Avijit Kumar Adak

Globotriose-Functionalized Gold Nanoparticles as Multivalent Probes for Shiga-like Toxin

Compared to monovalent carbohydrates, multivalent carbohydrate ligands exhibit significantly enhanced binding affinities to their interacting proteins. Here, we report globotriose (Pk ligand)‐functionalized gold nanoparticle (AuNP) probes for the investigation of multivalent interactions with the B5 subunit of Shiga‐like toxin I (B‐Slt). Six Pk‐ligand‐encapsulated AuNPs (Pk‐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‐Pk‐l‐AuNP, which had a relatively long linker showed a >108‐fold increase in affinity compared with the mono Pk 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 Pk‐AuNP‐based detection method for Slt‐I by combining the technique with silver enhancement.

Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application

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.

Bishydrazide glycoconjugates for lectin recognition and capture of bacterial pathogens.

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.

Advances in multifunctional glycosylated nanomaterials: preparation and applications in glycoscience

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.

Fabrication of Highly Stable Glyco‐Gold Nanoparticles and Development of a Glyco‐Gold Nanoparticle‐Based Oriented Immobilized Antibody Microarray for Lectin (GOAL) Assay

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.

Synthesis of sialic acid-containing saccharides.

Sialic acids are a diverse family of negatively charged monosaccharides with a shared nine-carbon carboxylated backbone, and they often serve as the terminal positions of cell surface glycoproteins and glycolipids. Sialic acids play essential roles in mediating or modulating numerous pathological, biological, and immunological recognition events. Advances in synthesis have provided chemically well-defined and structurally homogeneous sialic acid-containing carbohydrates that are crucial for studying glycobiology. This review highlights recent innovations in the chemical and chemoenzymatic synthesis of difficult α-sialosides, with a particular focus on methods developed for α-selective sialylation in the synthesis of O-linked and S-linked oligosialic acids.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

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.

DAST-Mediated Regioselective Anomeric Group Migration in Saccharides

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.

Synthesis and evaluation of a photoactive probe with a multivalent carbohydrate for capturing carbohydrate-lectin interactions.

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).

Label-Free Detection of Staphylococcus aureus Captured on Immutable Ligand Arrays

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.