Papillae revisited and the nature of the adhesive secreting collocytes.

Ascidian papillae (palps) constitute a transient sensory adhesive organ that assures larval settlement and the onset of metamorphosis to the filterfeeding adult. Despite the importance of papillae for the ascidian development, their cellular composition is only roughly described. For Ciona intestinalis/robusta, a clear definition of cell numbers and discriminative molecular markers for the different cell types is missing. While some attention was given to neural cell types and their connectivity little is known about the adhesive producing collocytes. We converge serial-section electron microscopy and confocal imaging with various marker combinations to document the 3D organization of the Ciona papillae. We show the papillar development with 4 axial columnar cells (ACCs), 4 lateral primary sensory neurons (PSNs) and 12 central collocytes (CCs). We propose molecular markers for each cell type including novel ones for collocytes. The subcellular characteristics are suggestive of their role in papillar function: the ACCs featuring apical protrusions and microvilli, also contain neuroactive and endocytic vesicles indicative of a chemosensory role. They are clearly distinct from the ciliated glutamatergic PSNs. CCs encircle the ACCs and contain microvilli, small endocytic vesicles and notably a large numbers of adhesive granules that, according to element analysis and histochemistry, contain glycoproteins. Interestingly, we detect two different types of collocyte granules, one of them containing fibrous material and larger quantities of polysaccharides. Consistently, carbohydrate specific lectins label the papillar apex, the granules within CCs and the adhesive plaques upon larval attachment. We further propose CCs to derive from an evolutionary ancient neurosecretory cell type. Our findings contribute to understanding the development of the anterior ('new head') region of the Ciona larva and notably the adhesive secreting cells which has implications for developmental biology, cell differentiation and evolution, but also bioadhesion.

Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle.

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

Identification of sperm equatorial segment protein 1 in the acrosome as the primary binding target of peanut agglutinin (PNA) in the mouse testis.

Peanut agglutinin (PNA), a plant lectin protein that recognizes the galactose ß (1 -> 3) N-acetylgalactosamine carbohydrate sequence, has been widely used as a sperm acrosome-specific marker; however, the acrosomal glycoproteins that specifically bind to PNA have yet to be identified. We herein purified and identified PNA-binding glycoproteins in the mouse testis using biotinylated PNA and streptavidin-coupled magnetic beads, and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. In six repeated experiments, sperm equatorial segment protein 1 (SPESP1) was detected most frequently as a PNA-binding glycoprotein, followed by dipeptidase 3, proacrosin-binding protein, and acrosin prepropeptide. The identification of SPEPS1 in the testis lysate and its PNA-bound fraction was verified with lectin and Western blot analyses, and the co-localization of PNA and SPEPS1 in acrosomes was confirmed with lectin- and immunohistochemistry. Since the PNA reactivity of sperm acrosomes was observed not only in normal mice, but also in SPESP1-deficient mice, although at lower levels, PNA was also considered to bind to other candidate glycoproteins. The present study identified SPESP1 in the acrosome as the primary binding target of PNA in the mouse testis. Further defining the specific lectin-glycoprotein relationships in individual cells will enhance the value of lectin histochemistry.

The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study.

The vision of multivalency as a strategy limited to achieve affinity enhancements between a protein receptor and its putative sugar ligand (glycotope) has proven too simplistic. On the one hand, binding of a glycotope in a dense glycocalix-like construct to a lectin partner has been shown to be sensitive to the presence of a third sugar entity (heterocluster effect). On the other hand, several carbohydrate processing enzymes (glycosidases and glycosyltransferases) have been found to be also responsive to multivalent presentations of binding partners (multivalent enzyme inhibition), a phenomenon first discovered for iminosugar-type inhibitory species (inhitopes) and recently demonstrated for multivalent carbohydrate constructs. By assessing a series of homo- and heteroclusters combining α-d-glucopyranosyl-related glycotopes and inhitopes, it was shown that multivalency and heteromultivalency govern both kinds of events, allowing for activation, deactivation or enhancement of specific recognition phenomena towards a spectrum of lectin and glycosidase partners in a multimodal manner. This unified scenario originates from the ability of (hetero)multivalent architectures to trigger glycosidase binding modes that are reminiscent of those harnessed by lectins, which should be considered when profiling the biological activity of multivalent architectures.

Adhesive organ regeneration in Macrostomum lignano.

BACKGROUND: Flatworms possess pluripotent stem cells that can give rise to all cell types, which allows them to restore lost body parts after injury or amputation. This makes flatworms excellent model systems for studying regeneration. In this study, we present the adhesive organs of a marine flatworm as a simple model system for organ regeneration. Macrostomum lignano has approximately 130 adhesive organs at the ventral side of its tail plate. One adhesive organ consists of three interacting cells: one adhesive gland cell, one releasing gland cell, and one modified epidermal cell, called an anchor cell. However, no specific markers for these cell types were available to study the regeneration of adhesive organs. RESULTS: We tested 15 commercially available lectins for their ability to label adhesive organs and found one lectin (peanut agglutinin) to be specific to adhesive gland cells. We visualized the morphology of regenerating adhesive organs using lectin- and antibody staining as well as transmission electron microscopy. Our findings indicate that the two gland cells differentiate earlier than the connected anchor cells. Using EdU/lectin staining of partially amputated adhesive organs, we showed that their regeneration can proceed in two ways. First, adhesive gland cell bodies are able to survive partial amputation and reconnect with newly formed anchor cells. Second, adhesive gland cell bodies are cleared away, and the entire adhesive organ is build anew. CONCLUSION: Our results provide the first insights into adhesive organ regeneration and describe ten new markers for differentiated cells and tissues in M. lignano. The position of adhesive organ cells within the blastema and their chronological differentiation have been shown for the first time. M. lignano can regenerate adhesive organs de novo but also replace individual anchor cells in an injured organ. Our findings contribute to a better understanding of organogenesis in flatworms and enable further molecular investigations of cell-fate decisions during regeneration.

Fabrication of Electrochemical Model Influenza A Virus Biosensor Based on the Measurements of Neuroaminidase Enzyme Activity.

Neuroaminidase (NA) enzyme is a kind of glycoprotein that is found on the influenza A virus. During infection, NA is important for the release of influenza virions from the host cell surface together with viral aggregates. It may also be involved in targeting the virus to respiratory epithelial cells. In this study, a model electrochemical influenza A viral biosensor in which receptor-binding properties have been based on NA was developed for the first time. The biosensor's working principle is based on monitoring the interactions between fetuin A and NA enzyme. The assay was monitored step by step by using electrochemical impedance spectroscopy.

Photoaffinity labeling studies of the carbohydrate-binding proteins with different affinities.

Photoaffinity labeling has been used as a promising approach to detection and isolation of carbohydrate-binding proteins, which are typically characterized by low binding affinity and selectivity. When there are several specific binding proteins, it is desirable that a photoaffinity probe is capable of simultaneously crosslinking them and that the crosslinking yields depend on the relative binding affinities. In this study, we describe the design and synthesis of carbohydrate photoaffinity probes and their ability to capture lectins of different binding affinities.

Comparison of the reactivity of carbohydrate photoaffinity probes with different photoreactive groups.

A judicious choice of photoreactive group is critical in successful photoaffinity labeling studies of small molecule-protein interactions. A set of carbohydrate-based photoaffinity probes was prepared to compare the effects of three major photoreactive groups on the efficiency and selectivity of crosslinking a binding protein with low affinity. We showed that, despite the low crosslinking yield, the diazirine probe displayed the high ligand-dependent reactivity consistent with the ideal mechanism of photoaffinity labeling. Moreover, we demonstrated that, among the three photoreactive groups, only the diazirine probe achieved highly selective crosslinking of a low-affinity binding protein in cell lysate.

Spontaneous activity promotes synapse formation in a cell-type-dependent manner in the developing retina.

Spontaneous activity is thought to regulate synaptogenesis in many parts of the developing nervous system. In vivo evidence for this regulation, however, is scarce and comes almost exclusively from experiments in which normal activity was reduced or blocked completely. Thus, whether spontaneous activity itself promotes synaptogenesis or plays a purely permissive role remains uncertain. In addition, how activity influences synapse dynamics to shape connectivity and whether its effects among neurons are uniform or cell-type-dependent is unclear. In mice lacking the cone-rod homeobox gene (Crx), photoreceptors fail to establish normal connections with bipolar cells (BCs). Here, we find that retinal ganglion cells (RGCs) in Crx⁻/⁻ mice become rhythmically hyperactive around the time of eye opening as a result of increased spontaneous glutamate release from BCs. This elevated neurotransmission enhances synaptogenesis between BCs and RGCs, without altering the overall circuit architecture. Using live imaging, we discover that spontaneous activity selectively regulates the rate of synapse formation, not elimination, in this circuit. Reconstructions of the connectivity patterns of three BC types with a shared RGC target further revealed that neurotransmission specifically promotes the formation of multisynaptic appositions from one BC type without affecting the maintenance or elimination of connections from the other two. Although hyperactivity in Crx⁻/⁻ mice persists, synapse numbers do not increase beyond 4 weeks of age, suggesting closure of a critical period for synaptic refinement in the inner retina. Interestingly, despite their hyperactivity, RGC axons maintain normal eye-specific territories and cell-type-specific layers in the dorsal lateral geniculate nucleus.

Quantum dot based fluorometric detection of cancer TF-antigen.

Cancer is a major global health challenge that would benefit from advances in screening methods for early detection that are rapid and low cost. TF-antigen is a tumor-associated antigen displayed on cell surface proteins of a high percentage of human carcinomas. Here we present a fluorometric bioassay for TF-antigen (galactose-ß-(1→3)-N-acetyl-d-galactosamine) that utilizes quantum dot (QD) technology coupled with magnetic beads for rapid detection of TF-antigen at high sensitivity (10(-7) M range). In the competitive bioassay, 4-aminophenyl ß-d-galactopyranoside (4-APG) conjugated to QDs competes with TF-antigen for binding sites on peanut agglutinin (PNA) that is immobilized on magnetic beads. The bioassay is specific and ultrasensitive in the environment of complex protein mixtures, demonstrating its potential applicability for the screening of clinical samples.

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy.

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5-70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.

Probing the nature of the cluster effect observed with synthetic multivalent galactosides and peanut agglutinin lectin.

We designed a set of multi-galactosides with valencies ranging from one to seven and different spacer-arm lengths. The compounds display a high structural homology for a strict assessment of multivalent phenomena. The multimers were first evaluated by an enzyme-linked lectin assay (ELLA) toward the peanut agglutinin (PNA). The binding affinity was shown to be dependent on the spacer-arm length, and cluster effects were observed for the galactosides bearing the shortest and the longest linkers. The latter compounds were shown to be much more potent PNA cross-linkers in a "sandwich assay". Dynamic light scattering (DLS) experiments also revealed the formation of soluble aggregates between heptavalent derivatives with medium or long linkers and the labeled PNA. ELLA experiments performed with valency-controlled clusters and labeled lectins are therefore not always devoid from aggregative processes. The precise nature of the multivalent interaction observed by ELLA for the compounds bearing the shortest linkers, which are unable to form PNA aggregates, was further investigated by atomic force microscopy (AFM). The galactosides were grafted onto the tip of a cantilever and the PNA lectin onto a gold surface. Similar unbinding forces were registered when the valency of the ligands was increased, thus showing that the multimers cannot interact more strongly with PNA. Multiple binding events to the PNA were also never observed, thus confirming that a chelate binding mode does not operate with the multivalent galactosides, probably because the linkers are too short. Altogether, these results suggest that the cluster effect that operates in ELLA with the multimers is not related to additional PNA stabilizations and can be ascribed to local concentration effects that favor a dynamic turnover of the tethered galactosides in the PNA binding sites.

Fullerene-sp2-iminosugar balls as multimodal ligands for lectins and glycosidases: a mechanistic hypothesis for the inhibitory multivalent effect.

Concerted functioning of lectins and carbohydrate-processing enzymes, mainly glycosidases, is essential in maintaining life. It was commonly assumed that the mechanisms by which each class of protein recognizes their cognate sugar partners are intrinsically different: multivalency is a characteristic feature of carbohydrate-lectin interactions, whereas glycosidases bind to their substrates or substrate-analogue inhibitors in monovalent form. Recent observations on the glycosidase inhibitory potential of multivalent glycomimetics have questioned this paradigm and led to postulate an inhibitory multivalent effect. Here the mechanisms at the origin of this phenomenon have been investigated. A D-gluco-configured sp(2)-iminosugar glycomimetic motif, namely 1-amino-5N,6O-oxomethylydenenojirimycin (1N-ONJ), behaving, simultaneously, as a ligand of peanut agglutinin (PNA) lectin and as an inhibitor of several glycosidases, has been identified. Both the 1N-ONJ-lectin- and 1N-ONJ-glycosidase-recognition processes have been found to be sensitive to multivalency, which has been exploited in the design of a lectin-glycosidase competitive assay to explore the implication of catalytic and non-glycone sites in enzyme binding. A set of isotropic dodecavalent C60-fullerene-sp(2)-iminosugar balls incorporating matching or mismatching motifs towards several glycosidases (inhitopes) was synthesized for that purpose, thereby preventing differences in binding modes arising from orientational preferences. The data supports that: 1) multivalency allows modulating the affinity and selectivity of a given inhitope towards glycosidases; 2) multivalent presentation can switch on the inhibitory capacity for some inhitope-glycosidase pairs, and 3) interactions of the multivalent inhibitors with non-glycone sites is critical for glycosidase recognition. The ensemble of results point to a shift in the binding mode on going from monovalent to multivalent systems: in the first case a typical ''key-lock'' model involving, essentially, the high-affinity active site can be assumed, whereas in the second, a lectin-like behavior implying low-affinity non-glycone sites probably operates. The differences in responsiveness to multivalency for different glycosidases can then be rationalized in terms of the structure and accessibility of the corresponding carbohydrate-binding regions.

Cone outer segment and Müller microvilli pericellular matrices provide binding domains for interphotoreceptor retinoid-binding protein (IRBP).

The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. In the IPM, retinoids and fatty acids are carried by interphotoreceptor retinoid-binding protein (IRBP). The fact that much of the retina's IRBP can be extracted by saline wash has led to the notion that IRBP does not bind to the retina, but freely distributes itself within the subretinal space. In this study, we challenge this idea by asking if there are specialized IPM domains that bind IRBP, perhaps facilitating its ability to target delivery/uptake of its ligands. Xenopus is an ideal animal model to study the role of the IPM in RPE-photoreceptor interactions. Here, we took advantage of the large size of its photoreceptors, ability to detach the retina in light, sustainability of the retina in short term organ culture, and the availability of recombinant full-length Xenopus IRBP and antisera directed against Xenopus IRBP. We compared the distribution of wash resistant native IRBP, and that of IRBP-Alexa 647 binding in Xenopus retina. IRBP and cone opsin were localized using anti-Xenopus IRBP serum, and monoclonal COS-1 respectively. Cone matrix sheath proteoglycans were localized with wheat germ agglutinin (WGA), and diffuse IPM proteoglycans with peanut agglutinin (PNA). Wholemounts and frozen sections were compared by immunofluorescence from retinas detached under Ringer's followed by additional washes, or detached directly under 4% paraformaldehyde without Ringer's wash. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). Immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments, filling the rod periciliary ridge, and associated with Müller microvilli. By immunofluorescence, Ringer's wash removed most of the diffuse IRBP, but not that coating the outer segments. IRBP-Alexa 647 bound to the cone outer segments and Müller villi region, and comparably less to rod outer segments. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and Alexa 647 dye alone showed no binding. Our data suggest that the pericellular matrix of the cone outer segments and Müller microvilli provide specialized domains that facilitate IRBP's functions.

Estimating glomerular number: why we do it and how.

There is currently much interest in determining the number of glomeruli, and thereby nephrons, in the kidney. Researchers have been trying to count glomeruli since the 19th century and currently four general approaches are available: (i) acid maceration; (ii) counting glomerular profiles in histological sections; (iii) model-based stereology; and (iv) design-based stereology. Although design-based stereological methods are generally considered the gold-standard method, all current methods have limitations. A new approach using magnetic resonance imaging has recently been described and may ultimately enable glomerular imaging and quantification in vivo. This report considers the advantages and disadvantages of current methods for counting glomeruli and describes the new magnetic resonance approach. In addition, a method for counting glomeruli in developing kidneys is described.

In vitro initiation of the acrosome reaction in the emu (Dromaius novaehollandiae).

1. An assessment of the efficiency of the acrosome reaction (AR) provides an important predictor of the fertilizing potential of semen and for diagnosis of the causes of infertility. A standardized protocol was therefore developed for initiation of the acrosome reaction in emu spermatozoa in vitro, and the role of CaCl2 or perivitelline membrane (PVM) proteins in determining the outcome of the reaction was investigated. 2. The acrosome reaction (assessed by FITC-PNA) was successfully induced in live spermatozoa by incubation for 2 min in NaCl-TES medium supplemented with 5 mM CaCl2. The maximum response was 32% live acrosome-reacted spermatozoa (LAR) achieved after 10 min incubation. 3. Compared to the outcome with 5 mM CaCl2 or PVM protein alone, the response was significantly better with a combination of PVM protein and CaCl2. 4. A significant variation in the percentage of LAR spermatozoa among individual males was observed. No treatment affected the percentage of dead acrosome-reacted spermatozoa. 5. The results emphasize the important role played by both PVM proteins and Ca(2+) in the in vitro initiation of the acrosome reaction.

Chromatic bipolar cell pathways in the mouse retina.

Like most mammals, mice feature dichromatic color vision based on short (S) and middle (M) wavelength-sensitive cone types. It is thought that mammals share a retinal circuit that in dichromats compares S- and M-cone output to generate blue/green opponent signals, with bipolar cells (BCs) providing separate chromatic channels. Although S-cone-selective ON-BCs (type 9 in mouse) have been anatomically identified, little is known about their counterparts, the M-cone-selective OFF-BCs. Here, we characterized cone connectivity and light responses of selected mouse BC types using immunohistochemistry and electrophysiology. Our anatomical data indicate that four (types 2, 3a/b, and 4) of the five mouse OFF-BCs indiscriminately contact both cone types, whereas type 1 BCs avoid S-cones. Light responses showed that the chromatic tuning of the BCs strongly depended on their position along the dorsoventral axis because of the coexpression gradient of M- and S-opsin found in mice. In dorsal retina, where coexpression is low, most type 2 cells were green biased, with a fraction of cells (≈ 14%) displaying strongly blue-biased responses, likely reflecting S-cone input. Type 1 cells were also green biased but did not comprise blue-biased "outliers," consistent with type 1 BCs avoiding S-cones. We therefore suggest that type 1 represents the green OFF pathway in mouse. In addition, we confirmed that type 9 BCs display blue-ON responses. In ventral retina, all BC types studied here displayed similar blue-biased responses, suggesting that color vision is hampered in ventral retina. In conclusion, our data support an antagonistically organized blue/green circuit as the common basis for mammalian dichromatic color vision.

Differential glycosylation of MUC1 and CEACAM5 between normal mucosa and tumour tissue of colon cancer patients.

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galß1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.

Microfluidic platform for the evaluation of multi-glycan expressions on living cells using electrochemical impedance spectroscopy and optical microscope.

A microfluidic platform to evaluate the expression of multi-glycans on a cell surface was developed using electrochemical impedance spectroscopy (EIS) and optical microscope technique. In the microfluidic channel, four indium tin oxide (ITO) electrodes were modified with three lectins and one passivation agent, respectively, to selectively recognize the corresponding carbohydrate epitopes on the cell surface. The binding of the cells on the electrode array was monitored by the electrochemical impedance to evaluate the expression of cell surface glycans. The excellent optical transparency of ITO electrode permitted the microscopic observation of the cell binding simultaneously to substantiate the impedance measurement. Compared with the individual technology, the double-check mode increased the sensitivity and accuracy of the assay. The experimental results using these two techniques indicated that the cell binding ability decreased in the order WGA > Con A > PNA, which was consistent with the expression difference of carbohydrate epitopes on K562 cell surface. The proposed strategy was further used for facile evaluating the variations of glycan expression on living cells in response to drugs. The consumption of cell sample for each sensing interface in the whole experiments is merely 5 × 10(3) cells. This platform offers great promise for cancer-associated glycol-biomarkers screening and further helps cancer diagnosis and treatment.

Elegant chemistry to directly anchor intact saccharides on solid surfaces used for the fabrication of bioactivity-conserved saccharide microarrays.

An easy chemical strategy was proposed and used to establish a method for direct anchoring of intact saccharides on solid surfaces with well conserved bioaffinity. The anchoring was achieved by temperature-modulated stepwise reactions with cyanuric chloride as a key linker, and was successfully applied to the fabrication of saccharide chips. To demonstrate, 15 intact reducing and nonreducing saccharides with various molecular sizes were dotted on a cyanuric-chloride-modified chip (1.0 × 1.0 cm(2)) and made to react with lectins. As expected, the anchored saccharides were capable of recognizing their target lectins, and more exciting were the perfect conservation of the specific recognizing ability of the anchored monosaccharides such as mannose, glucose, and even fructose (interacting only weakly with concanavalin A). This conservation was ascribed to the maintenance of the original structure (especially the anomeric configuration) of saccharides after immobilization and to the allowance of the anchored saccharides to rotate with and/or on the scaffold of cyanuric chloride, which makes them easily adapt to the recognition-preferred spatial position. The expected linkage of saccharides on cyanuric chloride and the maintenance of their anomeric configuration were characterized by mass spectrometry and nuclear magnetic resonance, respectively. The new method can be highlighted not only by its conservation of saccharide bioaffinity and universal applicability but also by its merits of easy manipulation or facile control of the reactions and cost-effectiveness due to the use of extremely cheap cyanuric chloride.