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Heterogeneous copper nanoparticles (CuNPs) have emerged as a promising alternative to complete the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) owing to better stability and biocompatibility ...

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.

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.

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. ...

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 Galβ(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.

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 (N

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.

A combination of boronic acid and a photoactivatable diazirine enables oriented conjugation of Fc-fused lectins and antibodies on nanoparticles., 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.

A straightforward synthesis of the α(2→5)-linked 9-O-sulfated Neu5Gc-capped O-linked tetrasaccharide (1) identified in the egg cell surface glycoproteins of the sea urchin Strongylocentrotus purpuratus receptor for sperm is reported. The construction of the α(2→5)Neu5Gc trimer by the formation of an amide linkage rather than a glycosidic bond avoids the requirement for α-stereoselective glycosylation. To highlight this amide bond formation strategy as a relatively facile method for synthesizing oligo-Neu5Gc containing O-linked glycans, its versatility is demonstrated by application to the coupling of SO3H-9Neu5Gcα(2→5)Neu5Gc glycolic acid and a sialyl-Tn-derived amine for achieving the target tetrasaccharide. This synthetic strategy may be implemented in the generation of other structurally similar O-sulfo Neu5Gc-capped α2→5-Oglycolyl-linked oligo-Neu5Gc chains, enabling additional biological and chemical investigations.

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.

Correction for ‘Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies’ by Chen-Yo Fan et al., Chem. Sci., 2019, 10, 8600–8609, DOI: 10.1039/c9sc01613a.

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.

A protocol for the microwave-assisted Heck coupling reaction of C-aryl glycosides and styrenes has been developed for the synthesis of mono- and bis(C-glycoside) stilbenes. This method exclusively provides C-glycoside trans-stilbenes in good yields. The Pd-catalyzed Heck coupling reaction was successfully applied to various functionalized C-glycosyl aryl iodides and various substituted styrenes to deliver several C-glycoside trans-stilbenes with high selectivity. The obtained C-glycosyl trans-stilbenes were also examined for human SGLT-2 inhibitory activity.

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 IC 50 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 IC 50 = 2.3 nM, and H3N2 IC 50 = 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.

The trifluoroacetic acid adduct of tetranuclear zinc cluster Zn4(OCOCF3)6O catalysis in per-O-acetylation and de-O-acetylation of carbohydrates at 70 °C can be tuned by adjusting the reaction medium. Per-O-acetylation of hexopyranoses with a near stoichiometric amount of acetic anhydride in toluene resulted in the exclusive formation of pyranosyl products as an anomeric mixture, whereas de-O-acetylation of acetates occurred in methanol in high yields. In the latter, methanol acts as both nucleophile and solvent, and the reaction conditions were compatible to acid- and base-sensitive groups and amino acid derivatives.

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.

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(2→8) GD3) that is resistant to enzymatic hydrolysis is reported. The key steps in the synthesis are a regio- and stereoselective α(2→3) 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(2→8) 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 immunoglobulin 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.

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3-phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA120 ( Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA120 and ovalbumin (OVA).

The rapid capture and label-free detection of Staphylococcus aureus , an opportunistic bacterium that can infect upon contact, can be performed using periodic microarrays of ligand-protein conjugates created by noncontact (inkjet) printing, darkfield imaging conditions, and a FFT-based readout method. Ink solutes were prepared using bovine serum albumin (BSA) conjugated to a glycan with high affinity for bacterial adhesins and printed as dot-matrix arrays with periodicities of 80-120 μm using a thermal injection method. Upon exposing the glycan-BSA microarrays to live strains of S. aureus , patterns emerge that can be detected under optical darkfield conditions. These patterns can be decoded by fast Fourier transform (FFT) analysis to generate fault-tolerant readout signals that correspond to the capture of S. aureus, with a limit of detection between 10(2) and 10(3) cfu/mL. Inkjet printing provides independent control over array periodicity, enabling FFT signals to be assigned to specific frequencies in reciprocal k-space.

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.

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.

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 sixteen bacteria and found capable of capturing multiple species, some with limits of detection as low as 103 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.

Bishydrazides are versatile linkers for attaching glycans to substrates for lectin binding and pathogen detection schemes. The α,ω-bishydrazides of carboxymethylated hexa(ethylene glycol) (4) can be conjugated at one end to unprotected oligosaccharides, then attached onto carrier proteins, tethered onto activated carboxyl-terminated surfaces, or functionalized with a photoactive cross-linking agent for lithographic patterning. Glycoconjugates of bishydrazide 4 can also be converted into dithiocarbamates (DTCs) by treatment with CS(2) under mild conditions, for attachment onto gold substrates. The immobilized glycans serve as recognition elements for cell-surface lectins and enable the detection and capture of bacterial pathogens such as Pseudomonas aeruginosa by their adsorption onto micropatterned substrates. A detection limit of 10³ cfu/mL is demonstrated, using a recently introduced method based on optical pattern recognition.

When saccharides bearing a sulfur, selenium, or oxygen substituent at the anomeric center and an unprotected hydroxyl group either at C-4 or C-6 were subjected to fluorination with DAST in dichloromethane, a regioselective migration of the anomeric substituent to the C-4 or C-6 position was observed. Certain saccharides gave a mixture of migration and normal fluorination products whereas others yielded mainly or exclusively migration products (beta-glycosyl fluorides). The high thermal and chemical stability of migrated glycosyl fluorides were demonstrated to be an important precursor for many significant carbohydrate analogies. It is therefore suggested that these migrations may have useful applications in organic synthesis.

This work establishes a general protocol for synthesizing phytosphingosines with various lengths of lipid chains. The synthetic strategy included the Wittig reaction and olefin cross-metathesis as key steps. Combining these two C–C bond formation methods provide rapid access to adequately protected phytosphingosine backbones.

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 (70 mol %) and an amphiphilic linker (30 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 Concanavalin 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 in 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.

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.

A method for the stereocontrolled synthesis of a bacterial phosphoglycolipid (PGL1) isolated from thermophilic bacteria is described. The key features of the synthesis include a highly α-selective glycosylation reaction between a trichloroacetimidate donor and a D-lyxose-derived primary alcohol acceptor and the late-stage incorporation of the phospholipid.

In an attempt to fabricate highly active immunoprobes for serum biomarker detection, we report a simple and effective method for site-specific and self-oriented immobilization of antibodies on magnetic nanoparticles (MNPs). Through boronate formation, the carbohydrate moiety within the constant domain, Fc, of the antibody can be specifically and covalently linked to a boronic acid-functionalized MNP (BA@MNP) without hindering the antigen binding domain, Fab. The performance was evaluated by immunoaffinity extraction of multiple serum antigens. Compared with the random immobilization of antibody on a MNP, the antibody self-oriented immunoprobe provides long-term stability (2 months) and 5-fold extraction efficiency. It also provides 5-fold improved sensitivity at a low nM range (0.4 nM), presumably through enhanced antibody@MNP activity. In addition, false-positive detections arising from nonspecific binding can be completely minimized by effective surface protection using concentration-dependent dextran blocking. Compared with conventional antibody site-specific immobilization through protein G, this new BA-mediated covalent antibody immobilization provides interference-free extraction resulting from noncovalent immobilization of antibody by protein G. The new immunoassay was applied in comparative profiling of serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP) in human serum. Our triple immunoassay revealed a distinct pattern among normal patients, patients with cancer, and patients with cardiovascular disease. Using the previously reported quantization capability of the MALDI MS readout, we expect that this site-specific immunonanoprobe-based immunoassay can be highly active, rapid, and accurate in nanodiagnosis.

Compared to monovalent carbohydrates, multivalent carbohydrate ligands exhibit significantly enhanced binding affinities to their interacting proteins. Here, we report globotriose (P(k) ligand)-functionalized gold nanoparticle (AuNP) probes for the investigation of multivalent interactions with the B(5) subunit of Shiga-like toxin I (B-Slt). Six P(k)-ligand-encapsulated AuNPs (P(k)-AuNPs) of varying particle size and linker length were synthesized and evaluated for their potential as multivalent affinity probes by using a surface plasmon resonance competition assay. The affinity of these probes for the interacting proteins was greatly affected by nanoparticle size, linker length, and ligand density on nanoparticle surface. For example, the 20-nm 20-P(k)-l-AuNP, which had a relatively long linker showed a >10(8)-fold increase in affinity compared with the mono P(k) ligand. This intrinsic high-affinity AuNP probe specifically captured the recombinant B-Slt from Escherichia coli lysate, and the resulting purity of the B-Slt was >95 %. We also developed a robust P(k)-AuNP-based detection method for Slt-I by combining the technique with silver enhancement.

The Cu(I)-catalyzed alkyne−azide [2 + 3] cycloaddition has been demonstrated to be an effective and orthogonal conjugation reaction to covalently immobilize biomolecules on magnetic nanoparticles (MNPs). The azido group on the MNP surface provides better conjugation efficiency with alkynated molecules. Moreover, the C-terminal alkynated protein was site-specifically immobilized on MNP. The protein binding activity presented by site-specific immobilization is higher than that by random amide bond formation.