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1.
Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time. 相似文献
2.
We report a new type of microarray, based on glyconanoparticles (GNPs), to study glycan-lectin interactions. GNPs, synthesized by conjugating carbohydrate ligands on silica nanoparticles, were printed on a photoactive surface followed by covalent immobilization by light activation. The GNP microarrays could be probed by lectins labeled with fluorescein as well as fluorescein-doped silica nanoparticles (FSNPs). Results showed that FSNP as the label enhanced the signals for the higher affinity ligands than the lower ones. 相似文献
3.
Carbohydrates constitute the most abundant organic matter in nature, serving as structural components and energy sources, and mediating a wide range of cellular activities. The emergence of nanomaterials with distinct optical, magnetic, and electronic properties has witnessed a rapid adoption of these materials for biomedical research and applications. Nanomaterials of various shapes and sizes having large specific surface areas can be used as multivalent scaffolds to present carbohydrate ligands. The resulting glyconanomaterials effectively amplify the glycan-mediated interactions, making it possible to use these materials for sensing, imaging, diagnosis, and therapy. In this review, we summarize the synthetic strategies for the preparation of various glyconanomaterials. Examples are given where these glyconanomaterials have been used in sensing and differentiation of proteins and cells, as well as in imaging glycan-medicated cellular responses. 相似文献
4.
Multifunctional Transmembrane Protein Ligands for Cell‐Specific Targeting of Plasma Membrane‐Derived Vesicles
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Chi Zhao David J. Busch Connor P. Vershel Jeanne C. Stachowiak 《Small (Weinheim an der Bergstrasse, Germany)》2016,12(28):3837-3848
Liposomes and nanoparticles that bind selectively to cell‐surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single‐domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP‐tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering. 相似文献
5.
Direct measurement of glyconanoparticles and lectin interactions by isothermal titration calorimetry
Glyconanomaterials have shown high potential in applications including bioanalysis and nanomedicine. Here, a quantitative analytical technique, based on isothermal titration calorimetry, was developed to characterize the interactions between glyconanoparticles and lectins. By titrating lectins into the glyconanoparticle solution, the apparent dissociation constant, thermodynamic parameters, and the number of binding sites were derived simultaneously. For the glyconanoparticles-lectin binding pairs investigated, a 3-5 order of magnitude affinity enhancement over the free ligand-lectin interactions was observed which can be attributed to the multivalent ligand presentation on the nanoparticles. The impact of ligand density was also studied, and results showed that the affinity increased with the number of glycans on the nanoparticle. 相似文献
6.
This paper describes the development of novel particle-based fluorescence resonance energy transfer (FRET) sensors to quantify the concentration and monitor the binding affinity of carbohydrates and glycoproteins to lectins, which are carbohydrate binding proteins. The sensing approach is based on FRET between fluorescein (donor)-labeled lectin molecules, adsorbed on the surface of micrometric polymeric beads, and polymeric dextran molecules labeled with Texas Red (acceptor). The FRET efficiency of the donor-acceptor pair decreases in the presence of carbohydrates or glycoproteins that compete with the Texas Red-labeled dextran molecules on the lectinic binding sites. The inhibitory effect is concentration and time dependent. The sensing technique enables the discrimination between carbohydrates and glycoproteins based on their binding affinity to the FRET sensing particles as well as quantitative analysis of carbohydrates and glycoproteins in aqueous samples. In the future, the newly developed sensors could enable screening glycoprotein-based drugs for their binding affinity toward selective receptors. 相似文献
7.
Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer 总被引:1,自引:0,他引:1
Yoshizako K Akiyama Y Yamanaka H Shinohara Y Hasegawa Y Carredano E Kikuchi A Okano T 《Analytical chemistry》2002,74(16):4160-4166
A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column. 相似文献
8.
A Huisgen 1,3-dipolar cycloaddition "click chemistry" was employed to immobilize azido sugars (mannose, lactose, alpha-Gal) to fabricate carbohydrate self-assembled monolayers (SAMs) on gold. This fabrication was based on preformed SAM templates incorporated with alkyne terminal groups, which could further anchor the azido sugars to form well-packed, stable, and rigid sugar SAMs. The clicked mannose, lactose, and alpha-Gal trisaccharide SAMs were used in the analysis of specific carbohydrate-protein interactions (i.e., mannose-Con A; ECL-lactose, alpha-Gal-anti-Gal). The apparent affinity constant of Con A binding to mannose was (8.7 +/- 2.8) x 10(5) and (3.9 +/- 0.2) x 10(6) M(-1) measured by QCM and SPR, respectively. The apparent affinity constants of lactose binding with ECL and alpha-Gal binding with polyclonal anti-Gal antibody were determined to be (4.6 +/- 2.4) x 10(6) and (6.7 +/- 3.3) x 10(6) M(-1), respectively by QCM. SPR, QCM, AFM, and electrochemistry studies confirmed that the carbohydrate SAM sensors maintained the specificity to their corresponding lectins and nonspecific adsorption on the clicked carbohydrate surface was negligible. This study showed that the clicked carbohydrate SAMs in concert with nonlabel QCM or SPR offered a potent platform for high-throughput characterization of carbohydrate-protein interactions. Such a combination should complement other methods such as ITC and ELISA in a favorable manner and provide insightful knowledge for the corresponding complex glycobiological processes. 相似文献
9.
Stefano Gottardi Kathrin Müller Juan Carlos Moreno‐Lpez Handan Yildirim Ute Meinhardt Milan Kivala Abdelkader Kara Meike Sthr 《Advanced Materials Interfaces》2014,1(1)
The self‐assembly of cyano‐substituted triarylamine derivatives on Au(111) is studied with scanning tunneling microscopy and density functional theory calculations. Two different phases, each stabilized by at least two different cyano bonding motifs are observed. In the first phase, each molecule is involved in dipolar coupling and hydrogen bonding, while in the second phase, dipolar coupling, hydrogen bonding and metal‐ligand interactions are present. Interestingly, the metal–ligand bond is already observed for deposition of the molecules with the sample kept at room temperature leaving the herringbone reconstruction unaffected. It is proposed that for establishing this bond, the Au atoms are slightly displaced out of the surface to bind to the cyano ligands. Despite the intact herringbone reconstruction, the Au substrate is found to considerably interact with the cyano ligands affecting the conformation and adsorption geometry, as well as leading to correlation effects on the molecular orientation. 相似文献
10.
Esther Amstad Stefan Zurcher Alireza Mashaghi Joyce Y. Wong Marcus Textor Erik Reimhult 《Small (Weinheim an der Bergstrasse, Germany)》2009,5(11):1334-1342
Magnetic resonance imaging (MRI), a non‐invasive, non‐radiative technique, is thought to lead to cellular or even molecular resolution if optimized targeted MR contrast agents are introduced. This would allow diagnosing progressive diseases in early stages. Here, it is shown that the high binding affinity of poly(ethylene glycol)‐gallol (PEG‐gallol) allows freeze drying and re‐dispersion of 9 ± 2‐nm iron oxide cores individually stabilized with ≈9‐nm‐thick stealth coatings, yielding particle stability for at least 20 months. Particle size, stability, and magnetic properties of PEGylated particles are compared to Feridex, a commercially available untargeted negative MR contrast agent. Biotin‐PEG(3400)‐gallol/methoxy‐PEG(550)‐gallol stabilized nanoparticles are further functionalized with biotinylated human anti‐VCAM‐1 antibodies using the biotin–neutravidin linkage. Binding kinetics and excellent specificity of these nanoparticles are demonstrated using quartz crystal microbalance with dissipation monitoring (QCM‐D). These MR contrast agents can be functionalized with any biotinylated ligand at controlled ligand surface density, rendering them a versatile research tool. 相似文献
11.
Hermes JP Sander F Peterle T Cioffi C Ringler P Pfohl T Mayor M 《Small (Weinheim an der Bergstrasse, Germany)》2011,7(7):920-929
The directed assembly of gold nanoparticles is essential for their use in many kinds of applications, such as electronic devices, biological labels, and sensors. Herein an atomic alteration in the molecular structure of ligand-stabilized gold nanoparticles that can shift the interparticle distance up to 1 nm upon covalent coupling to organic-inorganic superstructures is presented. Gold nanoparticles are stabilized by two octadentate thioether ligands and have a mean diameter of 1.1 nm. The ligands contain a central rigid rod varying in length and terminally functionalized with a protected acetylene. The two peripheral functional groups on each particle enable the directed assembly of nanoparticles to dimers, trimers, and tetramers by oxidative acetylene coupling. This is a wet chemical protocol resulting in covalently bound nanoparticles. These organic-inorganic hybrid superstructures are analyzed by transmission electron microscopy, small angle X-ray scattering, and UV/vis spectroscopy. The focus of the comparison here is the subunit, which is anchoring the bridgehead, either a pyridine or benzene moiety. The pyridine-based ligands reflect the calculated length of the rigid-rod spacer in their interparticle distances in the obtained hybrid structures. This suggests a perpendicular arrangement that results from the coordination of the pyridine's lone pair to the gold surface. An atomic variation in the ligand's center leads to smaller interparticle distances in the case of hybrid structures obtained from benzene ligands. This large difference in the spatial arrangement suggests a tangential arrangement of the interparticle bridging structure in the latter case. Consequently a rather flat arrangement parallel to the particle surface must be assumed for the central benzene unit of the benzene-based ligand. 相似文献
12.
When small ligands are immobilized onto a porous chromatography medium, only a limited number of binding sites contributes to the interaction with the target molecule. The main part of the ligand molecules is distributed on sites that are not accessible for the target protein due to steric hindrance. To direct the ligand into a well-accessible position, the ligand was conjugated to a large molecule that acted as a placeholder during the immobilization step. Then the placeholder molecule was cleaved off and washed out. Two linear peptides with affinity for lysozyme and human blood coagulation factor VIII, respectively, were studied as model systems. The protected peptide ligand was covalently linked to a 20-kDa poly(ethylene glycol) molecule containing an acid-labile linker. After selective deprotection of the peptide and purification, immobilization of this conjugate on a preactivated chromatography matrix was performed alternatively through the free N-terminus, the epsilon-amino group of lysine, or the sulfohydryl group of cysteine. After the immobilization reaction, the spacer molecule and remaining protecting groups were cleaved off and the gels were tested by affinity chromatography. This novel immobilization technique substantially increased the binding capacity and the ligand utilization for the target protein, and site-specific immobilization could be demonstrated. 相似文献
13.
Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose. 相似文献
14.
Immobilized lectins have now been incorporated into affinity surfaces that can be used to isolate broad classes of samples for mass spectrometric analysis. A carbohydrate and a bacterial species that displays the carbohydrate binding motif were isolated and concentrated out of solutions containing salt, urea, buffers, and other contaminants that are deleterious to MALDI mass spectrometry. Concanavalin A was immobilized to a gold foil via a self-assembled monolayer. Samples in phosphate buffer or urine were applied to the capture surface and allowed to interact. The capture surface was then washed to remove salts and other unbound components and subjected to matrix-assisted laser desorption/ionization on a time-of-flight mass spectrometer. The lectin-derivatized surface allowed samples to be concentrated and readily characterized at relatively low levels. 相似文献
15.
Yusko EC Johnson JM Majd S Prangkio P Rollings RC Li J Yang J Mayer M 《Nature nanotechnology》2011,6(4):253-260
Synthetic nanopores have been used to study individual biomolecules in high throughput, but their performance as sensors does not match that of biological ion channels. Challenges include control of nanopore diameters and surface chemistry, modification of the translocation times of single-molecule analytes through nanopores, and prevention of non-specific interactions with pore walls. Here, inspired by the olfactory sensilla of insect antennae, we show that coating nanopores with a fluid lipid bilayer tailors their surface chemistry and allows fine-tuning and dynamic variation of pore diameters in subnanometre increments. Incorporation of mobile ligands in the lipid bilayer conferred specificity and slowed the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. Lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (Aβ) oligomers and fibrils. Through combined analysis of their translocation time, volume, charge, shape and ligand affinity, different proteins were identified. 相似文献
16.
We have demonstrated that multi-walled carbon nanotubes (MWNTs) coated with a carbohydrate-carrying polymer for use as biological recognition signals can be easily prepared by a non-covalent method via hydrophobic interactions. Fluorescence observation by confocal laser scanning microscopy showed that the carbohydrate-carrying polymers were densely localized around the MWNTs. To evaluate biological recognition affinity, interactions of the MWNTs with lectins were examined by binding tests. The resultant MWNTs were found to acquire a selective binding affinity to the corresponding lectin without a non-specific interaction. On the other hand, bare MWNTs non-specifically interacted with lectins. These results showed that the MWNTs coated with a carbohydrate-carrying polymer have biological recognition signals. Modification of carbon nanotubes with various carbohydrate chains will be a useful protocol for molecular designs of biomaterials, nanoarchitecture and biosensors. 相似文献
17.
Lees EE Gunzburg MJ Nguyen TL Howlett GJ Rothacker J Nice EC Clayton AH Mulvaney P 《Nano letters》2008,8(9):2883-2890
Analytical ultracentrifugation (AUC) was used to characterize the size distribution and surface chemistry of quantum dots (QDs). AUC was found to be highly sensitive to nanocrystal size, resolving nanocrystal sizes that differ by a single lattice plane. Sedimentation velocity data were used to calculate the ligand packing density at the crystal surface for different sized nanocrystals. Dihydrolipoic acid poly(ethylene glycol) was found to bind between 66 and 60% of the surface cadmium atoms for CdSe nanocrystals in the 1.54-2.59 nm radius size regime. The surface ligand chemistry was found to affect QD sedimentation, with larger ligands decreasing the sedimentation rate through an increase in particle volume and increase in frictional coefficient. Finally, AUC was used to detect and analyze protein association to QDs. Addition of bovine serum albumin (BSA) to the QD sample resulted in a reduced sedimentation rate, which may be attributed to an associated frictional drag. We calculated that one to two BSA molecules bind per QD with an associated frictional ratio of 1.2. 相似文献
18.
Microfluidic Gradients Reveal Enhanced Neurite Outgrowth but Impaired Guidance within 3D Matrices with High Integrin Ligand Densities
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Nicole H. Romano Kyle J. Lampe Hui Xu Meghaan M. Ferreira Sarah C. Heilshorn 《Small (Weinheim an der Bergstrasse, Germany)》2015,11(6):722-730
The density of integrin‐binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell‐adhesive ligands on neurite chemoattraction is not well understood. A platform is presented here that combines gradient‐generating microfluidic devices with 3D protein‐engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia‐derived spheroids. Spheroids are encapsulated in elastin‐like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade‐off between neurite extension and neurite guidance is reminiscent of the well‐known trade‐off between adhesive forces at the leading and trailing edges of a migrating cell, implying that a similar matrix‐mediated balance of forces regulates neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels. 相似文献
19.
Penghui Ji Gianluca Galeotti Fabrizio De Marchi Daling Cui Kewei Sun Haiming Zhang Giorgio Contini Maryam Ebrahimi Oliver MacLean Federico Rosei Lifeng Chi 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(35)
While surface‐confined Ullmann‐type coupling has been widely investigated for its potential to produce π‐conjugated polymers with unique properties, the pathway of this reaction in the presence of adsorbed oxygen has yet to be explored. Here, the effect of oxygen adsorption between different steps of the polymerization reaction is studied, revealing an unexpected transformation of the 1D organometallic (OM) chains to 2D OM networks by annealing, rather than the 1D polymer obtained on pristine surfaces. Characterization by scanning tunneling microscopy and X‐ray photoelectron spectroscopy indicates that the networks consist of OM segments stabilized by chemisorbed oxygen at the vertices of the segments, as supported by density functional theory calculations. Hexagonal 2D OM networks with different sizes on Cu(111) can be created using precursors with different length, either 4,4″‐dibromo‐p‐terphenyl or 1,4‐dibromobenzene (dBB), and square networks are obtained from dBB on Cu(100). The control over size and symmetry illustrates a versatile surface patterning technique, with potential applications in confined reactions and host–guest chemistry. 相似文献
20.
Ligand‐Mediated Control of Dislocation Dynamics and Resulting Particle Morphology of GdOCl Nanocrystals
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While much progress has been achieved in the shape‐controlled synthesis of nanocrystals, chemical strategies to define morphology remain primarily empirical. Here, a mechanistic study of the influence of different coordinating ligands on the kinetics and thermodynamics of crystal growth during the preparation of GdOCl by the non‐hydrolytic condensation of GdCl3 and Gd(O i Pr)3 is reported. Growth using oleylamine, octadecylamine, trioctylamine, and didodecylamine yields 2D nanosheets with approximately square cross sections, whereas growth in trioctylphosphine oxide yields larger and thicker platelets. The nanostructures are characterized by the presence of spiral growth patterns and dislocations. Apart from preferential binding to specific crystallographic facets, the coordinating ligands are suggested to control the extent of supersaturation, thereby facilitating and tuning dislocation‐mediated growth. Upon depletion of monomers, thermodynamic surface energy considerations become of paramount importance and the nanocrystals are reshaped via mass transport from edges to sides yielding their eventual equilibrium shapes. The mechanisms developed here are thought to be broadly generalizable to the ligand‐directed growth of nanomaterials. 相似文献