Decoding the mechanism governing the structural stability of wheat germ agglutinin and its isolated domains: A combined calorimetric, NMR, and MD simulation study.

Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.

Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Precursor carboxy-silica for functionalization with interactive ligands. III. Carbodiimide assisted preparation of immobilized lectin stationary phases for high performance lectin affinity chromatography of sub-glycoproteomics from cancer and disease free human sera.

In this study, a precursor carboxy-silica support was demonstrated in the immobilization of two different lectins, namely concanavalin A (Con A) and wheat germ agglutinin (WGA) for use in high performance lectin affinity chromatography (LAC) for the selective capturing and enrichment of glycoproteins from healthy/disease free and cancer human sera. The lectin columns thus obtained (i.e., Con A- and WGA-columns) showed no nonspecific interactions toward some chosen standard glycoproteins and non-glycoproteins. Both columns were shown in sub-glycoproteomics enrichment from human sera including disease free and adenocarcinoma cancer sera. The collected fractions were subjected to LC-MS/MS for identification of the captured glycoproteins, whereby the total number of identified proteins using Con A column from disease-free and cancer sera were 164 and 188, respectively while 133 and 103 proteins were identified in the fractions captured by the WGA column from disease-free and cancer sera samples, respectively. Differentially expressed proteins (DEPs) between the disease free and cancer sera in both the Con A and WGA column fractions were identified via the plot of the abundance vs. the protein ratio whereby the binary logarithm of average intensities of cancer and disease free sera were plotted against the binary logarithm of cancer/disease free sera ratios. The proteins that exhibit log 2 (cancer/healthy) ratio values greater than +2 and less than -2 in both categories are considered as DEPs. Furthermore, for visualization of the data arrangement, Q-Q scatterplot were also used whereby the binary logarithm of cancer serum was plotted against the binary logarithm of disease-free serum for both Con A and WGA. For Con A column, 28 up-regulated and 10 down regulated proteins were identified with a total of 38 DEPs while only two being non-glycoproteins. Furthermore, the up-regulated, and down regulated proteins recorded for WGA column are 14 and 6, respectively, totaling 20 proteins including 3 non-glycoproteins. Some of the non-specific binding to lectin are most likely due to protein-protein interactions.

Human serum albumin nanoparticles as a versatile vehicle for targeted delivery of antibiotics to combat bacterial infections.

Urinary tract infections (UTIs) are among the most common bacterial infections. Despite a wide range of therapeutic options, treatment success is compromised by the efficient mechanism of tissue colonization of uropathogenic Escherichia coli. In advanced drug delivery systems, a similar, glycan-mediated targeting mechanism may be realized by conjugating the drug to a plant lectin, like wheat germ agglutinin (WGA). We introduce a drug delivery vehicle consisting of human serum albumin as nanoparticle shell, olive oil as core component, the active pharmaceutical ingredients (API) trimethoprim and rifampicin as well as WGA to facilitate cellular internalization. When WGA was embedded into the proteinaceous particle shell, cell binding studies revealed up to 60 % higher cell binding potential. Additionally, nanoparticles showed a good efficacy against gram-negative just as against gram-positive bacteria. The combination of the promising cell-associative properties and the proven antimicrobial potential might lead to an improved efficacy of advanced treatment of UTIs.

Surface-Templated Glycopolymer Nanopatterns Transferred to Hydrogels for Designed Multivalent Carbohydrate-Lectin Interactions across Length Scales.

Multivalent interactions between carbohydrates and proteins enable a broad range of selective chemical processes of critical biological importance. Such interactions can extend from the macromolecular scale (1-10 nm) up to much larger scales across a cell or tissue, placing substantial demands on chemically patterned materials aiming to leverage similar interactions in vitro. Here, we show that diyne amphiphiles with carbohydrate headgroups can be assembled on highly oriented pyrolytic graphite (HOPG) to generate nanometer-resolution carbohydrate patterns, with individual linear carbohydrate assemblies up to nearly 1 µm, and microscale geometric patterns. These are then photopolymerized and covalently transferred to the surfaces of hydrogels. This strategy suspends carbohydrate patterns on a relatively rigid polydiacetylene (persistence length ∼ 16 nm), exposed at the top surface of the hydrogel above the bulk pore structure. Transferred patterns of appropriate carbohydrates (e.g., N-acetyl-d-glucosamine, GlcNAc) enable selective, multivalent interactions (KD ∼ 40 nM) with wheat germ agglutinin (WGA), a model lectin that exhibits multivalent binding with appropriately spaced GlcNAc moieties. WGA binding affinity can be further improved (KD ∼ 10 nM) using diacetylenes that shift the polymer backbone closer to the displayed carbohydrate, suggesting that this strategy can be used to modulate carbohydrate presentation at interfaces. Conversely, GlcNAc-patterned surfaces do not induce specific binding of concanavalin A, and surfaces patterned with glucuronic acid, or with simple carboxylic acid or hydroxyl groups, do not induce WGA binding. More broadly, this approach may have utility in designing synthetic glycan-mimetic interfaces with features from molecular to mesoscopic scales, including soft scaffolds for cells.

LogP of N-acyl-gemcitabine and lectin-corona emerge as key parameters in nanoparticulate intravesical cancer therapy.

After surgical removal of the tumour tissue, bladder cancer is treated by intravesical instillation of cytotoxic drugs such as gemcitabine. Gemcitabine, however, is highly hydrophilic and possesses a short half-life due to fast enzymatic deamination. Additionally, continuous dilution by urine, a hardly permeable urothelial barrier and rapid excretion by urination make therapy difficult. To modify lipophilicity of the drug, N-acyl-gemcitabine derivatives with quite different solubility and logP were synthesized, purified and characterized. The loading of PLGA nanoparticles with the N-acyl-gemcitabine derivatives followed by release in artificial urine, revealed that the drug content increases but the subsequent release decreases with lipophilicity. Additionally, acylation increased cytotoxicity and opened passive diffusion as an additional pathway into cancer cells. To address physiological constraints, the surface of the monodisperse nanoparticles was grafted with bioadhesive wheat germ agglutinin. Cytoadhesion to artificial bladder cancer tissue and even uptake into the cells as indicated by microscopic imaging are expected to prolong the retention time in the bladder cavity as well as to promote uptake into the cells. By using N-caprylic-gemcitabine as most appropriate gemcitabine-derivative for drug loading and making use of the bioadhesive characteristics of wheat germ agglutinin for grafting the corona of PLGA-nanoparticles, an innovative strategy towards smart drug delivery for instillative therapy of bladder cancer is proposed.

Supramolecular Binding with Lectins: A New Route for Non-Covalent Functionalization of Polysaccharide Matrices.

The chemical functionalization of polysaccharides to obtain functional materials has been of great interest in the last decades. This traditional synthetic approach has drawbacks, such as changing the crystallinity of the material or altering its morphology or texture. These modifications are crucial when a biogenic matrix is exploited for its hierarchical structure. In this work, the use of lectins and carbohydrate-binding proteins as supramolecular linkers for polysaccharide functionalization is proposed. As proof of concept, a deproteinized squid pen, a hierarchically-organized ß-chitin matrix, was functionalized using a dye (FITC) labeled lectin; the lectin used was the wheat germ agglutinin (WGA). It has been observed that the binding of this functionalized protein homogenously introduces a new property (fluorescence) into the ß-chitin matrix without altering its crystallographic and hierarchical structure. The supramolecular functionalization of polysaccharides with protein/lectin molecules opens up new routes for the chemical modification of polysaccharides. This novel approach can be of interest in various scientific fields, overcoming the synthetic limits that have hitherto hindered the technological exploitation of polysaccharides-based materials.

Photoswitching Affinity and Mechanism of Multivalent Lectin Ligands.

Multivalent receptor-ligand binding is a key principle in a plethora of biological recognition processes. Immense binding affinities can be achieved with the correct spatial orientation of the ligands. Accordingly, the incorporation of photoswitches, which can be used to reversibly change the spatial orientation of molecules, into multivalent ligands is a means to alter the binding affinity and possibly also the binding mode of such ligands. We report a divalent ligand for the model lectin wheat germ agglutinin (WGA) containing an arylazopyrazole photoswitch. This switch, which has recently been introduced as an alternative to the more commonly used azobenzene moiety, is characterized by almost quantitative E/Z photoswitching in both directions, high quantum yields, and high thermal stability of the Z isomer. The ligand was designed in a way that only one of the isomers is able to bridge adjacent binding sites of WGA leading to a chelating binding mode. Photoswitching induces an unprecedentedly high change in lectin binding affinity as determined by isothermal titration calorimetry (ITC). Furthermore, additional dynamic light scattering (DLS) data suggest that the binding mode of the ligand changes from chelating binding of the E isomer to crosslinking binding of the Z isomer.

Lactoferrin modified by hypohalous acids: Partial loss in activation of human neutrophils.

Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are produced at high concentrations during inflammation and oxidative/halogenative stress by myeloperoxidase, an enzyme of azurophilic neutrophilic granules. Here we compared the effects of recombinant human lactoferrin (rhLTF) and its halogenated derivatives (rhLTF-Cl and rhLTF-Br) on functional responses of neutrophils. Our results demonstrated that after halogenative modification, rhLTF lost its ability to induce mobilization of intracellular calcium, actin cytoskeleton reorganization, and morphological changes in human neutrophils. Moreover, both forms of the halogenated rhLTF prevented binding of N-acetylglucosamine-specific plant lectin Triticum vulgaris agglutinin (WGA) to neutrophils and, in contrast to native rhLTF, inhibited respiratory burst of neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and by two plant lectins (WGA and PHA-L). However, we observed no differences between the effects of rhLTF, rhLTF-Cl, and rhLTF-Br on respiratory burst of neutrophils induced by phorbol 12-myristate 13-acetate (PMA), digitonin, and number of plant lectins with different glycan-binding specificity. Furthermore, all rhLTF forms interfered with PMA- and ionomycin-induced formation of neutrophil extracellular traps. Thus, halogenative modification of LTF is one of the mechanisms involved in modulating a variety of signaling pathways in neutrophils to control their pro-inflammatory activity.

Lectin from Triticum vulgaris (WGA) Inhibits Infection with SARS-CoV-2 and Its Variants of Concern Alpha and Beta.

Even in the face of global vaccination campaigns, there is still an urgent need for effective antivirals against SARS-CoV-2 and its rapidly spreading variants. Several natural compounds show potential as antiviral substances and have the advantages of broad availabilities and large therapeutic windows. Here, we report that lectin from Triticum vulgaris (Wheat Germ Agglutinin) displays antiviral activity against SARS-CoV-2 and its major Variants of Concern (VoC), Alpha and Beta. In Vero B4 cells, WGA potently inhibits SARS-CoV-2 infection with an IC50 of <10 ng/mL. WGA is effective upon preincubation with the virus or when added during infection. Pull-down assays demonstrate direct binding of WGA to SARS-CoV-2, further strengthening the hypothesis that inhibition of viral entry by neutralizing free virions might be the mode of action behind its antiviral effect. Furthermore, WGA exhibits antiviral activity against human coronavirus OC43, but not against other non-coronaviruses causing respiratory tract infections. Finally, WGA inhibits infection of the lung cell line Calu-3 with wild type and VoC viruses with comparable IC50 values. Altogether, our data indicate that topical administration of WGA might be effective for prophylaxis or treatment of SARS-CoV-2 infections.

Wheat Germ Agglutinin-Conjugated Disulfide Cross-Linked Alginate Nanoparticles as a Docetaxel Carrier for Colon Cancer Therapy.

PURPOSE: In chemotherapy, oral administration of drug is limited due to lack of drug specificity for localized colon cancer cells. The inability of drugs to differentiate cancer cells from normal cells induces side effects. Colonic targeting with polymeric nanoparticulate drug delivery offers high potential strategies for delivering hydrophobic drugs and fewer side effects to the target site. Disulfide cross-linked polymers have recently acquired high significance due to their potential to degrade in reducing colon conditions while resisting the upper gastrointestinal tract's hostile environment. The goal of this project is, therefore, to develop pH-sensitive and redox-responsive fluorescein-labeled wheat germ agglutinin (fWGA)-mounted disulfide cross-linked alginate nanoparticles (fDTP2) directly targeting docetaxel (DTX) in colon cancer cells. METHODS: fDTP2 was prepared by mounting fWGA on DTX-loaded nanoparticles (DTP2) using the two-step carbodiimide method. Morphology of fDTP2 was examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Dynamic light scattering (DLS) study was carried out to determine the mean diameter, polydispersity index (PDI) and zeta potential of fDTP2. Cellular uptake efficiency was examined using fluorescence microplate reader. Biocompatibility and active internalization of fDTP2 were conducted on HT-29. RESULTS: fDTP2 was found to exhibit a DTX loading efficiency of 19.3%. SEM and TEM tests revealed spherical nanoparticles. The in vitro DTX release test showed a cumulative release of 54.7%. From the DLS study, fDTP2 reported a 277.7 nm mean diameter with PDI below 0.35 and -1.0 mV zeta potential. HT-29 which was fDTP2-treated demonstrated lower viability than L929 with a half maximal inhibitory concentration (IC50) of 34.7 µg/mL. HT-29 (33.4%) internalized fDTP2 efficiently at 2 h incubation. The study on HT-29 active internalization of nanoparticles through fluorescence and confocal imaging indicated such. CONCLUSION: In short, fDTP2 demonstrated promise as a colonic drug delivery DTX transporter.

Separation of Glycosylated OmpA-Like Proteins from Porphyromonas gingivalis and Tannerella forsythia.

OmpA-like proteins located in the outer bacterial membrane are potential virulence factors from the major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia. Our previous studies have shown that OmpA-like proteins are glycosylated by O-linked N-acetylglucosamine (O-GlcNAc) and are strongly reactive to wheat germ agglutinin (WGA) lectin, which shows sugar specificity to GlcNAc. Utilizing this property, we have developed a separation method for OmpA-like proteins by affinity chromatography using WGA lectin-agarose. The purity of enriched native OmpA-like proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. More importantly, the purified OmpA-like proteins formed a unique trimeric structure keeping their bioactivity intact. In this chapter, we describe a detailed procedure to separate OmpA-like proteins, which may be used to further progress the biological studies of OmpA-like proteins.

A lectin-coupled porous silicon-based biosensor: label-free optical detection of bacteria in a real-time mode.

Accuracy and speed of detection, along with technical and instrumental simplicity, are indispensable for the bacterial detection methods. Porous silicon (PSi) has unique optical and chemical properties which makes it a good candidate for biosensing applications. On the other hand, lectins have specific carbohydrate-binding properties and are inexpensive compared to popular antibodies. We propose a lectin-conjugated PSi-based biosensor for label-free and real-time detection of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by reflectometric interference Fourier transform spectroscopy (RIFTS). We modified meso-PSiO2 (10-40 nm pore diameter) with three lectins of ConA (Concanavalin A), WGA (Wheat Germ Agglutinin), and UEA (Ulex europaeus agglutinin) with various carbohydrate specificities, as bioreceptor. The results showed that ConA and WGA have the highest binding affinity for E. coli and S. aureus respectively and hence can effectively detect them. This was confirmed by 6.8% and 7.8% decrease in peak amplitude of fast Fourier transform (FFT) spectra (at 105 cells mL-1 concentration). A limit of detection (LOD) of about 103 cells mL-1 and a linear response range of 103 to 105 cells mL-1 were observed for both ConA-E. coli and WGA-S. aureus interaction platforms that are comparable to the other reports in the literature. Dissimilar response patterns among lectins can be attributed to the different bacterial cell wall structures. Further assessments were carried out by applying the biosensor for the detection of Klebsiella aerogenes and Bacillus subtilis bacteria. The overall obtained results reinforced the conjecture that the WGA and ConA have a stronger interaction with Gram-positive and Gram-negative bacteria, respectively. Therefore, it seems that specific lectins can be suggested for bacterial Gram-typing or even serotyping. These observations were confirmed by the principal component analysis (PCA) model.

A novel method for live imaging of human airway cilia using wheat germ agglutinin.

Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

Rapid and sensitive glycan targeting by lectin-SERS assay.

Glycosylation is an important part of cell signalling that is implicated in many disease states in which glycans play an essential role. Therefore rapid and sensitive differentiation of glycans on proteins is highly desirable. Current technologies for glycan structural analysis normally involve the isolation of glycans from proteins, or enrichment of glycopeptides, and detection by mass spectrometry, which requires relatively large amounts of sample and is not able to be used by non-specialist laboratories. Herein we present a simple and new strategy for targeting the glycans on a protein (with IgG as a model glycoprotein) using surface-enhanced Raman scattering (SERS) coupled to glycan-binding WGA (wheat germ agglutinin) lectin, in a lectin-SERS assay. With one drop (1 µL) of glycoprotein solution, our lectin-SERS assay can detect as low as 10 ng IgG within two hours with high glycan specificity. We extend our technique to examine the surface glycan profiles on two human colorectal cancer cell lines, which show different and unique glycan signatures specific to the target cell lines. Thus, we believe that this method could be potentially used for the real-time and in situ monitoring of glycans on the surface of cells or tissue or in body fluids, and is thus a powerful tool for glycomics research.

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1.

The glycosylated mucin domain of the Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N-acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N-acetylglucosamine-decorated structures. Here, we tracked the localization of N-acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7- and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N-acetylglucosamine- and N-acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall.IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall.

Wheat germ agglutinin-conjugated fluorescent pH sensors for visualizing proton fluxes.

Small-molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes, because they bind to the cell's glycocalyx. Here, we describe the derivatization of WGA with a pH-sensitive rhodamine fluorophore (pHRho; pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform and did not appreciably alter the voltage gating of glycosylated ion channels, and the extracellular changes in pH correlated with proton channel activity. Using single-plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small-molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and nonelectrogenic events on the extracellular side of the plasma membrane.

Detection and phenotyping of extracellular vesicles by size exclusion chromatography coupled with on-line fluorescence detection.

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

Streptavidin-exposed magnetic nanoparticles for lectin magnetic separation (LMS) of Staphylococcus aureus prior to three quantification strategies.

A lectin magnetic separation (LMS) method for Staphylococcus aureus (S. aureus) was developed with the aim to improve the efficiency of magnetic nanoparticles and to expand the scope of bacterial recognition. Poly(ethylene glycol) (PEG)-mediated magnetic nanoparticles modified with streptavidin (MNP-PEG-SA) were synthesized and then applied to a two-step LMS based on the use of wheat germ agglutinin (WGA). Three specific methods for S. aureus detection (suitable for different requirements including detection time and sensitivity) were designed. The new LMS has improved anchoring efficiency (compared to two-step LMS methods) and requires a reduced number of magnetic particles. The Baird-Parker (B-P) method can detect S. aureus with a detection limit of 3 × 100 CFU·mL-1 within 15 h; the polymerase chain reaction (PCR) method can be finished within 4 h, with the lowest detection limit (LOD) of 3 × 102 CFU·mL-1. The LOD of HRP-pig IgG-based colorimetric method is 3 × 105 CFU·mL-1, and the method only lasts for 2 h. If combined with specific detection methods, it meets different needs for rapid detection of S. aureus. Graphical abstractSchematic representation of lectin magnetic separation (LMS) based on biotin-wheat germ agglutinin (WGA) and poly (ethylene glycol) (PEG)-mediated streptavidin-modified magnetic nanoparticles (MNP-PEG-SA) and three different quantification strategies (including B-P culture assay, PCR assay, and colorimetric assay) for S. aureus.