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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... 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.
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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,... 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...
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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.
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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... 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...
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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... 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...
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Research Interests: Engineering, Biochemistry, Biophysics, Materials Science, Materials Chemistry, and 15 moreMolecular Biology, Computational Biology, Biotechnology, Spectroscopy, Plant Biology, Cancer, Space Science, Medicine, Coating, Nmr, CHEMICAL SCIENCES, Quantum Dot, Device Design, Spectroscopic Techniques, and Nuclear Magnetic Resonance Spectroscopy
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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... 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.
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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... 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.
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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... 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 ...
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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... 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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... Mattoussi, H., Mauro, J., Goldman, E., Green, T., Anderson, G., Sundar, V. and Bawendi, M. (2001), Bioconjugation of Highly ... 3 Center for Materials Science and Engineering and Department ofChemistry, Massachusetts Institute of... more
... Mattoussi, H., Mauro, J., Goldman, E., Green, T., Anderson, G., Sundar, V. and Bawendi, M. (2001), Bioconjugation of Highly ... 3 Center for Materials Science and Engineering and Department ofChemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. ...
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We describe the design of high affinity metal-coordinating ligands for the effective coating of inorganic nanocrystals. The ligand design exploits the advantages of the nucleophilic addition reaction to introduce several metal-anchors,... more
We describe the design of high affinity metal-coordinating ligands for the effective coating of inorganic nanocrystals. The ligand design exploits the advantages of the nucleophilic addition reaction to introduce several metal-anchors, hydrophilic moieties and specific reactive groups along a poly(isobutylene-alt-maleic anhydride) short chain. Cap exchange with these ligands applied to luminescent quantum dots (QDs) yields homogeneous nanocrystal dispersions that exhibit long term colloidal stability over several biologically-relevant conditions. The introduction of a controllable number of reactive groups allows conjugation of the QDs to biomolecules via bio-orthogonal coupling chemistries including self-assembly of biotinylated QDs with streptavidin-dye, and coupling of azide-functionalized QDs to cyclooctyne-modified transferrin via copper-free click chemistry.
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We report the design and use of a multi-coordinating polymer ligand that is ideally suited for functionalizing gold nanoparticles and nanorods, and promoting their steric stabilization in buffer media. The ligand is prepared via a... more
We report the design and use of a multi-coordinating polymer ligand that is ideally suited for functionalizing gold nanoparticles and nanorods, and promoting their steric stabilization in buffer media. The ligand is prepared via a one-step nucleophilic addition reaction between poly(isobutylene-alt-maleic anhydride) and amine-modified anchoring groups and hydrophilic moieties. Surface functionalization of gold nanoparticles and nanorods with this polymer yields nanocrystals that exhibit excellent long-term colloidal stability over a broad range of conditions, including pH changes and in growth media, as verified using dynamic light scattering measurements combined with agarose gel electrophoresis. This polymer coating can also prevent the formation of protein corona. These features bode well for use in biological applications where small size, reduced nonspecific interactions and colloidal stability are highly desired. Furthermore, this design can be easily expanded to functionalize...
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Ligands made of lipoic acid (LA) appended with a polyethylene glycol (PEG) chain have been used in the aqueous phase growth of luminescent gold clusters with distinct emission from yellow to near-IR, using two different routes. In the... more
Ligands made of lipoic acid (LA) appended with a polyethylene glycol (PEG) chain have been used in the aqueous phase growth of luminescent gold clusters with distinct emission from yellow to near-IR, using two different routes. In the first route, the gold-ligand complex was chemically reduced using sodium borohydride in alkaline medium, which gave near- IR luminescent gold clusters with maximum emission around 745 nm. In the second method, LA-PEG ligand was photochemically modified to a mixture of thiols, oligomers and oxygenated species under UV-irradiation, which was then used as both reducing agent and stabilizing ligand. By adjusting the pH, temperature, and time of the reaction, we were able to obtain clusters with two distinct emission properties. Refluxing the gold-ligand complex in alkaline medium in the presence of excess ligand gave yellow emission within the first two hours and the emission shifted to red after overnight reaction. Mass spectrometry and chemical assay were used to understand the photo-chemical transformation of Lipoic Acid (LA). Mass spectroscopic studies showed the photo-irradiated product contains thiols, oligomers (dimers, trimers and tetramers) as well as oxygenated species. The amount of thiol formed under different conditions of irradiation was estimated using Ellman’s assay.
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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... more
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 ...
Research Interests: Chemistry, Organic Chemistry, Medicine, In Vitro, Surface modification, and 13 moreGold Nanoparticles, P-glycoprotein, Phase Change Heat Transfer, Proteins, Bioconjugate Chemistry, Macromolecule, Polyethylene Glycol, Sialic Acid, Carbohydrate, Bioconjugation, Biochemistry and cell biology, Driven, and Compact
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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...
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Luminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose... more
Luminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.
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We report on the ability of a chemically synthesized anticancer peptide, SVS-1, to promote the rapid uptake of gold nanorods (AuNRs) and gold nanoparticles (AuNPs) by live HeLa cells. For this, AuNPs and AuNRs, surface ligated with a... more
We report on the ability of a chemically synthesized anticancer peptide, SVS-1, to promote the rapid uptake of gold nanorods (AuNRs) and gold nanoparticles (AuNPs) by live HeLa cells. For this, AuNPs and AuNRs, surface ligated with a multicoordinating polymer that presents several amine groups per ligand, are simultaneously reacted with SVS-1 and Texas-Red dye; the latter allows fluorescence visualization of the nanocrystals. Using epifluorescence microscopy, we find that incubation of the SVS-1-conjugated AuNPs and AuNRs with a model cancer cell line yields extended staining throughout the cell cytoplasm, even at low conjugate concentrations (∼0.1 nM). Furthermore, uptake is specific to the SVS-1-conjugated nanocrystals. Additional endocytosis inhibition experiments, where cells have been incubated with the conjugates at 4 °C or in the presence of endocytic inhibitors, show that significant levels of conjugate uptake persist. These results combined indicate an uptake mechanism that...
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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... more
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...
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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... more
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.
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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... more
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 amo...
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The understanding of nanoparticle (NP) cytotoxicity is challenging because of incomplete information about physicochemical changes particles undergo once they come into contact with biological fluids. It is therefore essential to... more
The understanding of nanoparticle (NP) cytotoxicity is challenging because of incomplete information about physicochemical changes particles undergo once they come into contact with biological fluids. It is therefore essential to characterize changes in NP properties to better understand their biological fate and effects in mammalian cells. In this paper, we present a study on the effect of particle surface oxidation and dissolution rates of Cu NPs. Particle dissolution, cell-associated Cu doses, and oxidative stress responses in A549 luciferase reporter cells were examined for Cu NPs modified with mercaptocarboxylic acids with different carbon chain lengths and a thiotic acid appended-PEG ligand (TA). We found that these Cu NPs released ionic species together with small particles upon oxidation and that surface chemistry influenced the morphology and dissolution rate. The dissolution rate was also shown to impact both the cellular Cu dosimetry and associated oxidative stress respon...
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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... more
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...
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ABSTRACT