RESUMEN
Globo A is a neutral Globo-series glycosphingolipid (GSL) that shows natural properties of a cytotoxicity receptor NKp44 binding ligand. The highly complex heptasaccharide glycan structure of Globo A combined with its biological profile provides a unique target for the development of a synthetic method to facilitate its bioactivity studies. Here, a concise chemoenzymatic route to the synthesis of Globo A and its α1,3-galactose-linked congener Globo B is reported. The key to success was the use of a synthetic azido ß-Globo H sphingosine (Globo H-ßSph) as an acceptor substrate and two glycosyl transferases, an α1,3-N-acetylgalactosaminyltransferase from Helicobacter mustelae (BgtA) and a human blood group B α1,3-galactosyltransferase (h1,3GTB), for stereoselective construction of the terminal α1,3-GalNAc and α1,3-Gal linkages, respectively. The azido-Sph lipid sidechain is further elaborated by reduction and a chemoselective N-acylation to complete the total synthesis of the neutral Globo-series GSLs. In addition, the synthesis of Forssman and para-Forssman antigens were prepared. The strategy may be suitable for accessing other complex GSLs and related lipid-modified GSL derivatives.
Asunto(s)
Glicoesfingolípidos , Glicoesfingolípidos Neutros , Humanos , Glicoesfingolípidos/metabolismo , Unión ProteicaRESUMEN
Structural variants of α-galactosylceramide (α-GalCer) that stimulate invariant natural killer T (iNKT) cells constitute an emerging class of immunomodulatory agents in development for numerous biological applications. Variations in lipid chain length and/or fatty acids in these glycoceramides selectively trigger specific pro-inflammatory responses. Studies that would link a specific function to a structurally distinct α-GalCer rely heavily on the availability of homogeneous and pure materials. To address this need, we report herein a general route to the diversification of the ceramide portion of α-GalCer glycolipids. Our convergent synthesis commences from common building blocks and relies on the Julia-Kocienski olefination as a key step. A cleavable fluorous tag is introduced at the non-reducing end of the sugar that facilitates quick purification of products by standard fluorous solid-phase extraction. The strategy enabled the rapid generation of a focused library of 61 α-GalCer analogs by efficiently assembling various lipids and fatty acids. Furthermore, when compared against parent α-GalCer in murine cells, many of these glycolipid variants were found to have iNKT cell stimulating activity similar to or greater than KRN7000. ELISA assaying indicated that glycolipids carrying short fatty N-acyl chains (1fc and 1ga), an unsubstituted (1fh and 1fi) or CF3-substituted phenyl ring at the lipid tail, and a flexible, shorter fatty acyl chain with an aromatic ring (1ge, 1gf, and 1gg) strongly affected the activation of iNKT cells by the glycolipid-loaded antigen-presenting molecule, CD1d. This indicates that the method may benefit the design of structural modifications to potent iNKT cell-binding glycolipids.
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Interleucina-2 , Células T Asesinas Naturales , Ratones , Animales , Antígenos CD1d , Glucolípidos/farmacología , Ácidos GrasosRESUMEN
Naturally occurring N-glycans display much diversity in modifications, linkages, and peripheral presentation of the oligosaccharide chain. Despite continued advancements in oligosaccharide synthesis, synthetic access to these natural glycans remains challenging. Biologically relevant complex N-glycan mimetics with various natural and unnatural modifications are an alternate way for investigating glycan-protein interactions. Further supporting this pattern, we report here a new class of sialylated bi- and triantennary pseudo mannose N-glycans reproducing orientation of the underlying glycan chain and branching patterns and replacing the two inner mannopyranosyl units with 1,2,3-triazole rings. Such mimetics are straightforwardly generated by implementing multiple intermolecular Cu(I)-catalyzed azide-alkyne cycloaddition between chemoenzymatically synthesized azido sialosides and rationally designed C-3 and C-6 di-O- or C-2, C-3, and C-6 tri-O-alkynylated mannoside. Human recombinant Siglec-7-Fc fusion protein recognizes almost all sialylated pseudo mannose N-glycans in the microarray. However, a differential Sia-binding pattern was also observed. Given the library size, comparison of pairwise mannose N-glycan combinations showed that biantennary linear α(2,3)α(2,8)- and α(2,6)α(2,8)- or branched α(2,3)α(2,6)-, and triantennary branched α(2,3)α(2,6)-sialyl pseudo N-glycans possess similar binding capabilities and affinity to recombinant Siglec-7-Fc. While the full range of topological mannose arms remain elusive, the bi- and triantennary mimics are simpler structures for interrogating Siglec interactions.
Asunto(s)
Manosa , Polisacáridos , Humanos , Manosa/química , Oligosacáridos/química , Polisacáridos/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismoRESUMEN
Glycosphingolipids (GSLs) play essential roles in many important biological processes, making them attractive synthetic targets. In this paper, a viable chemoenzymatic method is described for the synthesis of globo-series GSLs, namely, Gb4, Gb5, SSEA-4, and Globo H. The strategy uses a chemically synthesized lactoside acceptor equipped with a partial ceramide structure that is uniquely extended by glycosyltransferases in a highly efficient one-pot multiple enzyme (OPME) procedure. A direct and quantitative conversion of Gb4 sphingosine to Globo H sphingosine is achieved by performing two-sequential OPME glycosylations. A reduction and N-acylation protocol allows facile incorporation of various fatty acids into the lipid portions of the GSLs. The chemically well-defined lipid-modified Globo H-GSLs displayed some differences in their immunosuppressive activities, which may benefit the structural modifications of Globo H ceramides in finding new types of immunosuppressive agents. The strategy outlined in this work should be applicable to the rapid access to other complex GSLs.
Asunto(s)
Glicoesfingolípidos , Esfingosina , Glicoesfingolípidos/química , Glicoesfingolípidos/metabolismo , Inmunosupresores/farmacologíaRESUMEN
[This corrects the article DOI: 10.1039/C9SC01613A.].
RESUMEN
The excellent molecular recognition capabilities of monoclonal antibodies (mAbs) have opened up exciting opportunities for biotherapeutic discovery. Taking advantage of the full potential of this tool necessitates affinity ligands capable of conjugating directly with small molecules to a defined degree of biorthogonality, especially when modifying natural Abs. Herein, a bioorthogonal boronate-affinity-based Ab ligand featuring a 4-(dimethylamino)pyridine and an S-aryl thioester to label full-length Abs is reported. The photoactivatable linker in the acyl donor facilitated purification of azide-labelled Ab (N3 -Ab) was quantitatively cleaved upon brief exposure to UV light while retaining the original Ab activity. Click reactions enabled the precise addition of biotin, a fluorophore, and a pharmacological agent to the purified N3 -Abs. The resulting immunoconjugate showed selectivity against targeted cells. Bioorthogonal traceless design and reagentless purification allow this strategy to be a powerful tool to engineer native antibodies amenable to therapeutic intervention.
Asunto(s)
Inmunoconjugados , Acilación , Anticuerpos Monoclonales , Azidas , Colorantes FluorescentesRESUMEN
Enzymatic synthesis that is commenced by the sugar nucleotide regeneration system (SNRS) protocol can minimize 1) the consumption of exorbitant sugar nucleotides, 2) the amount of transferases required, and 3) byproduct feedback inhibition. In this study, LacNAc extensions/modifications of the N-linked mannose core were carried out efficiently with SNRS with high yields and purities on all branches in a uniform manner. In addition, we demonstrate that with SNRS, bacterial glycosyltransferases exhibit a wide acceptor tolerance for bi- and triantennary mannose core structures as substrates for target oligosaccharides. The synthesized small library of mannose core-based glycans and linear O-glycans were screened for their binding affinity against h-Siglecs 2, 4, 7, 9, 14, 15, and m-Siglec-15 to explore their structure-based binding preferences. Microarray data revealed that each Siglec showed few distinct yet overlapping specificities. An increase in branching from mono to di or tri antennary did not necessarily lead to increasing affinity. Glycans with the disialoside sequence α(2,3)α(2,8)/α(2,6)α(2,8) showed high specificity and affinity for Siglec-7, and sLex α(2,3) exhibited a strong affinity for Siglec-9. Explicit recognition of α(2,6)α(2,3)- linear and α(2,3)α(2,6)-branched glycans by Siglecs-2, 4, and 15, respectively, suggests that these structures can act as potential candidates for the further development of high-affinity ligands.
Asunto(s)
Polisacáridos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Nucleótidos , Polisacáridos/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , AzúcaresRESUMEN
The total synthesis of the oligosaccharide moiety of disialosyl globopentaosylceramide (DSGb5 Cer), a dominant ganglioside isolated from malignant renal cell carcinoma tissues, is reported. The synthetic strategy relies on a chemical α(2,6)-sialylation at the internal GalNAc unit of a Gb5 pentasaccharide backbone that furnishes a Neu5Acα(2,6)GalNAc-linked hexasaccharide, suitable for an enzymatic α(2,3)-sialylation of the terminal Gal residue to construct a heptasaccharide glycan. Convergent access to this key α(2,6)-sialylated hexasaccharide was also achieved through a [3+3] glycosylation building upon a Galß(1,3)[Neu5Acα(2,6)]GalNAc-based trisaccharide donor and a Gb3 acceptor. The synthetic DSGb5 glycan bearing a 6-azidohexyl aglycon at the reducing end could undergo further regioselective functionalization. This approach represents a viable chemoenzymatic method for accessing complex ganglioside glycans and should be useful for the synthesis and biological investigation of DSGb5 derivatives.
Asunto(s)
Globósidos , Polisacáridos , Glicosilación , OligosacáridosRESUMEN
Antibodies have exquisite specificities for molecular recognition, which have led to their incorporation into array sensors that are crucial for research, diagnostic, and therapeutic applications. Many of these platforms rely heavily on surface-bound reactive groups to covalently tether antibodies to solid substrates; however, this strategy is hindered by a lack of orientation control over antibody immobilization. Here, we report a mild electrophilic phenylsulfonate (tosylate) ester-containing boronic acid affinity ligand for attaching antibodies to glass slides. A high level of antibody coupling located near the Fc region of the boronated antibody complex could be achieved by the proximal nucleophilic amino acid driven substitution reaction at the phenylsulfonate center. This enabled the full-length antibodies to be permanently tethered onto surfaces in an oriented manner. The advantages of this strategy were demonstrated through the individual and multiplex detection of protein and serum biomarkers. This strategy not only confers stability to the immobilized antibodies but also presents a different direction for the irreversible attachment of antibodies to solid supports in an orientation-controlled way.
RESUMEN
The utilization of immuno-magnetic nanoparticles (MNPs) for the selective capture, enrichment, and separation of specific glycoproteins from complicated biological samples is appealing for the discovery of disease biomarkers. Herein, MNPs were designed and anchored with abundant boronic acid (BA) and photoreactive alkyl diazirine (Diaz) functional groups to obtain permanently tethered Fc-fused Siglec-2 and antiserum amyloid A (SAA) mAb with the assistance of reversible boronate affinity and UV light activation in an orientation-controlled manner. The Siglec-2-Fc-functionalized MNPs showed excellent stability in fetal bovine serum (FBS) and excellent efficiency in the extraction of cell membrane glycoproteins. The anti-SAA mAb-functionalized MNPs maintained active Ab orientation and preserved antigen recognition capability in biological samples. Thus, the BA-Diaz-based strategy holds promise for the immobilization of glycoproteins, such as antibodies, with the original protein binding activity maintained, which can provide better enrichment for the sensitive detection of target proteins.
RESUMEN
Fluorous-modified surfaces have emerged as a powerful tool for the immobilization of fluorous-tagged biomolecules based on their specificity and the strength of fluorous-fluorous interactions. To fabricate a fluorous-based protein microarray, we designed two strategies for site-specific modification of proteins with a fluorous tag: attaching the fluorous tag to the C-termini of expressed proteins by native chemical ligation (NCL) or to the Fc domain of antibodies through boronic acid (BA)-diol interactions. The perfluoro-tagged proteins could be easily purified by fluorous-functionalized magnetic nanoparticles (MNPs) and immobilized on a fluorous chip with minimal non-specific adsorption. Importantly, proteins immobilized on the solid support through non-covalent fluorous-fluorous interactions were sufficiently stable to withstand continuous washing. We believe that this fluorous-fluorous immobilization strategy will be a highly valuable tool in protein microarray fabrication.
Asunto(s)
Flúor/metabolismo , Proteínas Inmovilizadas/metabolismo , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Coloración y Etiquetado/métodosRESUMEN
Replacing the interglycosidic oxygen atom of oligosaccharides with a nonhydrolyzable sulfur atom has attracted significant interest because it provides opportunities for developing new glycoconjugate vaccines. Herein, a stereocontrolled and highly convergent method to synthesize a non-reducing-end inter-S-glycosidic variant of the GD3 antigen (S-linked α(2â8) GD3) that is resistant to enzymatic hydrolysis is reported. The key steps in the synthesis are a regio- and stereoselective α(2â3) sialylation of a lactoside acceptor with a C8-iodide-derivatized sialyl donor and an anomeric S-alkylation, which enable stereoselective construction of a terminal S-linked α(2â8) disialyl residue. The sulfhydryl-reactive maleimide group was used as the linker for the well-defined conjugation of these antigens to the immunogenic protein keyhole limpet hemocyanin (KLH). Groups of mice were immunized with the GD3-KLH and S-linked GD3-KLH glycoconjugates in the presence of complete Freund's adjuvant. Microarray analysis of the sera showed the promise of the S-linked GD3-KLH vaccine: it stimulated a high immunoglobulinâ G response against S-linked GD3 and cross-reactivity with the O-linked GD3 antigen was low. The activity of the S-linked GD3-KLH vaccine was comparable to that of the O-linked GD3-KLH vaccine, which highlighted the effectiveness of generating glycoconjugate vaccines and immunotherapies by relatively simple means.
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Gangliósidos/química , Glicoconjugados/química , Hemocianinas/química , Animales , Antígenos/química , Antígenos/inmunología , Glicoconjugados/síntesis química , Glicoconjugados/inmunología , Glicoconjugados/metabolismo , Hemocianinas/inmunología , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Maleimidas/química , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/metabolismo , Compuestos de Sulfhidrilo/química , Vacunas Sintéticas/inmunología , Vibrio cholerae/enzimologíaRESUMEN
The interactions between soluble carbohydrates and/or surface displayed glycans and protein receptors are essential to many biological processes and cellular recognition events. Carbohydrate microarrays provide opportunities for high-throughput quantitative analysis of carbohydrate-protein interactions. Over the past decade, various techniques have been implemented for immobilizing glycans on solid surfaces in a microarray format. Herein, we describe a detailed protocol for fabricating carbohydrate microarrays that capitalizes on the intrinsic reactivity of boronic acid toward carbohydrates to form stable boronate diesters. A large variety of unprotected carbohydrates ranging in structure from simple disaccharides and trisaccharides to considerably more complex human milk and blood group (oligo)saccharides have been covalently immobilized in a single step on glass slides, which were derivatized with high-affinity boronic acid ligands. The immobilized ligands in these microarrays maintain the receptor-binding activities including those of lectins and antibodies according to the structures of their pendant carbohydrates for rapid analysis of a number of carbohydrate-recognition events within 30 h. This method facilitates the direct construction of otherwise difficult to obtain carbohydrate microarrays from underivatized glycans.
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Ácidos Borónicos/química , Carbohidratos/química , Análisis por Micromatrices/métodos , Proteínas/químicaRESUMEN
The design of potent metabolically stable neuraminidase (NA) inhibitors represents an attractive approach for treating influenza virus infection. In this study, we describe the exploitation of the 150-cavity in the active site of group 1 NA for the design, synthesis, and in vitro evaluation of new triazole-containing N-acyl derivatives related to Zanamivir. Inhibition studies with influenza virus NAs of group 1 (H1N1) and group 2 (H3N2) revealed that several of them are good inhibitors, with IC50 values in the low nanomolar (2.3 nM-31 nM) range. Substituents that form stable van der Waals interaction with the 150-cavity residues play crucial roles in NA inhibition as demonstrated by the potency of 6a (H1N1 IC50 = 2.3 nM, and H3N2 IC50 = 2.9 nM). Docking studies indicated that the cyclohexane-substituted triazole ring extended toward the hydrophobic region in the active site of group 1 NA in open form. The high potency observed for inhibitor 6a may be attributable to the highly favorable hydrophobic interactions in this region.
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Diseño de Fármacos , Neuraminidasa/antagonistas & inhibidores , Triazoles/química , Zanamivir/síntesis química , Zanamivir/farmacología , Antivirales/síntesis química , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Unión Competitiva , Dominio Catalítico , Técnicas de Química Sintética , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/enzimología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neuraminidasa/metabolismo , Zanamivir/química , Zanamivir/metabolismoRESUMEN
Hemin linked to hexa(ethylene glycol)bishydrazide was patterned by inkjet printing into periodic microarrays, and evaluated for their ability to capture bacterial pathogens expressing various hemin receptors. Bacterial adhesion was imaged under darkfield conditions with Fourier analysis, supporting a label-free method of pathogen detection. Hemin microarrays were screened against a panel of 16 bacteria and found capable of capturing multiple species, some with limits of detection as low as 10(3) cfu/mL. Several Gram-positive strains including Staphylococcus aureus and Bacillus anthracis also exhibited rapid adhesion, enabling pattern recognition within minutes of exposure. This can be attributed to differences in hemin acquisition systems: aggressively adherent bacteria express cell-surface hemin receptors (CSHRs) that enable direct hemin binding and uptake, whereas other types of bacteria including most Gram-negative strains rely on the secretion and recapture of soluble proteins (hemophores) for hemin acquisition, with consequently longer times for ligand binding and detection.
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Bacterias/aislamiento & purificación , Hemina/química , Análisis por Micromatrices/métodos , Hidrazinas/química , Tinta , Límite de Detección , Modelos Moleculares , Conformación Molecular , Polietilenglicoles/química , ImpresiónRESUMEN
The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.
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Biotina/química , Reactivos de Enlaces Cruzados/química , Sondas Moleculares/química , Fotólisis , Receptores de Superficie Celular/química , Animales , Ratones , Ratones Endogámicos C57BL , Sondas Moleculares/síntesis química , Estructura MolecularRESUMEN
The design of high-affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco-gold nanoparticles (glyco-AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco-AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70â mol %) and an amphiphilic linker (30â mol %) provide long-term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco-AuNPs enable the detection of model plant lectins such as Concanavalinâ A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop inâ situ glyco-AuNPs-based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle-based biosensors.
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Anticuerpos Inmovilizados/química , Anticuerpos/química , Proteínas Portadoras/química , Glicoproteínas/química , Oro/química , Lectinas/química , Polisacáridos/química , Bioensayo , Técnicas Biosensibles , Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Lectinas/metabolismo , Ligandos , Nanopartículas del MetalRESUMEN
Applications of glycosylated nanomaterials have gained considerable attention in recent years due to their unique structural properties and compatibility in biological systems. In this review, glyco-nanoparticles (glyco-NPs) are defined as compounds that contain a nano-sized metallic core, are composed of noble metals, magnetic elements, or binary inorganic nanoparticles, and that exhibit carbohydrate ligands on the surface in three dimensional polyvalent displays similar to the glycocalyx structures on cell membranes. Nanomaterials decorated with suitable biological recognition ligands have yielded novel hybrid nanobiomaterials with synergistic functions, especially in biomedical applications. This review focuses on strategies for building various types of glyco-NPs and highlights their potential in targeted drug delivery and molecular imaging as well as their uses in bioassays and biosensors. The most recent examples of glyco-NPs as vaccine candidates and probes for assaying enzymes with bond-forming activities are also discussed.
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Nanoestructuras/química , Nanotecnología/métodos , Animales , Bacterias/aislamiento & purificación , Glicosilación , Humanos , Sondas Moleculares/química , Sondas Moleculares/inmunología , Coloración y EtiquetadoRESUMEN
Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.
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Anticuerpos/química , Rayos Ultravioleta , Límite de Detección , Etiquetas de FotoafinidadRESUMEN
Carbohydrates are essential mediators of many important extracellular binding events. Multivalent effects are often required for these binding interactions that are generally weak. Biologically significant carbohydrate-modified nanomaterials are an appealing model system for a systematic investigation of multivalent binding effects. This review seeks to highlight recent striking examples of multivalent glycosylated nanomaterials concentrating on carbohydrate-protein interactions. The specific aim is to provide an overview of various aspects of glyco-nanoparticles in binding assays, targeted therapy, molecular imaging, enzyme activity assay, and bacterium detection.