Lectin Immobilization Research Articles

Characterization of the interaction of multivalent glycosylated ligands with bacterial lectins by biolayer interferometry.

The study of multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Biolayer interferometry is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous isothermal titration calorimetry experiments, and presented strong advantages because they require less quantity and purity for the biomolecules.

In English

The aim of the work was to study the peculiarities of interaction of the surface of bacterial lectin of Bacillus subtilis IMB B-7724 inthe native state and under different model conditions with water molecules by 1 H NMR; to create a composite system based on the studied lectin, in which the protein molecule is minimally affected by the surface of the carrier, because protein molecules are capable to bind a significant amount of water localized in the spaces between the polymer chains. A method of “dry” immobilization of bacterial lectin on the surface of hydrophobic silica has been developed. Hydration of native lectin and lectin fixed on the surface of hydrophobic silica AM-1-175 was studied by low-temperature 1 H NMR spectroscopy. It has been shown that the immobilization of lectin on the surface of AM1 is accompanied by an increase in the interfacial energy gS from 4.1 to 5.2 J/g. This is due to an increase in the concentration of strongly bound water. Analysis of changes in the distributions of radii R of clusters of adsorbed water allows us to state that in water adsorbed by native lectin, there are two main maxima at R = 1 and 3 nm. In the immobilized state, the maximum at R = 1 nm is present in both types of water (of different order), but the second maximum is observed only for more ordered associates. Chloroform medium slightly reduces the binding energy of water to native lectin molecules (from 4.3 to 4.1 J/g), but in the case of immobilized lectin in CDCl3 medium, the value of ΣgS increases from 5.2 to 7.4 J/g. That is, the weakly polar medium promotes to increase in the interaction of water with interfaces, which is manifested in a relative increase in the number of water clusters of smaller size (Fig. 4). It should be noted that weakly associated forms of water (signal 3) are also represented by several types of clusters that have a radius in the range R = 1–10 nm, and their size distribution changes significantly during immobilization of lectin on the surface of AM1. Probably, weakly associated types of water are formed both in cavities, between polymer chains of protein molecules, and on the surface of AM1, free of protein.

In Ukrainian

Composite systems with certain cytotoxic (AM1/lectin) and adsorption (AM1/gelatin) activity have been developed on the basis of methyl silica and protein molecules – lectin and gelatin. For both types of composites, mechanisms of water binding to the surface and methods of transferring of hydrophobic materials into the aquatic environment have been investigated. The state of interfacial water in air, organic and acid media was studied. It has been found that the presence of a hydrophobic component in composites stabilizes of surface water in a weakly associated state, when a significant part of water molecules does not form hydrogen bonds. Liquid hydrophobic medium enhances this effect, and the strong acid (trifluoroacetic), added to it, promotes the transition of water to a strongly associated state. It has been shown that the redistribution of water in the interparticle intervals of AM1 with protein molecules immobilized on their surface changes under the influence of mechanical loads. Mechanoactivated samples are characterized by the possibility of water penetration into the spaces between the primary particles of methyl silica. It has been shown that immobilization of lectin on the surface of AM1 is accompanied by an increase in the interfacial energy gS from 4.1 to 5.2 J/g. This is due to an increase in the concentration of strongly bound water. If we analyze the changes in the distributions of radii R of the clusters of adsorbed water, we can state that in the water adsorbed by native lectin molecules, there are two main maxima at R = 1 and 3 nm. In the immobilized state, the maximum at R = 1 nm is present in both types of water (of different order), but the second maximum is observed only for more ordered associates.

In English

Сombination of properties of lectins and magnetically sensitive iron-containing nanocomposites (NC) for use in oncology is actual and promising from scientific and applied point of view. The aim of the research is to synthesize and to study new iron-containing NC and magnetic fluids containing bioactive bacterial lectin, promising for use as prototypes of new effective antitumor vector systems for targeted drug delivery and combined local therapy of cancer with minimized side effects on the body and improved compatibility with other remedies. To create vector systems, nanodisperse magnetite was synthesized by the Elmore reaction. The synthesis of aluminum-containing coating on the surface of Fe3O4 was carried out by double chemical modification with aluminum isopropylate. The obtained Fe3O4/Al2O3 NC was impregnated with sucrose solutions. Carbonization of the carbohydrate shell of NC was carried out in argon (500 °C). As a result, Fe3O4/Al2O3/C NC was obtained. The magnetic properties of nanostructures were measured using a laboratory vibration magnetometer of Foner type at room temperature. Adsorption immobilization of lectin was performed in 0.9 % NaCl solution in a dynamic mode at room temperature. Bacterial cytotoxic lectin of B. subtilis IMB B-7724 was used in the experiments. The amount of adsorbed substance (A) on the surface of nanocomposites was determined by measuring the concentration of lectin in the contact solutions before and after adsorption using a calibration graph. Measuring of the optical density and absorption spectra of lectin was performed on a spectrometer Lambda 35 UV/vis Perkin Elmer Instruments at λ = 280 nm. Standard techniques and equipment were used for biological research. The processes of adsorption immobilization of cytotoxic bacterial lectin of B. subtilis IMB B-7724 from physiologic saline on the surface of magnetite and carbon-containing Fe3O4/Al2O3/C NC were studied at room temperature. It has been found that the adsorption capacity of lectin on the surface of magnetite is 25.3 mg/g, and Fe3O4/Al2O3/C NC – 36.3 mg/g (at initial concentrations of lectin 0.06–0.4 mg/mL). The extraction extent of lectin R (%) was 12–38 % for magnetite and 46–67 % for Fe3O4/Al2O3/C NC. The dependence of the adsorption capacity on time was studied. A magnetic fluid (MF) based on single-domain Fe3O4, containing lectin was synthesized and investigated. Immobilization of lectin on MF particles was carried out in a dynamic mode at room temperature for 3 hours. The concentration of lectin in the composition of MF was 0.2 mg/mL. MF with immobilized lectin was further modified with PEG-2000. The synthesis of Fe3O4/ol.Na/lectin/PEG (ol.Na – sodium oleate) vector system was carried out in a dynamic mode for 3 hours. Modification of the surface of nanoparticles with polyethylene glycol was performed in order to increase the stability of the magnetic fluid, reducing the aggregation of particles. To determine the effect of experimental samples on the viability of MCF-7 cells in vitro, the following samples were prepared: Fe3O4/ol.Na/PEG (MF), CFe3O4 = 3 mg/mL; cytotoxic lectin of B. subtilis IMB B-7724 (CL), CCL = 0.2 mg/mL; nanobiocomposite (NBC). Nanobiocomposite based on MF and bacterial lectin was found to have a synergistic cytotoxic effect on MCF-7 human breast cancer cells, causing up to 40 % cell death. The IC50 values for the nanobiocomposite and lectin in relation to MCF-7 cells were 100 and 125 μg/mL, respectively. The results of research show that the combination of properties of lectins and magnetically sensitive iron-containing NC for use in oncology is a promising direction in creating new effective antitumor vector systems for targeted drug delivery and combined local therapy of cancer. The use of natural components in vector systems is a way to minimize the side effects on the body and improve compatibility with other antitumor remedies.

Enhanced sugar chain detection by oriented immobilization of Fc-fused lectins.

We report a novel scaffold for clustering and oriented immobilization of human IgG1 Fc-fused lectins on biosensors without chemical modifications. This approach uses a bio-nanocapsule (BNC) displaying a tandem form of IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). Incorporating ZZ-BNC effectively increased both the sensitivity and sugar chain-binding capacity compared with the condition without ZZ-BNC.

Oriented immobilization of rBC2LCN lectin for highly sensitive detection of human pluripotent stem cells using cell culture supernatants

Human pluripotent stem cells (hPSCs) are considered ideal cell sources for regenerative medicine, but their clinical and industrial applications are hindered by their tumorigenic potential. We previously identified an hPSC-specific lectin, rBC2LCN, that recognizes the podocalyxin glycoprotein secreted by undifferentiated hPSCs into the culture media. Using biotinylated rBC2LCN and a peroxidase-labeled R-10G antibody, we developed a sandwich assay for the detection of tumorigenic hPSCs. In this assay, the lectin is randomly immobilized on streptavidin-coated microplates to capture hPSC-derived podocalyxin. In the present study, rBC2LCN was genetically fused with polystyrene-binding peptides (PS-tags) for direct, site-specific, and oriented immobilization on polystyrene microplates. rBC2LCN lectins fused with PS-tags at the C-terminus were successfully overexpressed as a soluble form in Escherichia coli and then purified by affinity chromatography. We optimized the various parameters (protein and NaCl concentration, buffer pH, and blocking agents) of the sandwich assay by using PS-tagged rBC2LCN and the R-10G antibody. Finally, the lower limit of detection (LLOD) of the sandwich assay for hPSCs was examined. The LLOD was 2.2-fold lower than that achieved with the previous method. Considering that the developed method does not require the precoating of polystyrene microplates with streptavidin, it provides a cost-effective approach for the highly sensitive detection of hPSCs residing in hPSC-derived cell therapeutics.

Application of Porous Materials to Carbohydrate Chemistry and Glycoscience.

There is a growing interest in using a range of porous materials to meet research needs in carbohydrate chemistry and glycoscience in general. Among the applications of porous materials reviewed in this chapter, enrichment of glycans from biological samples prior to separation and analysis by mass spectrometry is a major emphasis. Porous materials offer high surface area, adjustable pore sizes, and tunable surface chemistry for interacting with glycans, by boronate affinity, hydrophilic interactions, molecular imprinting, and polar interactions. Among the materials covered in this review are mesoporous silica and related materials, porous graphitic carbon, mesoporous carbon, porous polymers, and nanoporous gold. In some applications, glycans are enzymatically or chemically released from glycoproteins or glycopeptides, and the porous materials have the advantage of size selectivity admitting only the glycans into the pores and excluding proteins. Immobilization of lectins onto porous materials of suitable pore size allows for the use of lectin-carbohydrate interactions in capture or separation of glycoproteins. Porous material surfaces modified with carbohydrates can be used for the selective capture of lectins. Controlled release of therapeutics from porous materials mediated by glycans has been reported, and so has therapeutic targeting using carbohydrate-modified porous particles. Additional applications of porous materials in glycoscience include their use in the supported synthesis of oligosaccharides and in the development of biosensors for glycans.

<b>PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography

Immobilized lectins are a powerful biotechnological tool for separation and isolation of glycoconjugates. In the present study, polyvinyl alcohol (PVA) and glutaraldehyde (GA) were used as a support for Concanavalin A (Con A) covalent immobilization and for entrapment of Parkia pendula seed gum (PpeG). Con A immobilization yielded approximately 30% and 0.6 M glucose solution was the minimum concentration able to elute fetuin from column. PVA-GA-PpeG column was efficiently recognized by pure P. pendula lectin (PpeL). These findings indicate that PVA-GA interpenetrated network showed to be an efficient support for lectin covalent immobilization and as affinity chromatography matrix after trapping of PpeG.

Carboxybetaine Modified Interface for Electrochemical Glycoprofiling of Antibodies Isolated from Human Serum.

Impedimetric lectin biosensors capable of recognizing two different carbohydrates (galactose and sialic acid) in glycans attached to antibodies isolated from human serum were prepared. The first step entailed the modification of a gold surface by a self-assembled monolayer (SAM) deposited from a solution containing a carboxybetaine-terminated thiol applied to the subsequent covalent immobilization of lectins and to resist nonspecific protein adsorption. In the next step, Sambucus nigra agglutinin (SNA) or Ricinus communis agglutinin (RCA) was covalently attached to the SAM, and the whole process of building a bioreceptive layer was optimized and characterized using a diverse range of techniques including electrochemical impedance spectroscopy, cyclic voltammetry, quartz crystal microbalance, contact angle measurements, zeta-potential assays, X-ray photoelectron spectroscopy, and atomic force microscopy. In addition, the application of the SNA-based lectin biosensor in the glycoprofiling of antibodies isolated from the human sera of healthy individuals and of patients suffering from rheumatoid arthritis (RA) was successfully validated using an SNA-based lectin microarray. The results showed that the SNA lectin, in particular, is capable of discriminating between the antibodies isolated from healthy individuals and those from RA patients based on changes in the amount of sialic acid present in the antibodies. In addition, the results obtained by the application of RCA and SNA biosensors indicate that the abundance of galactose and sialic acid in antibodies isolated from healthy individuals is age-related.

Magnetic Parkia pendula seed gum as matrix for Concanavalin A lectin immobilization and its application in affinity purification.

The present work aimed to magnetize Parkia pendula seeds gum and use it as a matrix for Concanavalin A covalent immobilization. This composite was applied in affinity purification of glycoconjugates. Parkia pendula seeds were hydrated and the gum provenient from the supernatant was precipitated and washed with ethanol and dried. The gum was magnetized in co-precipitation using solutions of Fe+2 and Fe+3. Matrix activation was accomplished with NaIO4. Magnetized Parkia pendula seeds gum with covalently immobilized Concanavalin A was used as an affinity matrix for the recognition of bovine serum fetuin glycoprotein. Fetuin elution was carried out with a solution of glucose (300mM) and evaluated through SDS-PAGE. The efficiency of lectin immobilization and fetuin purification were 63% and 14%, respectively. These results indicate that the composite produced is a promising magnetic polysaccharide matrix for lectins immobilization. Thus, such system can be applied for affinity purification allowing an easy recovery by magnetic field.

Perspectives in glycomics and lectin engineering.

This chapter would like to provide a short survey of the most promising concepts applied recently in analysis of glycoproteins based on lectins. The first part describes the most exciting analytical approaches used in the field of glycoprofiling based on integration of nanoparticles, nanowires, nanotubes, or nanochannels or using novel transducing platforms allowing to detect very low levels of glycoproteins in a label-free mode of operation. The second part describes application of recombinant lectins containing several tags applied for oriented and ordered immobilization of lectins. Besides already established concepts of glycoprofiling several novel aspects, which we think will be taken into account for future, more robust glycan analysis, are described including modified lectins, peptide lectin aptamers, and DNA aptamers with lectin-like specificity introduced by modified nucleotides. The last part of the chapter describes a novel concept of a glycocodon, which can lead to a better understanding of glycan-lectin interaction and for design of novel lectins with unknown specificities and/or better affinities toward glycan target or for rational design of peptide lectin aptamers or DNA aptamers.

Optimized immobilization of lectins using self-assembled monolayers on polysilicon encoded materials for cell tagging

Self-assembled monolayers (SAMs) have been used for the preparation of functional microtools consisting of encoded polysilicon barcodes biofunctionalized with proteins of the lectin family. These hybrid microtools exploit the lectins ability for recognizing specific carbohydrates of the cell membrane to give an efficient system for cell tagging. This work describes how the control of the methodology for SAM formation on polysilicon surfaces followed by lectin immobilization has a crucial influence on the microtool biofunction. Several parameters (silanization time, silane molar concentration, type of solvent or deposition methodology) have been studied to establish optimal function. Furthermore, silanes incorporating different terminal groups, such as aldehyde, activated ester or epoxide groups were tested in order to analyze their chemical coupling with the biomolecules, as well as their influence on the biofunctionality of the immobilized protein. Two different lectins – wheat germ agglutinin (WGA) and phytohemagglutinin (PHA-L) – were immobilized, because they have different and specific cell recognition behaviour and exhibit different cell toxicity. In this way we can assess the effect of intrinsic bulk toxicity with that of the cell compatibility once immobilized as well as the importance of cell affinity. A variety of nanometrical techniques were used to characterize the active surfaces, and lectin immobilization was quantified using ultraviolet-visible absorption spectroscopy (UV–vis) and optical waveguide light mode spectroscopy (OWLS). Once the best protocol was found, WGA and PHA were immobilized on polysilicon coded barcodes, and these microtools showed excellent cell tagging on living mouse embryos when WGA was used.

Facile Preparation of Well-Defined Hydrophilic Core–Shell Upconversion Nanoparticles for Selective Cell Membrane Glycan Labeling and Cancer Cell Imaging

Molecular imaging enables in situ visualization of biomolecules in living organisms and creates numerous opportunities for basic biological research and early disease diagnosis. As luminescent probes for molecular imaging, lanthanide-doped upconversion nanoparticles (UCNPs) exhibit superior performance compared to conventional fluorescent dyes in many ways, including high tissue penetration depth and minimized autofluorescence and photobleaching, making them particularly advantageous for imaging analysis. Although various synthesis methods have been reported, the preparation of high quality, water-soluble UCNPs remains challenging. For in situ imaging, glycans on the cell surface are particularly attractive due to their key roles in cellular activity and disease occurrence and development. However, glycan imaging is a challenging task due to their diverse structures and incompatibility with genetically encoded fluorescent tagging techniques. Herein, we report a new type of highly water-soluble, lectin-functionalized core-shell UCNP synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) for selective cell membrane glycan labeling and cancer cell imaging. SI-ATRP modification results in controlled growth of hydrophilic polymers on the UCNP surface and well-defined core-shell structure, producing UCNPs with improved biocompatibility and intact luminance property. Furthermore, the numerous functional groups on the polymer brush shell provide a large number of binding sites and 3D support for lectin immobilization. The increased loading density and diversified architecture of the immobilized lectins facilitates multivalent binding between the lectins and the glycans on the cell surface and leads to selective labeling of highly metastatic hepatocellular carcinoma cells (HCCHM3) in vitro and successful in vivo imaging of HCCHM3 inoculated mice.

Ultrasensitive impedimetric lectin biosensors with efficient antifouling properties applied in glycoprofiling of human serum samples.

Ultrasensitive impedimetric lectin biosensors recognizing different glycan entities on serum glycoproteins were constructed. Lectins were immobilized on a novel mixed self-assembled monolayer containing 11-mercaptoundecanoic acid for covalent immobilization of lectins and betaine terminated thiol to resist nonspecific interactions. Construction of biosensors based on Concanavalin A (Con A), Sambucus nigra agglutinin type I (SNA), and Ricinus communis agglutinin (RCA) on polycrystalline gold electrodes was optimized and characterized with a battery of tools including electrochemical impedance spectroscopy, various electrochemical techniques, quartz crystal microbalance (QCM), Fourier transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) and compared with a protein/lectin microarray. The lectin biosensors were able to detect glycoproteins from 1 fM (Con A), 10 fM (Ricinus communis agglutinin (RCA), or 100 fM (SNA) with a linear range spanning 6 (SNA), 7 (RCA), or 8 (Con A) orders of magnitude. Furthermore, a detection limit for the Con A biosensor down to 1 aM was achieved in a sandwich configuration. A nonspecific binding of proteins for the Con A biosensor was only 6.1% (probed with an oxidized invertase) of the signal toward its analyte invertase and a negligible nonspecific interaction of the Con A biosensor was observed in diluted human sera (1000×), as well. The performance of the lectin biosensors was finally tested by glycoprofiling of human serum samples from healthy individuals and those having rheumatoid arthritis, which resulted in a distinct glycan pattern between these two groups.

Brush polymer modified and lectin immobilized core–shell microparticle for highly efficient glycoprotein/glycopeptide enrichment

Protein glycosylation regulates numerous important biological processes and plays key roles in many diseases including cancer, diabetes and inflammation. The ability to efficiently profile variation of protein glycosylation in biological samples is very useful for identifying new diagnostic biomarkers or developing new therapeutic approaches. Due to the low availability of glycoprotein/glycopeptide from natural sources, enrichment before mass spectrometry (MS) analysis is usually a prerequisite. Affinity enrichment using lectins is currently one of the most widely adopted approaches. Conventionally, lectins are immobilized on solid supporting materials for sample recovery. However, the limited specific surface area, high steric hindrance and rigid nature of such supporting materials restricts lectin loading amount and results in low flexibility as well as accessibility of the immobilized lectins. Therefore, we proposed using core–shell microparticles composed of silica core and brush-like polymer chains shell for improved lectin immobilization. The surface bound brush-like polymer are synthesized by in situ growth of polymer chains from microparticle surface using surface initiated atom transfer radical polymerization (SI-ATRP). The flexible non-crosslinked polymer chains not only provide numerous binding sites, but also work as three-dimensional support for lectin immobilization, which leads to high loading amount and good accessibility of the immobilized lectin. Successful enrichment which facilitated glycoprotein/glycopeptide identification is demonstrated.

Ring‐opening metathesis polymerization‐derived, lectin‐functionalized monolithic supports for affinity separation of glycoproteins

Lectin-functionalized monolithic columns were prepared within polyether ether ketone (PEEK) columns (150 × 4.6 mm id) via transition metal-catalyzed ring-opening metathesis polymerization of norborn-2-ene (NBE) and trimethylolpropane-tris(5-norbornene-2-carboxylate) (CL) using the first-generation Grubbs initiator RuCl2 (PCy3 )2 (CHPh) (1, Cy = cyclohexyl) in the presence of a macro- and microporogen, i.e. of 2-propanol and toluene. Postsynthesis functionalization was accomplished via in situ grafting of 2,5-dioxopyrrolidin-1-yl-bicyclo[2.2.1]hept-5-ene-2-carboxylate to the surface of the monoliths followed by reaction with α,ω-diamino-poly(ethyleneglycol). The pore structure of the poly(ethyleneglycol)- derivatized monoliths was investigated by electron microscopy and inverse-size exclusion chromatography, respectively. The amino-poly(ethyleneglycol) functionalized monolithic columns were then successfully used for the immobilization of lectin from Lens culinaris hemagglutinin. The thus prepared lectin-functionalized monoliths were applied to the affinity chromatography-based purification of glucose oxidase. The binding capacity of Lens culinaris hemagglutinin-immobilized monolithic column for glucose oxidase was found to be 2.2 mg/column.

Binding of d-mannose-containing glycoproteins to d-mannose-specific lectins studied by surface plasmon resonance

Binding of selected glycoproteins containing α- d-mannose sites (invertase, glucoamylase, transferrin, glucose oxidase, and albumin α- d-mannopyranosylphenyl isothiocyanate) with two lectins selective for α- d-mannose branching glycans, concanavalin A (ConA), and Lens culinaris agglutinin (LCA) was studied on lectin biochips by microfluidic surface plasmon resonance (SPR). Lectin-containing biochips were prepared by covalent immobilization of lectins on a flat carboxymethylated gold surface. Both measurement as well as regeneration conditions were optimized. The determined dissociation constants of lectin–glycoprotein interactions were 10 −5 to 10 −7 mol/l. Dissociation constants K D of studied bindings were estimated by the steady state specific binding models based on both a binding to one site and the multiple binding sites model using Hill slope.

Concanavalin A and polyvinyl butyral use as a potential dengue electrochemical biosensor

Immobilization of concanavalin A on gold electrode by means of gold nanoparticles and polyvinyl butyral was carried out and investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The system was tested with sera from patients infected by dengue fever (DF) and dengue hemorrhagic fever (DHF). Electrochemical impedance spectroscopy (in the frequency range from 100mHz to 100KHz), and cyclic voltammetry (from −0.2 to 0.7V vs. Ag/AgCl), was performed in phosphate buffer solution containing 10mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe. As biomolecules accumulated on the electrode surface the voltammetric response changed from a clear diffusional to an irreversible behavior. Impedance spectroscopy showed a clear increase of the electron-transfer resistance when the sensor is exposed to contaminated sera (DF or DHF) as compared to exposure to uncontaminated serum (NDF). The results were analyzed through an equivalent circuit and values of charge transfer resistance and capacitance were obtained. Variations in charge transfer resistance were used to distinguish the sensor response for the different sera investigated (DF, DHF and NDF). Alternatively, a three-dimensional graph gave the best response for differentiation of all three blood sera. The distinctive patterns of impedimetric responses observed were ascribed to different glycoprotein patterns in the sera investigated. Therefore, the lectin immobilization on electrode surface with gold nanoparticles and polyvinyl butyral, combined with the three-dimensional impedance analysis introduced herein are valuable tools in the development of a biosensor for immunological response to diseases.

Monitoring microbial populations of sulfate-reducing bacteria using an impedimetric immunosensor based on agglutination assay

An impedimetric immunosensor was fabricated for rapid and non-labeled detection of sulfate-reducing bacteria, Desulforibrio caledoiensis (SRB) by immobilizing lectin-Concanavalin A using an agglutination assay. The immobilization of lectin was conducted using amine coupling on the surface of a gold (Au) electrode assembled with 11-Mercaptoundecanoic acid. Electrochemical impedance spectroscopy (EIS) was used to verify the stepwise assembly of the sensor system. The work conditions of the impedimetric immunosensor, such as pH of the buffer solutions and the incubation time of lectin, were optimized. Faradic impedance spectra for charge transfer for the redox probe Fe(CN) 6 3−/4−were measured to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance ( R ct) increased with increasing SRB concentration. A linear relationship between R ct and SRB concentration was obtained in SRB concentration range of 1.8 to 1.8 × 10 7 cfu/ml. The variation of the SRB population during the growth process was also monitored using the impedimetric immunosensor. This approach has great potential for simple, low-cost, and time-saving monitoring of microbial populations.

Kinetic Analysis of Glycoprotein–Lectin Interactions by Label-Free Internal Reflection Ellipsometry

Abstract Introduction Glycoproteomics is undergoing rapid development, largely as a result of advances in technologies for isolating glycoproteins and analyzing glycan structures. However, given the number and diversity of glycans, there is need for new technologies that can more rapidly provide differential carbohydrate–protein structural information on a large scale. We describe a new microarray platform based on a label-free imaging ellipsometry technique, which permits simultaneous detection of multiple glycoprotein–lectin interactions without the need for reporter labels, while still providing high throughput kinetic information at much lower cost. Our results demonstrate the utility of LFIRE™ (Label-Free Internal Reflection Ellipsometry) for the rapid kinetic screening of carbohydrate–lectin recognition. The technology was also used to evaluate the benefits of the lectin immobilization format using multi-lectin affinity chromatography (M-LAC) to capture glycoproteins (with enhanced binding strength or avidity) from biological samples. Using a printed panel of lectins, singly or in combination, we examined the binding characteristics of standard glycoproteins. Results and Discussion Using kinetic measurements, it was observed that the binding strength of lectins to carbohydrates is enhanced using a multi-lectin strategy, suggesting that improved selectivity and specificity can lead to increased functional avidity. The data presented confirm that this label-free technology can be used to effectively screen single or combinations of lectins. Furthermore, the combination of LFIRE™ and M-LAC may permit more rapid and sensitive identification of novel biomarkers based on carbohydrate changes in glycoproteins, and lead to a better understanding of the connections of glycan function in cellular mechanisms of health and disease.