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1.
Langmuir ; 38(6): 2038-2045, 2022 02 15.
Article in English | MEDLINE | ID: mdl-35119286

ABSTRACT

Amphiphilic block copolymers with weak polyelectrolyte blocks can assemble stimulus-responsive nanostructures and interfaces. Applications of these materials in drug delivery, biomimetics, and sensing largely rely on the well-understood swelling of polyelectrolyte chains upon deprotonation, often induced by changes in pH or ionic strength. This deprotonation can also tune interfacial interactions between the polyelectrolyte blocks and surrounding solution, an effect which is less studied than morphological swelling of polyelectrolytes but can be just as critical for intended function. Here, we investigate whether the pH-driven morphological response of polyelectrolyte-bearing nanostructures also affects the interactions of these nanostructures with molecules in solution, using micelles of a short-chain polybutadiene-block-poly(acrylic acid) (pBd-pAA) as a model system. We introduce a Förster resonance energy transfer (FRET) approach to probe interactions between micelles and fluorescent molecular solutes as a function of solution pH. As expected, the pAA corona of these pBd-pAA micelles increases in thickness monotonically as a function of pH. However, FRET efficiency, which provides a metric of the spatial proximity of fluorescently labeled micelles and freely diffusing fluorophores, exhibits complex nonmonotonic behavior as a function of pH, indicating that the average separation of micelles and acceptor fluorophores is not strictly correlated with micelle swelling. Dialysis experiments quantify the affinity of fluorophores for micelles as a function of pH, confirming that changes in FRET are driven almost entirely by the pH-dependent affinity of the pAA block for the investigated molecular fluorophores, not simply by a shape change of the pAA corona. This study provides key insights into the interfacial interactions between weak-polyelectrolyte-bearing nanostructures and molecular solutes, of importance for the development of their stimulus-responsive applications.


Subject(s)
Micelles , Polymers , Drug Delivery Systems , Hydrogen-Ion Concentration , Polyelectrolytes , Polymers/chemistry
3.
Materials (Basel) ; 12(4)2019 Feb 17.
Article in English | MEDLINE | ID: mdl-30781571

ABSTRACT

Polystyrene-b-polyethylene glycol (PS-b-PEG) amphiphilic block copolymers featuring a terminal tridentate N,N,N-ligand (terpyridine) were synthesized for the first time through an efficient route. In this approach, telechelic chain-end modified polystyrenes were produced via reversible addition-fragmentation chain-transfer (RAFT) polymerization by using terpyridine trithiocarbonate as the chain-transfer agent, after which the hydrophilic polyethylene glycol (PEG) block was incorporated into the hydrophobic polystyrene (PS) block in high yields via a thiol-ene process. Following metal-coordination with Mn2+, Fe2+, Ni2+, and Zn2+, the resulting metallo-polymers were self-assembled into spherical, vesicular nanostructures, as characterized by dynamic light scattering and transmission electron microscopy (TEM) imaging.

4.
J Drug Deliv ; 2018: 2851579, 2018.
Article in English | MEDLINE | ID: mdl-30534433

ABSTRACT

Cationic liposomes with DNA-transportation properties have attracted considerable attention for their ability to deliver medicinal oligonucleotides to mammalian cells. Amongst these are metalloliposomes that use transition metal ions to confer the lipid molecules cationic charge and unique advantages such as redox- and ligand-exchange triggered DNA-release properties. In this study, lipophilic copper (II) and zinc (II) complexes of 1-alkyl-1,4,7-triazacyclononane were prepared to investigate their ability to bind and transfect double stranded DNA with mammalian cells in vitro and in vivo. The copper(II)-surfactant complexes Cu(TACN-C8)2 (1), Cu(TACN-C10)2 (2), Cu(TACN-C12)2 (3), Cu(TACN-C14)2 (4), Cu(TACN-C16)2 (5), and Cu(TACN-C18)2 (6) that comprise ligands that vary in the length of the alkyl group and the zinc (II)-surfactant complex of Zn(TACN-C12)2 (7) were synthesized. The critical micelle concentration (CMC) for 1-7 was measured using fluorescence spectroscopy and an evaluation of the transfection efficiency of the complexes was assessed using the pEGFP-N1 plasmid and HEK 293-T cells. An inverse relationship between DNA transfection efficiency and CMC of the Cu(II) metallosurfactants was observed. The highest transfection efficiency of 38% was observed for Cu(TACN-C12)2 corresponding to the surfactant with dodecyl alkyl chain having a CMC of 50 µM. Further, an in vivo experiment using mice models was conducted to test the Cu(TACN-C12)2 (3) and Zn(TACN-C12)2 (7) metallosurfactants delivering a DNA vaccine designed for protection against leishmaniasis disease and the study revealed that the Cu-lipoplex elicited the production of significantly more T cells than the Zn-lipoplex and the control group in vivo.

5.
ACS Omega ; 3(1): 503-508, 2018 Jan 31.
Article in English | MEDLINE | ID: mdl-31457908

ABSTRACT

A magnetically active Fe3O4/poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD) nanocomposite is formed by the encapsulation of magnetite nanoparticles with a short-chain amphiphilic block copolymer. This material is then incorporated into the self-assembly of higher order polymer architectures, along with an organic pigment, to yield biosynthetic, bifunctional optical and magnetically active Fe3O4/bacteriochlorophyll c/PEO-b-PBD polymeric chlorosomes.

6.
Autism ; 22(1): 6-19, 2018 01.
Article in English | MEDLINE | ID: mdl-29034696

ABSTRACT

The population of adults on the autism spectrum continues to increase, and vocational outcomes are particularly poor. Longitudinal studies of adults with autism spectrum and without intellectual disability have shown consistent and persistent deficits across cognitive, social, and vocational domains, indicating a need for effective treatments of functional disabilities as each impact employment. This initial pilot study is an open trial investigation of the feasibility, acceptability, and initial estimates of outcomes for the newly developed Supported Employment, Comprehensive Cognitive Enhancement, and Social Skills intervention, a manualized "soft skills" curriculum, to enhance both cognitive and social development in adults with autism spectrum. A total of eight adults with autism spectrum, without intellectual disability (78% males), participated in the study. Results support the original hypothesis that adults with autism spectrum can improve both cognitive (i.e. executive functioning) and social cognitive (i.e. social thinking and social communication) abilities. Further Supported Employment, Comprehensive Cognitive Enhancement, and Social Skills was found to be feasible, acceptable, and highly satisfactory for participants and parents. Employment rates more than doubled post-intervention, with an increase from 22% to 56% of participants employed. Conclusion is that Supported Employment, Comprehensive Cognitive Enhancement, and Social Skills has promise as an intervention that can be easily embedded into exiting supported employment vocational training programs to improve cognitive, social, and vocational outcomes.


Subject(s)
Autism Spectrum Disorder/therapy , Cognition , Social Skills , Vocational Education , Adolescent , Adult , Autism Spectrum Disorder/psychology , Curriculum , Employment , Executive Function , Female , Humans , Male , Pilot Projects , Young Adult
7.
ACS Appl Mater Interfaces ; 9(35): 30185-30195, 2017 Sep 06.
Article in English | MEDLINE | ID: mdl-28809101

ABSTRACT

We report a versatile microsphere-supported lipid bilayer system that can serve as a general-purpose platform for implementing DNA nanotechnologies on a fluid surface. To demonstrate our platform, we implemented both toehold-mediated strand displacement (TMSD) and DNAzyme reactions, which are typically performed in solution and which are the cornerstone of DNA-based molecular logic and dynamic DNA nanotechnology, on the surface. We functionalized microspheres bearing supported lipid bilayers (µSLBs) with membrane-bound nucleic acid components. Using functionalized µSLBs, we developed TMSD and DNAzyme reactions by optimizing reaction conditions to reduce nonspecific interactions between DNA and phospholipids and to enhance bilayer stability. Additionally, the physical and optical properties of the bilayer were tuned via lipid composition and addition of fluorescently tagged lipids to create stable and multiplexable µSLBs that are easily read out by flow cytometry. Multiplexed TMSD reactions on µSLBs enabled the successful operation of a Dengue serotyping assay that correctly identified all 16 patterns of target sequences to demonstrate detection of DNA strands derived from the sequences of all four Dengue serotypes. The limit of detection for this assay was 3 nM. Furthermore, we demonstrated DNAzyme reactions on a fluid lipid surface, which benefit from free diffusion on the surface. This work provides the basis for expansion of both TMSD and DNAzyme based molecular reactions on supported lipid bilayers for use in molecular logic and DNA nanotechnology. As our system is multiplexable and results in fluid surfaces, it may be of use in compartmentalization and improved kinetics of molecular logic reactions and as a useful building block in a variety of DNA nanotechnology systems.


Subject(s)
Lipid Bilayers/chemistry , DNA , Microspheres , Nanotechnology
8.
Langmuir ; 33(25): 6427-6438, 2017 06 27.
Article in English | MEDLINE | ID: mdl-28585832

ABSTRACT

Chlorosomes are one of the characteristic light-harvesting antennas from green sulfur bacteria. These complexes represent a unique paradigm: self-assembly of bacteriochlorophyll pigments within a lipid monolayer without the influence of protein. Because of their large size and reduced complexity, they have been targeted as models for the development of bioinspired light-harvesting arrays. We report the production of biohybrid light-harvesting nanocomposites mimicking chlorosomes, composed of amphiphilic diblock copolymer membrane bodies that incorporate thousands of natural self-assembling bacteriochlorophyll molecules derived from green sulfur bacteria. The driving force behind the assembly of these polymer-chlorosome nanocomposites is the transfer of the mixed raw materials from the organic to the aqueous phase. We incorporated up to five different self-assembling pigment types into single nanocomposites that mimic chlorosome morphology. We establish that the copolymer-BChl self-assembly process works smoothly even when non-native combinations of BChl homologues are included. Spectroscopic characterization revealed that the different types of self-assembling pigments participate in ultrafast energy transfer, expanding beyond single chromophore constraints of the natural chlorosome system. This study further demonstrates the utility of flexible short-chain, diblock copolymers for building scalable, tunable light-harvesting arrays for technological use and allows for an in vitro analysis of the flexibility of natural self-assembling chromophores in unique and controlled combinations.


Subject(s)
Nanocomposites , Bacterial Proteins , Bacteriochlorophylls , Energy Transfer , Organelles , Polymers
9.
PLoS One ; 11(5): e0156295, 2016.
Article in English | MEDLINE | ID: mdl-27227979

ABSTRACT

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. We also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.


Subject(s)
Cell Membrane/metabolism , Lipid Bilayers/metabolism , Lipopolysaccharides/metabolism , O Antigens/metabolism , Shiga-Toxigenic Escherichia coli/metabolism , Animals , Cattle , Cell Membrane/chemistry , Escherichia coli Proteins/metabolism , Food Microbiology , Lipid Bilayers/chemistry , Lipopolysaccharides/chemistry , Serogroup , Shiga-Toxigenic Escherichia coli/chemistry
10.
Sci Rep ; 5: 10331, 2015 May 27.
Article in English | MEDLINE | ID: mdl-26015293

ABSTRACT

Supported lipid bilayers have proven effective as model membranes for investigating biophysical processes and in development of sensor and array technologies. The ability to modify lipid bilayers after their formation and in situ could greatly advance membrane technologies, but is difficult via current state-of-the-art technologies. Here we demonstrate a novel method that allows the controlled post-formation processing and modification of complex supported lipid bilayer arrangements, under aqueous conditions. We exploit the destabilization effect of lipopolysaccharide, an amphiphilic biomolecule, interacting with lipid bilayers to generate voids that can be backfilled to introduce desired membrane components. We further demonstrate that when used in combination with a single, traditional soft lithography process, it is possible to generate hierarchically-organized membrane domains and microscale 2-D array patterns of domains. Significantly, this technique can be used to repeatedly modify membranes allowing iterative control over membrane composition. This approach expands our toolkit for functional membrane design, with potential applications for enhanced materials templating, biosensing and investigating lipid-membrane processes.


Subject(s)
Lipid Bilayers/chemistry , Lipopolysaccharides/chemistry , Lipid Bilayers/metabolism , Lipopolysaccharides/metabolism , Liposomes/chemistry , Liposomes/metabolism , Microarray Analysis , Microscopy, Atomic Force , Microscopy, Confocal , Phosphatidylcholines/chemistry
11.
Nano Lett ; 15(4): 2422-8, 2015 Apr 08.
Article in English | MEDLINE | ID: mdl-25719733

ABSTRACT

We report generation of modular, artificial light-harvesting assemblies where an amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(butadiene), serves as the framework for noncovalent organization of BODIPY-based energy donor and bacteriochlorin-based energy acceptor chromophores. The assemblies are adaptive and form well-defined micelles in aqueous solution and high-quality monolayer and bilayer films on solid supports, with the latter showing greater than 90% energy transfer efficiency. This study lays the groundwork for further development of modular, polymer-based materials for light harvesting and other photonic applications.

12.
Langmuir ; 30(28): 8481-90, 2014 Jul 22.
Article in English | MEDLINE | ID: mdl-24988144

ABSTRACT

A simple and robust nanolithographic method that allows sub-100 nm chemical patterning on a range of oxide surfaces was developed in order to fabricate nanoarrays of plant light-harvesting LHCII complexes. The site-specific immobilization and the preserved functionality of the LHCII complexes were confirmed by fluorescence emission spectroscopy. Nanopatterned LHCII trimers could be reversibly switched between fluorescent and quenched states by controlling the detergent concentration in the imaging buffer. A 3-fold quenching of the average fluorescence intensity was accompanied by a decrease in the average (amplitude-weighted) fluorescence lifetime from approximately 2.24 ns to approximately 0.4 ns, attributed to the intrinsic ability of LHCII to switch between fluorescent and quenched states upon changes in its conformational state. The nanopatterning methodology was extended by immobilizing a second protein, the enhanced green fluorescent protein (EGFP), onto LHCII-free areas of the chemically patterned surfaces. This very simple surface chemistry, which allows simultaneous selective immobilization and therefore sorting of the two types of protein molecules on the surface, is a key underpinning step toward the integration of LHCII into switchable biohybrid antenna constructs.


Subject(s)
Light-Harvesting Protein Complexes/chemistry , Chlorophyll/chemistry , Photosystem II Protein Complex/chemistry , Spectrometry, Fluorescence , Spinacia oleracea/metabolism
13.
Biophys J ; 106(11): 2395-407, 2014 Jun 03.
Article in English | MEDLINE | ID: mdl-24896118

ABSTRACT

Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na(+) leads to the formation of 100-µm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca(2+) gives rise to 100-µm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions.


Subject(s)
Lipid Bilayers/chemistry , Lipopolysaccharides/pharmacology , Liposomes/chemistry , Calcium/chemistry , Calcium/pharmacology , Cell Membrane/drug effects , Lipopolysaccharides/chemistry , Liposomes/ultrastructure , Microscopy, Atomic Force , Microscopy, Fluorescence , Sodium/chemistry , Sodium/pharmacology
14.
Langmuir ; 28(50): 17396-403, 2012 Dec 18.
Article in English | MEDLINE | ID: mdl-23163515

ABSTRACT

Electrostatic interaction plays a leading role in nanoparticle interactions with membrane architectures and can lead to effects such as nanoparticle binding and membrane disruption. In this work, the effects of nanoparticles (NPs) interacting with mixed lipid systems were investigated, indicating an ability to tune both NP binding to membranes and membrane disruption. Lipid membrane assemblies (LBAs) were created using a combination of charged, neutral, and gel-phase lipids. Depending on the lipid composition, nanostructured networks could be observed using in situ atomic force microscopy representing an asymmetrical distribution of lipids that rendered varying effects on NP interaction and membrane disruption that were domain-specific. LBA charge could be localized to fluidic domains that were selectively disrupted when interacting with negatively charged Au nanoparticles or quantum dots. Disruption was observed to be related to the charge density of the membrane, with a maximum amount of disruption occurring at ∼40% positively charged lipid membrane concentration. Conversely, particle deposition was determined to begin at charged lipid concentrations greater than 40% and increased with charge density. The results demonstrate that the modulation of NP and membrane charge distribution can play a pivitol role in determining NP-induced membrane disruption and NP surface assembly.


Subject(s)
Gold/chemistry , Lipid Bilayers/chemistry , Metal Nanoparticles/chemistry , Quantum Dots , Metal Nanoparticles/ultrastructure , Microscopy, Atomic Force , Particle Size , Static Electricity
15.
Chem Commun (Camb) ; 48(40): 4863-5, 2012 May 18.
Article in English | MEDLINE | ID: mdl-22497007

ABSTRACT

Cooperative binary ionic (CBI) solids comprise a versatile new class of opto-electronic and catalytic materials consisting of ionically self-assembled pairs of organic anions and cations. Herein, we report CBI nanocomposites formed by growing nanoparticles of one type of porphyrin CBI solid onto a second porphyrin CBI substructure with complementary functionality.


Subject(s)
Nanoparticles/chemistry , Porphyrins/chemistry , Cobalt/chemistry , Ions/chemistry , Nanoparticles/ultrastructure , Tin/chemistry , Zinc/chemistry
16.
ACS Nano ; 6(2): 1532-40, 2012 Feb 28.
Article in English | MEDLINE | ID: mdl-22251101

ABSTRACT

The deposition of amphiphilic poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD) copolymer micelles is demonstrated on solid substrates. Depending upon surface chemistry, micelle adsorption creates either monolayer or bilayer films. Lateral diffusion measurements reveal that strong coupling between hydrophilic surfaces and PEO blocks creates immobile bilayers, while monolayers retain the fluidity previously observed in vesicular assemblies.


Subject(s)
Biomimetic Materials/chemistry , Butadienes/chemistry , Cell Membrane/chemistry , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Micelles , Polyethylene Glycols/chemistry , Adsorption
17.
Tuberculosis (Edinb) ; 92(1): 38-47, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22033469

ABSTRACT

Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers.


Subject(s)
Lipid Bilayers/metabolism , Lipopolysaccharides/metabolism , Mannose/metabolism , Mycobacterium tuberculosis/metabolism , Animals , Humans , Mycobacterium tuberculosis/isolation & purification , Sensitivity and Specificity , Spectrometry, Mass, Fast Atom Bombardment
18.
Chem Commun (Camb) ; 47(35): 9858-60, 2011 Sep 21.
Article in English | MEDLINE | ID: mdl-21776520

ABSTRACT

Interferometric lithographically fabricated porous carbon acts as active substrates for Surface Enhanced Raman Scattering (SERS) applications with enhancement factors ranging from 7 to 9 orders of magnitude.

19.
Langmuir ; 27(15): 9484-9, 2011 Aug 02.
Article in English | MEDLINE | ID: mdl-21699157

ABSTRACT

We used positively charged lipids to prepare lipid bilayer assemblies (LBAs) upon which we assembled negatively charged gold nanoparticles (AuNPs). Treatment of the assembly with zirconium chloride resulted in the formation of nanorings of the diameters inversely related to the zirconium ion concentration. The nanorings were attributed to the zirconium ion coordinated AuNPs formed during the lipid bilayer budding process promoted by the acid effect of zirconium chloride. Nanoring organization was also dependent on the fluidity of lipid bilayers, an indication of LBA-assisted nanomaterials organization. We suggest that such bioorganic-inorganic hybrid assemblies coupled to unique topological and morphological variations might be useful as stimuli-responsive sensors or storage compartments for proteins or drugs.


Subject(s)
Chlorides/chemistry , Coordination Complexes/chemical synthesis , Gold/chemistry , Lipid Bilayers/chemistry , Metal Nanoparticles/chemistry , Zirconium/chemistry , Coordination Complexes/chemistry , Ions/chemistry , Particle Size , Surface Properties
20.
J Colloid Interface Sci ; 358(2): 635-8, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21477809

ABSTRACT

Understanding the interactions of nanoparticles with lipid membranes is crucial in establishing the mechanisms that govern assembly of membrane-based nanocomposites, nanotoxicology, and biomimetic inspired self-assembly. In this study, we explore binding of charged nanoparticles to lipid bilayers, both as liposomes and substrate supported assemblies. We find that the presence of a solid-support, regardless of curvature, eliminates the ability of zwitterionic fluid phase lipids to bind charged nanoparticles.


Subject(s)
Lipid Bilayers/chemistry , Nanoparticles/chemistry , Static Electricity , Binding Sites , Biomimetics/methods
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