the QD surfaces in hydrophilic media will be highly valuable; it has great implications for the use of QDs in targeting and bioconjugation, cellular imaging as well as energy and charge transfer interactions.
We have designed a new set of coordinating ligands made of a lipoic acid (LA) anchor and poly(eth... more We have designed a new set of coordinating ligands made of a lipoic acid (LA) anchor and poly(ethylene glycol) (PEG) hydrophilic moiety appended with a terminal aldehyde for the surface functionalization of QDs. This ligand design was combined with a recently developed photoligation strategy to prepare hydrophilic CdSe-ZnS QDs with good control over the fraction of intact aldehyde (-CHO) groups per nanocrystal. We further applied the efficient hydrazone ligation to react aldehyde-QDs with 2-hydrazinopyridine (2-HP). This covalent modification produces QD-conjugates with a well-defined absorption feature at 350 nm ascribed to the hydrazone chromophore. We exploited this unique optical signature to accurately measure the number of aldehyde groups per QD when the fraction of LA-PEG-CHO per nanocrystal was varied. This allowed us to extract an estimate for the number of LA-PEG ligands per QD. These results suggest that hydrazone ligation has the potential to provide a simple and general analytical method to estimate the number of surface ligands for a variety of nanocrystals such as metal, metal oxide and semiconductor nanocrystals.
Publisher Summary This chapter reviews the progress made in bio-related applications of luminesce... more Publisher Summary This chapter reviews the progress made in bio-related applications of luminescent colloidal quantum dots (QDs). The material reviewed represents only the prologue of an unfolding story, as quantum dots are a relatively recent discovery and their biological applications are newer still. Nonetheless, a significant body of research literature exists pointing the way toward future advances. We begin with a basic introduction to quantum dots, including their synthesis and some characteristic physical properties, followed by a review of bio-related work involving semiconductor nanocrystals published to date. Work involving the preparation and use of QD-protein conjugates in cellular imaging, quantitative immunoassays, and in early-stage energy transfer applications is reviewed, in addition to the uses of QD-DNA conjugates as nanoscale building blocks. A listing of early patents in this area is also included for those who contemplate utilizing these materials in the commercial arena. Advantages and limitations in bio-related applications are discussed based on the current state-of-the-art in QD technology.
Abstract A method for the preparation and characterization of bioinorganic conjugates made with h... more Abstract A method for the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe–ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays is presented. The conjugation strategy employs ...
We describe a new quantum dot (QD)-conjugate prepared with a lytic peptide, derived from a nonenv... more 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 perf...
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