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1.
Chem Biomed Imaging ; 1(3): 251-259, 2023 Jun 26.
Article in English | MEDLINE | ID: mdl-37388960

ABSTRACT

Quantum dots (QDs) can be used as fluorescent probes in single molecule localization microscopy to achieve subdiffraction limit resolution (super-resolution fluorescence imaging). However, the toxicity of Cd in the prototypical CdSe-based QDs can limit their use in biological applications. Furthermore, commercial CdSe QDs are usually modified with relatively thick shells of both inorganic and organic materials to render them in the 10-20 nm size range, which is relatively large for biological labels. In this report, we present compact (4-6 nm) CuInS2/ZnS (CIS/ZnS) and compare them to commercially sourced CdSe/ZnS QDs for their blinking behavior, localization precision and super-resolution imaging. Although commercial CdSe/ZnS QDs are brighter than the more compact Cd-free CIS/ZnS QD, both give comparable results of 4.5-5.0-fold improvement in imaging resolution over conventional TIRF imaging of actin filaments. This likely results from the fact that CIS/ZnS QDs show very short on-times and long off times which leads to less overlap in the point spread functions of emitting CIS/ZnS QD labels on the actin filaments at the same labeling density. These results demonstrate that CIS/ZnS QDs are an excellent candidate to complement and perhaps even replace the larger and more toxic CdSe-based QDs for robust single- molecule super-resolution imaging.

2.
Anal Biochem ; 640: 114524, 2022 03 01.
Article in English | MEDLINE | ID: mdl-34933004

ABSTRACT

Human fibroblast growth factor-1 (hFGF1) binding to its receptor and heparin play critical roles in cell proliferation, angiogenesis and wound healing but is also implicated in cancer. Fluorescence imaging is a powerful approach to study such protein interactions, but it is not always obvious if the site chosen will be efficiently labeled, often relying on trial-and-error. To provide a more systematic approach towards an efficient site-specific labeling strategy, we labeled two structurally distinct regions of the protein - the flexible N-terminus and a rigid loop. Several dyes were chosen to cover the visible region and to investigate how the structure of the dye affects the labeling efficiency. Flexibility in either the protein labeling site or the dye structure was found to result in high labeling efficiency, but flexibility in both resulted in a significant decrease in labeling efficiency. Conversely, too much rigidity in both can result in dye-protein interactions that can aggregate the protein. Importantly, site-specifically labeling hFGF1 in these regions maintained biological activity. These results could be applicable to other proteins by considering the flexibility of both the protein labeling site and the dye structure.


Subject(s)
Fibroblast Growth Factor 1
3.
Biophys J ; 120(22): 4992-5004, 2021 11 16.
Article in English | MEDLINE | ID: mdl-34662559

ABSTRACT

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsible for binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single-molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site-directed mutagenesis, trypsin digestion assays, and all-atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3-Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter-residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3-Cterm for the LHC-loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3-Oxa1-YidC family of insertases.


Subject(s)
Intrinsically Disordered Proteins/chemistry , Membrane Proteins/chemistry , Plant Proteins/chemistry , Pisum sativum , Protein Structure, Secondary , Protein Transport , Thylakoids/metabolism
4.
ACS Nano ; 11(3): 2905-2916, 2017 03 28.
Article in English | MEDLINE | ID: mdl-28221750

ABSTRACT

The majority of quantum dot (QD) blinking studies have used a model of switching between two distinct fluorescence intensity levels, "on" and "off". However, a distinct intermediate intensity level has been identified in some recent reports, a so-called "grey" or "dim" state, which has brought this binary model into question. While this grey state has been proposed to result from the formation of a trion, it is still unclear under which conditions it is present in a QD. By performing shell-dependent blinking studies on CdSe QDs, we report that the populations of the grey state and the on state are strongly dependent on both the shell material and its thickness. We found that adding a ZnS shell did not result in a significant population of the grey state. Using ZnSe as the shell material resulted in a slightly higher population of the grey state, although it was still poorly resolved. However, adding a CdS shell resulted in the population of a grey state, which depended strongly on its thickness up to 5 ML. Interestingly, while the frequency of transitions to and from the grey state showed a very strong dependence on CdS shell thickness, the brightness of and the dwell time in the grey state did not. Moreover, we found that the grey state acts as an on-pathway intermediate state between on and off states, with the thickness of the shell determining the transition probability between them. We also identified two types of blinking behavior in QDs, one that showed long-lived but lower intensity on states and another that showed short-lived but brighter on states that also depended on the shell thickness. Intensity-resolved single QD fluorescence lifetime analysis was used to identify the relationship between the various exciton decay pathways and the resulting intensity levels. We used this data to propose a model in which multiple on, grey, and off states exist whose equilibrium populations vary with time that give rise to the various intensity levels of single QDs and which depends on shell composition and thickness.


Subject(s)
Cadmium Compounds/chemistry , Luminescent Agents/chemistry , Luminescent Measurements , Quantum Dots/chemistry , Sulfides/chemistry , Luminescence , Photochemical Processes
5.
Biophys J ; 111(6): 1151-1162, 2016 Sep 20.
Article in English | MEDLINE | ID: mdl-27653474

ABSTRACT

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Förster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post- and cotranslational signaling processes.


Subject(s)
GTP Phosphohydrolases/chemistry , Signal Recognition Particle/chemistry , Animals , Arabidopsis , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chloroplasts/metabolism , Dogs , Escherichia coli , Fluorescence Resonance Energy Transfer , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Methanocaldococcus , Molecular Dynamics Simulation , Mutation , Protein Domains , Scattering, Small Angle , Signal Recognition Particle/genetics , Signal Recognition Particle/metabolism , Structural Homology, Protein , Sulfolobus solfataricus , Thermus , X-Ray Diffraction
6.
ACS Nano ; 10(4): 4072-82, 2016 04 26.
Article in English | MEDLINE | ID: mdl-27058120

ABSTRACT

Currently, the most common way to reduce blinking in quantum dots (QDs) is accomplished by using very thick and/or perfectly crystalline CdS shells on CdSe cores. Ideally, a nontoxic material such as ZnS is preferred to be the outer material in order to reduce environmental and cytotoxic effects. Blinking suppression with multishell configurations of CdS and ZnS has been reported only for "giant" QDs of 15 nm or more. One of the main reasons for the limited progress is that the role that interfacial trap states play in blinking in these systems is not very well understood. Here, we show a "Goldilocks" effect to reduce blinking in small (∼7 nm) QDs by carefully controlling the thicknesses of the shells in multishell QDs. Furthermore, by correlating the fluorescence lifetime components with the fraction of time that a QD spends in the on-state, both with and without applying a threshold, we found evidence for two types of blinking that separately affect the average fluorescence lifetime of a single QD. A thorough characterization of the time-resolved fluorescence at the ensemble and single-particle level allowed us to propose a detailed physical model involving both short-lived interfacial trap states and long-lived surface trap states that are coupled. This model highlights a strategy of reducing QD blinking in small QDs by balancing the magnitude of the induced lattice strain, which results in the formation of interfacial trap states between the inner shell and the outer shell, and the confinement potential that determines how accessible the interfacial trap states are. The combination of reducing blinking while maintaining a small overall QD size and using a Cd-free outer shell of ZnS will be useful in a wide array of applications, particularly for advanced bioimaging.


Subject(s)
Fluorescent Dyes/chemistry , Nanoshells/chemistry , Quantum Dots/chemistry , Cadmium Compounds/chemistry , Fluorescence , Fluorescent Dyes/chemical synthesis , Particle Size , Selenium Compounds/chemistry , Sulfides/chemistry , Surface Properties , Zinc Compounds/chemistry
7.
Small GTPases ; 7(1): 1-11, 2016.
Article in English | MEDLINE | ID: mdl-26828437

ABSTRACT

The Ras-related protein Cell division cycle 42 (Cdc42) is important in cell-signaling processes. Protein interactions involving Cdc42 occur primarily in flexible "Switch" regions that help regulate effector binding. We studied the kinetics of intrinsic GTP hydrolysis reaction in the absence and presence of a biologically active peptide derivative of a p21-activated kinase effector (PBD46) for wt Cdc42 and compared it to the Switch 1 variant Cdc42(T35A). While the binding of PBD46 to wt Cdc42 results in complete inhibition of GTP hydrolysis, this interaction in Cdc42(T35A) does not. Comparison of the crystal structure of wt Cdc42 in the absence of effector (1AN0.pdb), as well as the NMR structure of wt Cdc42 bound to an effector in the Switch 1 region (1CF4.pdb) ( www.rcsb.org ) suggests that the orientation of T(35) with bound Mg(2+) changes in the presence of effector, resulting in movement of GTP away from the catalytic box leading to the inhibition of GTP hydrolysis. For Cdc42(T35A), molecular dynamics simulations and structural analyses suggest that the nucleotide does not undergo the conformational shift observed for the wt Cdc42-effector interaction. Our data suggest that change in dynamics in the Switch 1 region of Cdc42 caused by the T35A mutation (Chandrashekar, et al. 2011, Biochemistry, 50, p. 6196) fosters a conformation for this Cdc42 variant that allows hydrolysis of GTP in the presence of PBD46, and that alteration of the conformational dynamics could potentially modulate Ras-related over-activity.


Subject(s)
Guanosine Triphosphate/chemistry , Peptides/pharmacology , cdc42 GTP-Binding Protein/chemistry , cdc42 GTP-Binding Protein/genetics , p21-Activated Kinases/chemistry , Animals , Binding Sites , Catalytic Domain , Genetic Variation , Humans , Hydrolysis , Mice , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Tertiary
8.
Chemphyschem ; 17(5): 731-40, 2016 Mar 03.
Article in English | MEDLINE | ID: mdl-26693950

ABSTRACT

Choosing the composition of a shell for QDs is not trivial, as both the band-edge energy offset and interfacial lattice mismatch influence the final optical properties. One way to balance these competing effects is by forming multishells and/or gradient-alloy shells. However, this introduces multiple interfaces, and their relative effects on quantum yield and blinking are not yet fully understood. Here, we undertake a systematic, comparative study of the addition of inner shells of a single component versus gradient-alloy shells of cadmium/zinc chalogenides onto CdSe cores, and then capping with a thin ZnS outer shell to form various core/multishell configurations. We show that architecture of the inner shell between the CdSe core and the outer ZnS shell significantly influences both the quantum yield and blinking dynamics, but that these effects are not correlated-a high ensemble quantum yield doesn't necessarily equate to reduced blinking. Two mathematical models have been proposed to describe the blinking dynamics-the more common power-law model and a more recent multiexponential model. By binning the same data with 1 and 20 ms resolution, we show that the on times can be better described by the multiexponential model, whereas the off times can be better described by the power-law model. We discuss physical mechanisms that might explain this behavior and how it can be affected by the inner-shell architecture.


Subject(s)
Quantum Dots , Cadmium Compounds/chemistry , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Oxides/chemistry , Selenium Compounds/chemistry
9.
J Colloid Interface Sci ; 461: 225-231, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26402781

ABSTRACT

Non-covalent incorporation of hydrophobic drugs into polymeric systems is a commonly-used strategy for drug delivery because non-covalent interactions minimize modification of the drug molecules whose efficacy is retained upon release. The behaviors of the drug-polymer delivery system in the biological environments it encounters will affect the efficacy of treatment. In this report, we have investigated the interaction between a hydrophobic drug and its encapsulating polymer in model biological environments using a photosensitizer encapsulated in a polymer-coated nanoparticle system. The photosensitizer, 3-(1'-hexyloxyethyl)-3-devinylpyropheophorbide-a (HPPH), was non-covalently incorporated to the poly(ethylene glycol) (PEG) layer coated on Au nanocages (AuNCs) to yield AuNC-HPPH complexes. The non-covalent binding was characterized by Scatchard analysis, fluorescence lifetime, and Raman experiments. The dissociation constant between PEG and HPPH was found to be ∼35 µM with a maximum loading of ∼2.5×10(5) HPPHs/AuNC. The release was studied in serum-mimetic environment and in vesicles that model human cell membranes. The rate of protein-mediated drug release decreased when using a negatively-charged or cross-linked terminus of the surface-modified PEG. Furthermore, the photothermal effect of AuNCs can initiate burst release, and thus allow control of the release kinetics, demonstrating on-demand drug release. This study provides insights regarding the actions and release kinetics of non-covalent drug delivery systems in biological environments.


Subject(s)
Chlorophyll/analogs & derivatives , Gold/metabolism , Metal Nanoparticles/chemistry , Models, Biological , Photosensitizing Agents/metabolism , Polyethylene Glycols/metabolism , Porphyrins/metabolism , Cell Membrane , Chlorophyll/chemistry , Chlorophyll/metabolism , Drug Delivery Systems , Gold/chemistry , Humans , Kinetics , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Polyethylene Glycols/chemistry , Porphyrins/chemistry , Surface Properties
10.
J Biol Chem ; 290(25): 15462-15474, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-25918165

ABSTRACT

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Intracellular Membranes/metabolism , Signal Recognition Particle/metabolism , Thylakoids/metabolism , Amino Acid Motifs , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Intracellular Membranes/chemistry , Protein Binding/physiology , Protein Transport/physiology , Signal Recognition Particle/chemistry , Signal Recognition Particle/genetics , Thylakoids/chemistry , Thylakoids/genetics
11.
J Phys Chem B ; 118(49): 14103-9, 2014 Dec 11.
Article in English | MEDLINE | ID: mdl-25036275

ABSTRACT

Ligand cross-linking is known to improve the colloidal stability of nanoparticles, particularly in aqueous solutions. However, most cross-linking is performed chemically, in which it is difficult to limit interparticle cross-linking, unless performed at low concentrations. Photochemical cross-linking is a promising approach but usually requires ultraviolet (UV) light to initiate. Using such high-energy photons can be harmful to systems in which the ligand-nanoparticle bond is fairly weak, as is the case for the commonly used semiconductor quantum dots (QDs). Here, we introduce a novel approach to cross-link thiolated ligands on QDs by utilizing the photocatalytic activity of QDs upon absorbing visible light. We show that using visible light leads to better ligand cross-linking by avoiding the problem of ligand dissociation that occurs upon UV light exposure. Once cross-linked, the ligands significantly enhance the colloidal stability of those same QDs that facilitated cross-linking.


Subject(s)
Acetylene/chemistry , Colloids/chemistry , Cross-Linking Reagents/chemistry , Quantum Dots/chemistry , Sulfhydryl Compounds/chemistry , Water/chemistry , Catalysis , Ligands , Light , Luminescence , Semiconductors
12.
Nano Lett ; 13(10): 4746-52, 2013 Oct 09.
Article in English | MEDLINE | ID: mdl-24016073

ABSTRACT

Coordinating ligands are widely used to vary the solubility and reactivity of nanoparticles for subsequent bioconjugation. Although long-term colloidal stability is enhanced by using bidentate coordinating ligands over monodentate ones, other properties such as nonspecific adsorption of target molecules and ligand exchange have not been quantified. In this study, we modified a near-infrared dye to serve as a highly sensitive reporter for nonspecific binding of thiolated target molecules to nanoparticle surfaces that are functionalized with monodentate or bidentate coordinated ligands. Specifically, we analyzed nonspecific binding mechanisms to quantum dots (QDs) by fitting the adsorption profiles to the Hill equation and the parameters are used to provide a microscopic picture of how ligand density and lability control nonspecific adsorption. Surprisingly, bidentate ligands are worse at inhibiting adsorption to QD surfaces at low target/QD ratios, although they become better as the ratio increases, but only if the nanoparticle surface area is large enough to overcome steric effects. This result highlights that a balance between ligand density and lability depends on the dentate nature of the ligands and controls how molecules in solution can coordinate to the nanoparticle surface. These results will have major implications for a range of applications in nanobiomedicine, bioconjugation, single molecule spectroscopy, self-assembly, and nano(photo)catalysis where both nonspecific and specific surface interactions play important roles. As an example, we tested the ability of monodentate and bidentate functionalized nanoparticles to resist nonspecific adsorption of IgG antibodies that contained free thiol groups at a 1:1 QD/IgG ratio and found that QDs with monodentate ligands did indeed result in lower nonspecific adsorption.


Subject(s)
Adsorption , Metal Nanoparticles/chemistry , Nanotechnology , Quantum Dots/chemistry , Antibodies/chemistry , Antibodies/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Ligands , Solubility , Sulfhydryl Compounds/chemistry
13.
J Phys Chem C Nanomater Interfaces ; 117(5): 2317-2327, 2013 Feb 07.
Article in English | MEDLINE | ID: mdl-23543893

ABSTRACT

CdTe quantum dots have unique characteristics that are promising for applications in photoluminescence, photovoltaics or optoelectronics. However, wide variations of the reported quantum yields exist and the influence of ligand-surface interactions that are expected to control the excited state relaxation processes remains unknown. It is important to thoroughly understand the fundamental principles underlying these relaxation processes to tailor the QDs properties to their application. Here, we systematically investigate the roles of the surface atoms, ligand functional groups and solvent on the radiative and non-radiative relaxation rates. Combining a systematic synthetic approach with X-ray photoelectron, quantitative FT-IR and time-resolved visible spectroscopies, we find that CdTe QDs can be engineered with average radiative lifetimes ranging from nanoseconds up to microseconds. The non-radiative lifetimes are anticorrelated to the radiative lifetimes, although they show much less variation. The density, nature and orientation of the ligand functional groups and the dielectric constant of the solvent play major roles in determining charge carrier trapping and excitonic relaxation pathways. These results are used to propose a coupled dependence between hole-trapping on Te atoms and strong ligand coupling, primarily via Cd atoms, that can be used to engineer both the radiative and non-radiative lifetimes.

14.
Anal Chem ; 85(9): 4414-22, 2013 May 07.
Article in English | MEDLINE | ID: mdl-23537496

ABSTRACT

Redox magnetohydrodynamics (MHD) is a promising technique for developing new electrochemical-based microfluidic flow devices with unique capabilities, such as easily switching flow direction and adjusting flow speeds and flow patterns as well as avoiding bubble formation. However, a detailed description of all the forces involved and predicting flow patterns in confined geometries is lacking. In addition to redox-MHD, density gradients caused by the redox reactions also play important roles. Flow in these devices with small fluid volumes has mainly been characterized by following microbead motion by optical microscopy either by particle tracking velocimetry (PTV) or by processing the microbead images by particle image velocimetry (PIV) software. This approach has limitations in spatial resolution and dimensionality. Here we use fluorescence correlation spectroscopy (FCS) to quantitatively and accurately measure flow speeds and patterns in the ~5-50 µm/s range in redox-MHD-based microfluidic devices, from which 3D flow maps are obtained with a spatial resolution down to 2 µm. The 2 µm spatial resolution flow speeds map revealed detailed flow profiles during redox-MHD in which the velocity increases linearly from above the electrode and reaches a plateau across the center of the cell. By combining FCS and video-microscopy (with PTV and PIV processing approaches), we are able to quantify a vertical flow of ~10 µm/s above the electrodes as a result of density gradients caused by the redox reactions and follow convection flow patterns. Overall, combining FCS, PIV, and PTV analysis of redox-MHD is a powerful combination to more thoroughly characterize the underlying forces in these promising microfluidic devices.


Subject(s)
Electrochemical Techniques , Hydrodynamics , Microfluidic Analytical Techniques , Electrochemical Techniques/instrumentation , Electrodes , Microfluidic Analytical Techniques/instrumentation , Oxidation-Reduction
15.
Chem Commun (Camb) ; 49(6): 624-6, 2013 Jan 21.
Article in English | MEDLINE | ID: mdl-23223513

ABSTRACT

We report cadmium-free, biocompatible (Zn)CuInS(2) quantum dots with long fluorescence lifetimes as superior bioimaging probes using time-gated detection to suppress cell autofluorescence and improve the signal : background ratio by an order of magnitude. These results will be important for developing non-toxic fluorescence imaging probes for ultrasensitive biomedical diagnostics.


Subject(s)
Fluorescent Dyes/chemistry , Quantum Dots , Antibodies/immunology , Breast Neoplasms , Cadmium/chemistry , Cell Line, Tumor , Copper/chemistry , Female , Humans , Indium/chemistry , Microscopy, Confocal , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/metabolism , Sulfur/chemistry , Zinc/chemistry
16.
J Neurosci ; 32(37): 12915-20, 2012 Sep 12.
Article in English | MEDLINE | ID: mdl-22973015

ABSTRACT

The subunit stoichiometry of heteromeric glycine-gated channels determines fundamental properties of these key inhibitory neurotransmitter receptors; however, the ratio of α1- to ß-subunits per receptor remains controversial. We used single-molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2ß. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.


Subject(s)
Oocytes/chemistry , Oocytes/metabolism , Protein Subunits/analysis , Receptors, Glycine/chemistry , Receptors, Glycine/metabolism , Animals , Cells, Cultured , Female , Humans , Receptors, Glycine/classification , Xenopus laevis
17.
ACS Nano ; 5(11): 9062-73, 2011 Nov 22.
Article in English | MEDLINE | ID: mdl-22023370

ABSTRACT

The optical properties of core-shell CdSe-ZnS quantum dots (QDs) are characterized by complex photophysics leading to difficulties in interpreting quantitative measurements based on QD emission. By comparing the pH dependence of fluorescence of single QDs to that of an ensemble, we have been able to propose a molecular scale model of how QD surface chemical and physical processes are affected by protons and oxygen. We show that the connection between the ensemble fluorescence intensity and the single QD fluorescence properties such as dark fraction, blinking, particle brightness, and a multiexponential fluorescence lifetime decay is not trivial. The ensemble fluorescence intensity is more weakly dependent on pH than the single particle fluorescence which, together with fluorescence lifetime analysis, provided evidence that the dark fraction of QDs emits photons with low quantum efficiency and long lifetime. We uncovered two surface-dependent mechanisms that affected the fluorescence emission: an immediate physical effect of charges surrounding the QD and an irreversible chemical effect from reaction of the H(+) and O(2) with the QD shell surface. These results will have important implications for those using QD-based fluorescence lifetime imaging as well as for proper implementation of these probes for quantitative cellular imaging applications.


Subject(s)
Quantum Dots , Spectrometry, Fluorescence/methods , Cadmium Compounds/chemistry , Hydrogen-Ion Concentration , Optical Phenomena , Selenium Compounds/chemistry , Sulfides/chemistry , Zinc Compounds/chemistry
18.
ACS Nano ; 3(9): 2573-80, 2009 Sep 22.
Article in English | MEDLINE | ID: mdl-19719085

ABSTRACT

Applications of water-soluble quantum dots (QDs) in the life sciences are limited by their poor colloidal stability in physiological media and nonspecific interaction with biomatter, particularly cell membranes. We have studied colloidal stability and nonspecific interactions with living cells for zwitterionic d-penicillamine-coated QDs (DPA-QDs) and the traditionally used carboxylated 11-mercaptoundecanoic acid-coated QDs (MUA-QDs) and found clear advantages of DPA-QDs. In single molecule fluorescence experiments, DPA-QDs showed no aggregation over the physiologically relevant pH range of 5-9, whereas MUA-QDs showed significant aggregation below pH 9. Upon exposure to living Mono Mac 6 cells, DPA-QDs, which possess overall charge-neutral surfaces, exhibited weak interactions with the cell membrane and were easily removed by flushing with buffer. By contrast, the highly charged MUA-QDs strongly associated with the cells and could not be removed even by extensive rinsing with buffer solution. DPA-QDs exhibit a high chemical stability even in strongly oxidizing conditions, in contrast to cysteine-coated QDs reported earlier. This beneficial property may arise from reduced interactions between DPA ligands due to steric effects of the methyl groups on their beta-carbon atoms.


Subject(s)
Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Monocytes/metabolism , Quantum Dots , Adsorption , Animals , Cell Line, Tumor , Fatty Acids/chemistry , Fatty Acids/metabolism , Humans , Hydrogen-Ion Concentration , Microscopy, Fluorescence , Penicillamine/chemistry , Penicillamine/metabolism , Solubility , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolism , Water/chemistry
19.
ACS Nano ; 3(5): 1167-75, 2009 May 26.
Article in English | MEDLINE | ID: mdl-19385605

ABSTRACT

CdSe quantum dots (QDs) are known to exhibit both power-law blinking dynamics and a dark fraction. A complete description of the mechanistic origins of these properties is still lacking. We show that a change in the pH of the QD environment systematically changes both the dark fraction and the blinking statistics. As pH is lowered, shorter "on" times and longer "off" times, as well as an increase in the permanent dark fraction, are observed. The increase in the dark fraction is preceded by a decrease in the emission intensity of a single QD. Interestingly, the form of the probability distribution function describing blinking changes when the QDs are taken from an air-exposed environment into an aqueous one. These results are used to propose a coupled role for H(+) ions by which they first reduce the intensity of the emitting state as well as affect the probabilities of the QD to switch between "on" and "off" states and eventually trap the QD in a permanent "off" state. We discuss and extend two theoretical blinking models to account for the effect of H(+) ions as well as to highlight their common principle of a diffusion-controlled mechanism governing blinking.


Subject(s)
Lighting/methods , Models, Theoretical , Quantum Dots , Spectrometry, Fluorescence/methods , Computer Simulation , Models, Statistical
20.
Mol Biosyst ; 3(6): 419-30, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17533455

ABSTRACT

Poly(ethylene) glycol (PEG) is an excellent material to modify surfaces to resist non-specific protein adsorption. Linear PEG has been extensively studied both theoretically and experimentally and it has been found that resistance of PEG-coated surfaces to protein adsorption depends mainly on the molecular weight of the polymer and the surface grafting density. End-functionalized star-shaped PEGs allow for interpolymer crosslinking to form a dense layer. An excellent example of such a system consists of a 6-arm PEG/PPG (4 : 1) star polymer functionalized with isocyanate using IPDI. The end functionalization may be further biofunctionalized to recognize specific biomolecules such as streptavidin, His-tagged proteins, amino-terminated oligonucleotides and cell receptors. This functionalization may be patterned into specific geometries using stamping techniques or randomly distributed by statistical reaction of the end group with the biofunctional molecule in solution. The surface preparation uses simple spin-, dip- or spray-coating and produces smooth layers with low background fluorescence. These properties, together with the advantageous chemical properties of PEG, render the surfaces ideal for immobilizing proteins on surfaces with detection limits down to the single molecule level. Proteins immobilized on such surfaces are able to maintain their folded, functional form and are able to completely refold if temporarily exposed to denaturing conditions. Immobilized enzyme molecules were able to perform their function with the same activity as the enzyme in solution. Future directions of using surfaces coated with such crosslinked star polymers in highly sensitive and robust biotechnology applications will be discussed.


Subject(s)
Polyethylene Glycols/chemistry , Polyethylene Glycols/chemical synthesis , Adsorption , Cross-Linking Reagents , Proteins/chemistry , Proteins/metabolism , Surface Properties
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