Interaction of lectin Sambucus nigra with sialylated trisaccharides in the presence of osmolytes. Chronopotentiometric sensing.

Trisaccharides bind to their interaction partners-lectins relatively weakly, which makes detection of their complexes challenging. In this work, we show that an osmolyte presence improves the distinguishing complexes of lectin Sambucus nigra with trisialyllactoses with various binding affinities. The addition of osmolyte, non-binding sugar mannose significantly improved the precision of binding experiments performed using chronopotentiometric stripping at the electrode surface and fluorescence analysis in solution. Osmolytes minimized nonspecific interactions between binding sugar and lectin. Obtained findings can be utilized in any in vitro methods studying interactions of carbohydrates, respectively their conjugates with proteins. The study of carbohydrate interactions appears important since they play essential roles in a variety of biological processes including carcinogenesis.

Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.

Chemical Characterization of Sambucus nigra L. Flowers Aqueous Extract and Its Biological Implications.

The main goal of this study was to chemically characterize an aqueous S. nigra flower extract and validate it as a bioactive agent. The elderflower aqueous extraction was performed at different temperatures (50, 70 and 90 °C). The extract obtained at 90 °C exhibited the highest phenolic content and antiradical activity. Therefore, this extract was analyzed by GC-MS and HPLC-MS, which allowed the identification of 46 compounds, being quercetin and chlorogenic acid derivatives representative of 86% of the total of phenolic compounds identified in hydrophilic fraction of the aqueous extract. Naringenin (27.2%) was the major compound present in the lipophilic fraction. The antiproliferative effects of the S. nigra extract were evaluated using the colon cancer cell lines RKO, HCT-116, Caco-2 and the extract's antigenotoxic potential was evaluated by the Comet assay in RKO cells. The RKO cells were the most susceptible to S. nigra flower extract (IC50 = 1250 µg mL-1). Moreover, the extract showed antimicrobial activity against Gram-positive bacteria, particularly Staphylococcus aureus and S. epidermidis. These results show that S. nigra-based extracts can be an important dietary source of bioactive phenolic compounds that contribute to health-span improving life quality, demonstrating their potential as nutraceutical, functional foods and/or cosmetic components for therapeutic purposes.

A glutaminyl cyclase-catalyzed α-synuclein modification identified in human synucleinopathies.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79-α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may-in analogy to pGlu-Aß peptides in Alzheimer's disease-act as a seed for pathogenic protein aggregation.

Mass Spectrometry-Based Shotgun Glycomics for Discovery of Natural Ligands of Glycan-Binding Proteins.

Glycans attached to lipids and membrane-bound and secreted proteins and peptides mediate many important physiological and pathophysiological processes through interactions with glycan-binding proteins (GBPs). However, uncovering functional glycan ligands is challenging due to the large number of naturally occurring glycan structures, the limited availability of glycans in their purified form, the low affinities of GBP-glycan interactions, and limitations in existing binding assays. This work explores the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening libraries of N-glycans derived from natural sources. The assay was tested by screening a small-defined library of complex N-glycans at equimolar concentrations against plant and human GBPs with known specificities for either α2-3- or α2-6-linked sialosides, with affinities in the millimolar to micromolar range. Validation experiments, performed in negative ion mode, revealed that bound N-glycan ligands are readily released, as intact deprotonated ions, from GBPs in the gas phase using collision-induced dissociation. Moreover, the relative abundances of the released ligands closely match their solution affinities. The results obtained for a natural N-glycan library produced from cultured immune cells serve to highlight the ease with which CaR-ESI-MS can screen complex mixtures of N-glycans for interactions. Additionally, scaling the relative abundances of released glycan ligands according to their relative abundances in solution, as determined by hydrophilic interaction-ultrahigh-performance liquid chromatography of the fluorescently labeled library, allows the relative affinities of glycan ligands to be ranked.

Lectin-Based Method for Deciphering Human Milk IgG Sialylation.

In light of the immunoprotective function of human milk and the incontestable impact of IgG glycosylation on its immune functions, characterization of the sialylation profile of human milk IgG is needed. Lectins as a molecular probe were applied in lectin-IgG-ELISA to analyze the sialylation and galactosylation pattern of skim milk IgG of mothers who delivered at term and prematurely. Well-defined biotinylated lectins were used: Maackia amurensis II (MAA II), Sambucus nigra (SNA), Ricinus communis I (RCA I), and Griffonia simplicifolia II (GSL II) specific to α2,3-Neu5Ac, α2,6-Neu5Ac, Gal(ß1,4)GlcNAc, and agalactosylated glycans, respectively. The sialylation pattern of milk IgG differs qualitatively and quantitatively from maternal plasma IgG and is related to lactation stage and perinatal risk factors. Expression of MAA-, SNA-, and GSL-reactive glycotopes on term milk IgG showed a positive correlation with milk maturation from days 1 to 55. Preterm birth was associated with an increase of MAA-reactive and a decrease of RCA-reactive IgG glycotopes. Moreover, higher SNA- and GSL-reactive and lower RCA-reactive glycoform levels of milk IgG were associated with infection of lactating mothers. Application of a specific and simple method, lectin-IgG-ELISA, reveals the sialylation pattern of milk IgG over milk maturation. However, further investigations are needed in this area.

Engineering of spectator glycocalyx structures to evaluate molecular interactions at crowded cellular boundaries.

In the mucosal epithelium, the cellular glycocalyx can project tens to hundreds of nanometers into the extracellular space, erecting a physical barrier that provides protective functions, mediates the exchange of nutrients and regulates cellular interactions. Little is understood about how the physical properties of the mucosal glycocalyx influence molecular recognition at the cellular boundary. Here, we report the synthesis of PEG-based glycopolymers with tunable glycan composition, which approximate the extended architecture of mucin glycoproteins, and tether them to the plasma membranes of red blood cells (RBC) to construct an artificial mucin brush-like glycocalyx. We evaluated the association of two lectins, ConA and SNA, with their endogenous glycan ligands on the surface of the remodelled cells. The extended glycocalyx provided protection against agglutination of RBCs by both lectins; however, the rate and magnitude of ConA binding were attenuated to a greater degree in the presence of the glycopolymer spectators compared to those measured for SNA. The different sensitivity of ConA and SNA to glycocalyx crowding likely arises from the distinct presentation of their mannoside and sialoside receptors, respectively, within the native RBC glycocalyx.

Staining for viability testing, germination and maturation of Sambucus nigra L. pollen in vitro.

Freshly released pollen of black elderberry (Sambucus nigra L.) was incubated under various culture conditions until germination was achieved. Optimal conditions for germination were determined and used for maturation of unicellular microspores in vitro. Staining with 5-diphenyltetrazolium bromide, propidium iodide and iodine potassium iodide was used to assess pollen viability, nuclear phase and maturation, respectively. The germination rate was highest when fresh pollen was agitated at 40 rpm in Petri dishes containing a liquid medium consisting of Brewbaker and Kwack salts, 15% (w/v) sucrose, 500 mg/l MES sodium salt, at pH 5.0; germination reached nearly 70% after only 1 h in culture. Under these conditions, and with addition of 200 mg/l glutamine, 260 mg/l cytidine and 500 mg/l uridine, uninucleate microspores developed into mature pollen at a 12% germination rate. Our report is the first demonstration of maturation of S. nigra microspores in vitro.

The higher the better? Differences in phenolics and cyanogenic glycosides in Sambucus nigra leaves, flowers and berries from different altitudes.

BACKGROUND: Elderberry (Sambucus nigra L.) possesses high antioxidant activity and has been used to treat numerous medicinal disorders. In addition to their antioxidant properties, elderberry parts accumulate toxic cyanogenic glycosides (CGG). It has been proven that altitude influences the biosynthesis of many secondary metabolites. In the present study we investigated the change of phenolics and CGG in elder leaves, flowers, and berries induced by different altitudes and locations. RESULTS: The data indicate that the accumulation of CGG and phenolics is affected by the altitude of the growing site. An increase of anthocyanin content was recorded in elder berries collected at higher elevations in both locations. Fruit collected at the foothills of location 2 contained 3343 µg g-1 anthocyanins as opposed to fruit from the hilltop, which contained 7729 µg g-1 . Elder berries contained the lowest levels of harmful CGG compared to other analysed plant parts. However, more cyanogenic glycosides were always present in plant parts collected at the hilltop. Accordingly, berries accumulated 0.11 µg g-1 CGG at the foothills and 0.59 µg g-1 CGG at the hilltop. CONCLUSION: Elder berries and flowers collected at the foothill were characterised by the lowest levels of both beneficial (phenolics) and harmful compounds (CGG) and are suitable for moderate consumption. © 2016 Society of Chemical Industry.

Shifts in plant foliar and floral metabolomes in response to the suppression of the associated microbiota.

BACKGROUND: The phyllospheric microbiota is assumed to play a key role in the metabolism of host plants. Its role in determining the epiphytic and internal plant metabolome, however, remains to be investigated. We analyzed the Liquid Chromatography-Mass Spectrometry (LC-MS) profiles of the epiphytic and internal metabolomes of the leaves and flowers of Sambucus nigra with and without external antibiotic treatment application. RESULTS: The epiphytic metabolism showed a degree of complexity similar to that of the plant organs. The suppression of microbial communities by topical applications of antibiotics had a greater impact on the epiphytic metabolome than on the internal metabolomes of the plant organs, although even the latter changed significantly both in leaves and flowers. The application of antibiotics decreased the concentration of lactate in both epiphytic and organ metabolomes, and the concentrations of citraconic acid, acetyl-CoA, isoleucine, and several secondary compounds such as terpenes and phenols in the epiphytic extracts. The metabolite pyrogallol appeared in the floral epiphytic community only after the treatment. The concentrations of the amino acid precursors of the ketoglutarate-synthesis pathway tended to decrease in the leaves and to increase in the foliar epiphytic extracts. CONCLUSIONS: These results suggest that anaerobic and/or facultative anaerobic bacteria were present in high numbers in the phyllosphere and in the apoplasts of S. nigra. The results also show that microbial communities play a significant role in the metabolomes of plant organs and could have more complex and frequent mutualistic, saprophytic, and/or parasitic relationships with internal plant metabolism than currently assumed.

A switched catalysis qualified sealers capped one-step synthesis biocompatibility bimetallic scaffold film for Neu5Acα(2-6)Gal ß MP Glycoside specific detection.

In this work, a novel label-free biosensor was designed for the sensitive and selective determination of Neu5Acα(2-6)Gal ß MP Glycoside using AuPt-PPy(polypyrrole) conductive nanocomposite film as the sensor platform. The introduced AuPt-PPy nanocomposite provided a large surface area for the immobilization of Sambucus nigra agglutinis (SNA) through a coupling agent for specifically recognizing analytes and exhibited high electrocatalytic activity toward the reduction of hydrogen peroxide (H2O2) as an analytical signal. Subsequently, to block the non-specific sites of the modified electrode, GOx was employed instead of the usual sealers. Most importantly, in the presence of glucose, these localized GOx further enhanced the electrochemical signal, which was achieved by the efficient catalysis of glucose. This study is the first that demonstrates the specific detection of Neu5Acα(2-6)Gal ß MP Glycoside using AuPt-PPy as the electrocatalytic. Under optimal conditions, the electrochemical biosensor exhibited a wide linear range of 0.01 pgmL(-1)-800 ngmL(-1) with a low detection limit of 0.003 pgmL(-1) (S/N=3), due to the affinity between SNA and Neu5Acα(2-6)Gal ß MP Glycoside. Therefore, the co-catalysis signal amplification approach has considerable potential in clinical applications and is suitable for the quantification of other biomarkers.

Total phenolic contents, antioxidant activities, and lipid fractions from berry pomaces obtained by solid-state fermentation of two Sambucus species with Aspergillus niger.

The aim of this study was to investigate the effect of solid-state fermentation (SSF) by Aspergillus niger on phenolic contents and antioxidant activity in Sambucus nigra L. and Sambucus ebulus L. berry pomaces. The effect of fermentation time on the total fats and major lipid classes (neutral and polar) was also investigated. During the SSF, the extractable phenolics increased with 18.82% for S. ebulus L. and 11.11% for S. nigra L. The levels of antioxidant activity of methanolic extracts were also significantly enhanced. The HPLC-MS analysis indicated that the cyanidin 3-sambubioside-5-glucoside is the major phenolic compound in both fermented Sambucus fruit residues. In the early stages of fungal growth, the extracted oils (with TAGs as major lipid fraction) increased with 12% for S. nigra L. and 10.50% for S. ebulus L. The GC-MS analysis showed that the SSF resulted in a slight increase of the linoleic and oleic acids level.

The distribution of sialic acid receptors of avian influenza virus in the reproductive tract of laying hens.

The susceptibility of the host to influenza virus is determined by the distribution of the sialic acid (SA) receptors on host cell membrane. Avian influenza virus (AIV) preferentially binds to SA α-2,3-galactose (SA α2,3-gal) linked receptors, while human strains bind to sialic acid α2,6-galactose (SA α2,6-gal) linked receptors. Here, we describe the SA patterns and distributions in the reproductive tract of hens by employing two specific lectins, Maackia amurensis agglutinin (MAA) for SA α2,3-gal and sambucus nigra agglutinin (SNA) for SA α 2,6-gal receptors. Our results revealed that both SA α2,3-gal and SA α2,6-gal receptors exist in the reproductive tract of hens, including magnum, isthmus, uterus and vagina except for infundibulum. The distribution of SAα-2,3-gal receptor was more abundantly in the columnar epithelium cells of magnum, isthmus and uterus. Only minimal positive results for SA α-2,6-gal receptors were detected in the columnar epithelium cells of magnum, isthmus, uterus and vagina. Furthermore, AIV in tissues of the reproductive tract tissues of laying hens were detected by SYBR green-based reverse transcription and polymerase chain reaction (RT-PCR). Results showed that both viral loads and pathological changes in different parts of the reproductive tract were positively correlated with the expression of both receptors. Our results revealed that the reproductive tract of hens may provide an environment for the replication of both avian and human influenza viruses.

Glycoprofiling as a novel tool in serological assays of systemic sclerosis: a comparative study with three bioanalytical methods.

Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient's quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics.

Construction and validation of a Sambucus nigra biosensor for cancer-associated STn antigen.

A label-free electrochemical impedance spectroscopy biosensor for selective detection and discrimination of the cancer-associated sialyl-Tn (STn) antigen was developed by using Sambucus nigra agglutinin type I (SNA-I) as the recognition element. The SNA-I biosensor was constructed by immobilizing the lectin on screen-printed gold electrodes. The formation of a complex between SNA-I and STn-containing glycoproteins (transferrin and bovine submaxillary mucin) was monitored by measuring the impedance increase of the biosensor. The increase in electron transfer resistance was linearly proportional to the concentration of the glycoproteins up to 70 ng of transferrin and 40 ng of bovine submaxillary mucin, with a limit of detection of 20 ng for transferrin. Albumin, the most abundant serum protein, did not interfere in the detection of the STn-glycoproteins up to a concentration of 0.2 mg ml(-1). The developed lectin-based biosensor was used to evaluate the STn-expression in serum samples and discriminate samples from healthy individuals and patients with different types of malignant tumors, mostly carcinomas, where the increased expression of STn aberrant glycans is well established. This work demonstrates the feasibility of employing SNA-I to selectively recognize the STn epitope in glycoproteins and the use of the constructed biosensor was effective in the analysis of serum samples with the ability to discriminate in a fast way between cancer and healthy status. The proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated STn glycan expression in serum for diagnosis and therapy monitoring.

Assessment of extracts of Helichrysum arenarium, Crataegus monogyna, Sambucus nigra in photoprotective UVA and UVB; photostability in cosmetic emulsions.

The aim of our study was to investigate the photoprotective activity and photostability efficacy of sunscreen formulations containing Helichrysum arenarium, Sambucus nigra, Crataegus monogyna extracts and their combination. UV transmission of the emulsion films was performed by using diffuse transmittance measurements coupling to an integrating sphere. In vitro photoprotection and photostability efficacy were evaluated according to the following parameters: sun protection factor (SPF), UVA protection factor (PF-UVA), UVA/UVB ratio and critical wavelength (λc) before and after UV irradiation. The results obtained show that the formulations containing polyphenols fulfill the official requirements for sunscreen products due to their broad spectrum of UV protection combined with their high photostability and remarkable antioxidant properties. Therefore H. arenarium, S. nigra, C. monogyna extracts represent useful additives for cosmetic formulation.

Use of glycan microarrays to explore specificity of glycan-binding proteins.

Microarrays of defined glycans represent a high throughput approach to determining the specificity of lectins, or more generally glycan-binding proteins (GBPs). The utility of a glycan microarray is directly related to the number and variety of the glycans available on the printed surface for interrogation by GBPs. The Consortium for Functional Glycomics (CFG), funded by the National Institute of General Medical Sciences (NIGMS), has generated a glycan microarray available to the public as an investigator-driven resource, where hundreds of GBPs have been analyzed. Here we describe the methods generally used by the CFG to prepare glycan arrays and interrogate them with GBPs. We also describe our new approach to normalizing glycan microarray data derived from concentration-dependent analyses of GBP binding, and the application of this approach with the plant lectin Sambucus nigra agglutinin (SNA-I) and human galectin-8. The use of glycan microarrays with this approach readily generates a prediction of the glycan determinants required for high affinity binding by a GBP.

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra.

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.

Expression of Sambucus nigra agglutinin (SNA-I') from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species.

Tobacco plants (Nicotiana tabacum cv Samsun NN) have been transformed with the gene encoding the type-2 ribosome-inactivating protein (RIP) SNA-I' from elderberry (Sambucus nigra) under the control of the Cauliflower Mosaic Virus 35S promoter. Previous research confirmed that these plants synthesize, correctly process and assemble a fully active RIP. Variability in protein expression was observed within the transgenic lines. The effects of the type-2 RIP SNA-I' delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae) and Lepidoptera, the beet armyworm (Spodoptera exigua). In the experiment with aphids, significant effects were observed on the life parameters, such as survival, intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring, whereas with caterpillars significant reduction in fresh weight as well as retardation in development were observed. In addition, significant increases in mortality were noted for insects fed on the transgenic lines as compared to wild type plants. This information provides further support for RIPs having a role in plant resistance to insect pest species.

Fabrication and characterization of a sialoside-based carbohydrate microarray biointerface for protein binding analysis with surface plasmon resonance imaging.

Monitoring multiple biological interactions in a multiplexed array format has numerous advantages. However, converting well-developed surface chemistry for spectroscopic measurements to array-based high-throughput screening is not a trivial process and often proves to be the bottleneck in method development. This paper reports the fabrication and characterization of a new carbohydrate microarray with synthetic sialosides for surface plasmon resonance imaging (SPRi) analysis of lectin-carbohydrate interactions. Contact printing of functional sialosides on neutravidin-coated surfaces was carried out and the properties of the resulting elements were characterized by fluorescence microscopy and atomic force microscopy (AFM). Sambucus nigra agglutinin (SNA) was deposited on four different carbohydrate functionalized surfaces and differential binding was analyzed to reveal affinity variation as a function of headgroup sialic acid structures and linking bonds. SPRi studies indicated that this immobilization method could result in high quality arrays with RSD < 5% from array element to array element, superior to the conventional covalent linkage used for protein cholera toxin (CT) in a comparison experiment, which yields nonuniform array elements with RSD > 15%. Multiplexed detection of SNA/biotinylated sialoside interactions on arrays up to 400 elements has been performed with good data correlation, demonstrating the effectiveness of the biotin-neutravidin-based biointerface to control probe orientation for reproducible and efficient protein binding to take place. Additionally, the regeneration of the array surface was demonstrated with a glycine stripping buffer, rendering this interface reusable. This in-depth study of array surface chemistry offers useful insight into experimental conditions that can be optimized for better performance, allowing many different protein-based biointeractions to be monitored in a similar manner.