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Real-time detection of glycosylation on label-free cancer cell surfaces is of significance for the diagnosis and treatment of cancer. In this work, we have successfully developed a novel dynamic reversible sensor based on pH-sensitive phenylboronic esters to determine in real-time the binding kinetics of protein-carbohydrate interactions on suspension cancer cell surfaces using a quartz crystal microbalance (QCM) technique.

The plasma protein histidine-rich glycoprotein (HRG) is implicated in the polarization of macrophages to an M1 antitumoral phenotype. The broadly expressed secreted protein stanniocalcin 2 (STC2), also implicated in tumor inflammation, is an HRG interaction partner. With the aim to biochemically characterize the HRG and STC2 complex, binding of recombinant HRG and STC2 preparations to each other and to cells was explored using the quartz crystal microbalance (QCM) methodology. The functionality of recombinant proteins was tested in a phagocytosis assay, where HRG increased phagocytosis by monocytic U937 cells while STC2 suppressed HRG-induced phagocytosis. The binding of HRG to STC2, measured using QCM, showed an affinity between the proteins in the nanomolar range, and both HRG and STC2 bound individually and in combination to vitamin D3-treated, differentiated U937 monocytes. HRG, but not STC2, also bound to formaldehyde-fixed U937 cells irrespective of their differentiation stage in part through the interaction with heparan sulfate. These data show that HRG and STC2 bind to each other as well as to U937 monocytes with high affinity, supporting the relevance of these interactions in monocyte/macrophage polarity.

The plasma protein Histidine-rich glycoprotein (HRG), is implicated in macrophage polarization to an M1 antitumoral phenotype. The broadly expressed, secreted protein Stanniocalcin 2 (STC2), also implicated in tumor inflammation, is an HRG interaction partner. With the aim to biochemically characterize the HRG and STC2 complex, binding of recombinant HRG and STC2 preparations to each other and to cells was explored using quartz crystal microbalance (QCM) methodology. Protein functionality was tested in a phagocytosis assay, where HRG increased phagocytosis by monocytic U937 cells while STC2 suppressed HRG-induced phagocytosis. Binding of HRG to STC2 measured using QCM showed an affinity between the proteins in the nanomolar range, which occurred in a conformation-dependent manner. Both HRG and STC2 bound individually and in combination to vitamin D3-treated, differentiated U937 monocytes. HRG, but not STC2, also bound to formaldehyde-fixed U937 cells irrespective of their differentiation stage, involving both a high affinity interaction and binding to heparan sulfate. These data show that binding of HRG to STC2 occurs with high affinity and that HRG and STC2 bind to separate sites on U937 monocytes, suggesting that they exert their effects through distinct cell surface entities.

The quartz crystal microbalance (QCM) has been used to study how the interfacial layer of an ionic liquid dissolved in a polar oil at low weight percentages responds to changes in applied potential. The changes in surface composition at the QCM gold surface depend on both the magnitude and sign of the applied potential. The time-resolved response indicates that the relaxation kinetics are limited by the diffusion of ions in the interfacial region and not in the bulk, since there is no concentration dependence. The measured mass changes cannot be explained only in terms of simple ion exchange; the relative molecular volumes of the ions and the density changes in response to ion exclusion must be considered. The relaxation behavior of the potential between the electrodes upon disconnecting the applied potential is more complex than that observed for pure ionic liquids, but a measure of the surface charge can be extracted from the exponential decay when the rapid initial potential drop is accounted for. The adsorbed film at the gold surface consists predominantly of ionic liquid despite the low concentration, which is unsurprising given the surtactant-like structures of (some of) the ionic liquid ions. Changes in response to potential correspond to changes in the relative numbers of cations and anions, rather than a change in the oil composition. No evidence for an electric field induced change in viscosity is observed. This work shows conclusively that electric potentials can be used to control the surface composition, even in an oil-based system, and paves the way for other ion solvent studies.

12 p.-4 fig.-4 tab., Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Gal beta(1,4) Glc beta, whereas VAA recognized the Gala(1,4) Gal beta(1,4) Glc beta epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding., We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness(Grants BFU2015-70052-R, CTQ2015-64597-C2-2-P and SAF2015-66520-R), the Department of Health of the Navarra Government (ref 03/2016), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO(Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012-317297), and WntsApp (Grant PITN-GA-2013-608180).

The role of sulfur in glycosidic bonds has been evaluated using quartz crystal microbalance methodology. Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-glycosidic bonds have been developed, and the recognition properties assessed in competition binding assays with the cognate lectin concanavalin A. Mannose-presenting QCM sensors were produced using photoinitiated, nitrene-mediated immobilization methods, and the subsequent binding study was performed in an automated flow-through instrumentation, and correlated with data from isothermal titration calorimetry. The recorded Kd-values corresponded well with reported binding affinities for the O-linked dimannosides with affinities for the α1-2-linked dimannosides in the lower micromolar range. The S-linked analogs showed slightly disparate effects, where the α1-6-linked analog showed weaker affinity than the O-linked dimannoside, as well as positive apparent cooperativity, whereas the α1-2-analog displayed very similar binding compared to the O-linked structure.

In biological fluids, proteins and other biomolecules bind to the surface of nanoparticles to form a coating known as the protein corona which in turn becomes primary determinant of the nanoparticles’ fate and behaviour. Here we develop a QCM-based platform and methodology to obtain data from real-time interactions of nanoparticles with selected human plasma proteins. Polystyrene particles coated with transferrin are immobilized on QCM sensor chips and by means of a ‘sandwich’ format binding assay, specific epitopes on the particles can be quantified as measured by the increase of the sensor's resonant frequency. Cell binding experiments where adherent cells are directly grown on the sensor surface are also performed. Interaction of nanoparticles injected over the cell surface is observed only in the case of particle-transferrin complexes demonstrating that it is the nanoparticle-corona complex, rather than the native nanoparticle, “what the cell sees”, with the corona being the interface between the nanoparticle and the cellular system. Our data highlight the potential of the proposed QCM-based platform and methodology for characterization of the bio-nano-interface and tracking the interaction of nanoparticles with biological cells in the presence of a realistic milieu.

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated na&iuml, ve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

Mussel-inspired polydopamine (PDA) has been widely applied for functionalizing various material surfaces through a simple and versatile approach. Several studies have demonstrated the adhesion-promotion properties of the PDA surface for adherent cells. However, few reports are associated with suspension cells. In this study, the attachment property of suspension cells on the PDA surface was investigated for the first time, where we found that the suspension cells can be directly attached to the PDA coating without any secondary modifications. To exemplify the potential of this property, a PDA-based suspension-cell QCM biosensor was fabricated. The biosensor showed a high degree of repeatability and stability as well as low nonspecific binding to the irrelevant protein. The real-time and label-free evaluation of the binding of a diverse range of lectins on three different types of suspension cells indicated the variation of the glycosylation on these cell surfaces. Furthermore, the kinetic evaluation of the protein-carbohydrate interactions on the suspension cell surface was also performed. This work reports a new strategy for fabrication of PDA-based suspension-cell QCM biosensor via simple and efficient immobilization of suspension cells, which provides a simple way for the study of the complex molecular recognition on suspension cell surfaces.

Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface., We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grants BFU2012-36825, BFU2015-70052-R, SAF2012-31166, and SAF2015-66520-R), the Department of Health of the Navarra Government (ref 359/2012), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO (Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012-317297), and WntsApp (GA-No. 608180, FP7-PEOPLE-2013). I.K. and D.P. were funded by Marie Curie contracts from the European Commission.

A two-dimensional (2D) and a three-dimensional (3D) His-tag capture surfaces were fabricated for oriented and reversible immobilization of His-tagged proteins on quartz crystal microbalance (QCM) biosensor surfaces, which can be used for label-free and real-time detection of the interactions between His-tagged protein and its interacting protein (analyte). His-tagged proteins immobilized on the 2D His-tag capture surface maintained a higher binding activity than those immobilized on a 2D carboxyl surface via amine coupling. The 3D His-tag capture surface has about twice the amount of immobilization capacity as the 2D His-tag capture surface, which enables a higher sensitivity for detection. His-tag capture surface can be optionally regenerated to remove the His-tagged protein as well as the analyte for the next cycle of His-tagged protein immobilization, or to only selectively remove the analyte, leaving the His-tagged protein on the surface for the next cycle of analyte binding. Furthermore, the kinetic and affinity studies of the interactions between the His-tagged protein and its interacting protein were performed. This study provides an efficient way to study protein–protein interactions by oriented and reversible immobilization of His-tagged proteins on QCM biosensor surfaces.

A novel approach for preparing carbohydrate chips based on polydopamine (PDA) surface to study carbohydrate&ndash, lectin interactions by quartz crystal microbalance (QCM) biosensor instrument has been developed. The amino-carbohydrates were immobilized on PDA-coated quartz crystals via Schiff base reaction and/or Michael addition reaction. The resulting carbohydrate-chips were applied to QCM biosensor instrument with flow-through system for real-time detection of lectin&ndash, carbohydrate interactions. A series of plant lectins, including wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europaeus agglutinin I (UEA-I), soybean agglutinin (SBA), and peanut agglutinin (PNA), were evaluated for the binding to different kinds of carbohydrate chips. Clearly, the results show that the predicted lectin selectively binds to the carbohydrates, which demonstrates the applicability of the approach. Furthermore, the kinetics of the interactions between Con A and mannose, WGA and N-Acetylglucosamine were studied, respectively. This study provides an efficient approach to preparing carbohydrate chips based on PDA for the lectin&ndash, carbohydrate interactions study.

We present a method to study the interaction between biomolecules and receptors present on the cell surface. This enables studies of molecular interactions in a natural biological context. As the analyte interacts with the receptors still intact on the cell surface, the experimental data provides complete dynamics and complexity of the interaction, thereby generating highly informative data. Attana's cell-based biosensor platform can be used to obtain this information from a diverse range of interactions as described in these protocols, which detail how to grow or capture cells on a surface, how to stabilize and visualize the cells on the surface, and how to set up assays to measure detailed interaction kinetics directly on the cell surface.

Nanomedicine, nanotargeting and nanotherapeutics have in the last few years faced several difficulties in translating the promising results obtained in vitro to an in vivo scenario. The origin of this discrepancy might be found in the lack of a detailed and realistic characterization of the biological surface of nanoparticles. Despite the capability to engineer nanomaterials with a great variety and a precise control of the surface functionalization, the targeting capability is lost when the nanoparticles are embedded in complex biological media, due to the formation of a biological layer (biomolecular corona). This biological layer represents the ultimate nanoparticle surface, likely to interact with the cell machinery. Therefore, in addition to traditional nanoparticle characterization techniques, a more insightful investigation of the biomolecular corona is needed, including the capability to assess the orientation and functionality of specific key molecular features. Here we present a method for the rapid screening of exposed protein recognition motifs on the surface of nanoparticles exploiting quartz crystal microbalance (QCM). We quantify accessible functional epitopes of transferrin-coated nanoparticles and correlate them to differences in nanoparticle size and functionalization. The target recognition occurs label free in flow, thereby pushing our investigation into a more in vivo-like scenario. Our method is applicable to a wide array of nanoparticles and therefore holds the potential to become an advanced technique for the classification of all kinds of nanobioconstructs based on their biological external functionality.

When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time.

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria–host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment., We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grants BFU2012-36825, BFU2015-70052-R, SAF2012-31166, and SAF2015-66520-R), the Department of Health of the Navarra Government (ref. 359/2012), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO (Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012- 317297), and WntsApp (GA-No. 608180, FP7-PEOPLE-2013). I.K. and D.P. were beneficiaries of Marie Curie contracts from the European Commission.

We present a method to study the interaction between biomolecules and receptors present on the cell surface. This enables studies of molecular interactions in a natural biological context. As the analyte interacts with the receptors still intact on the cell surface, the experimental data provides complete dynamics and complexity of the interaction, thereby generating highly informative data. Attana's cell-based biosensor platform can be used to obtain this information from a diverse range of interactions as described in these protocols, which detail how to grow or capture cells on a surface, how to stabilize and visualize the cells on the surface, and how to set up assays to measure detailed interaction kinetics directly on the cell surface.

Analytical methods founded upon whole cell-based assays are of importance in early stage drug development and in fundamental studies of biomolecular recognition. Here we have studied the binding of the monoclonal antibody trastuzumab to human epidermal growth factor receptor 2 (HER2) on human ovary adenocarcinoma epithelial cancer cells (SKOV3) using quartz crystal microbalance (QCM) technology. An optimized procedure for immobilizing the cells on the chip surface was established with respect to fixation procedure and seeding density. Trastuzumab binding to the cell decorated sensor surface was studied, revealing a mean dissociation constant, KD, value of 7 ± 1 nM (standard error of the mean). This study provides a new perspective on the affinity of the antibody-receptor complex presented a more natural context compared to purified receptors. These results demonstrate the potential for using whole cell-based QCM assay in drug development, the screening of HER2 selective antibody-based drug candidates, and for the study of biomolecular recognition. This real time, label free approach for studying interactions with target receptors present in their natural environment afforded sensitive and detailed kinetic information about the binding of the analyte to the target.

Quartz crystal microbalance (QCM) has been extensively applied in real-time and label-free biomolecular interaction studies. However, the sensitive detection by QCM technology remains challenging, mainly due to the limited surface immobilization capacity. Here, a three-dimensional (3D) carboxymethyl dextran coated gold sensor chip surface was successfully fabricated with dextran of different molecular weight (100, 500 and 2000 kDa, respectively). To evaluate the 3D carboxymethyl dextran surface immobilization capacity, the 3D surface was used for studying antigen–antibody interactions on the QCM biosensor. The results showed that the protein immobilization capacity of the 3D carboxymethyl dextran (2000 kDa) surface exceeded more than 4 times the capacity of the 2D carboxyl surface, and 2 times the capacity of the traditional 3D carboxymethyl dextran (500 kDa) surface. Furthermore, the kinetic and affinity properties of antigen–antibody interactions were performed. Most notably, the optimized 3D carboxymethyl dextran (2000 kDa) surface could be used for small molecule detection, where the binding of biotinylated oligo (0.67 kDa) reached 8.1 Hz. The results confirmed that a 3D carboxymethyl dextran (2000 kDa) surface can be exploited for sensitive detection of low molecular weight analytes, which have great potential applications for characterizing the interactions between small molecule drugs and proteins.

Quartz crystal microbalance (QCM) methodology has been adopted to unravel important factors contributing to the “cluster glycoside effect” observed in carbohydrate-lectin interactions. Well-defined, glycosylated nanostructures of precise sizes, geometries and functionalization patterns were designed and synthesized, and applied to analysis of the interaction kinetics and thermodynamics with immobilized lectins. The nanostructures were based on Borromean rings, dodecaamine cages, and fullerenes, each of which carrying a defined number of carbohydrate ligands at precise locations. The synthesis of the Borromeates and dodecaamine cages was easily adjustable due to the modular assembly of the structures, resulting in variations in presentation mode. The binding properties of the glycosylated nanoplatforms were evaluated using flow-through QCM technology, as well as hemagglutination inhibition assays, and compared with dodecaglycosylated fullerenes and a monovalent reference. With the QCM setup, the association and dissociation rate constants and the associated equilibrium constants of the interactions could be estimated, and the results used to delineate the multivalency effects of the lectin-nanostructure interactions.

The signal enhancement properties of QCM sensors based on dynamic, biotinylated poly(acrylic acid) brushes has been studied in interaction studies with an anti-biotin Fab fragment. The poly(acrylic acid) sensors showed a dramatic increase in signal response with more than ten times higher signal than the carboxyl-terminated self-assembled monolayer surface.

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.

The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein–carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.

According to a current concept, copper canisters of thickness 0.05 m will be safe for nuclear waste containment for 100,000 years. We show that more than 1 m copper thickness might be required for 100,000 years durability based on water exposures of copper for 20 h, 7 weeks, 15 years, and 333 years. An observed evolution of hydrogen which involves heterogeneous catalysis of molecular hydrogen, first principles simulations, thermodynamic considerations and corrosion product characterization provide further evidence that water corrodes copper resulting in the formation of a copper hydroxide. These findings cast additional doubt on copper for nuclear waste containment and other important applications.

Dynamic acrylamide/acrylate polymeric brushes were synthesized at gold-plated quartz crystal surfaces. The crystals were initially coated with polystyrene-type thin films, derivatized with photolabile iniferter groups, and subsequently subjected to photoinitiated polymerization in acrylamide/acrylate monomer feeds. This surface-confined polymerization method enabled direct photocontrol over the polymerization, as followed by increased frequency responses of the crystal oscillations in a quartz crystal microbalance (QCM). The produced polymer layers were also found to be highly sensitive to external acid/base stimuli. Large oscillation frequency shifts were detected when the brushes were exposed to buffer solutions of different pH. The dynamic behavior of the resulting polymeric brushes was evaluated, and the extent of expansion and contraction of the films was monitored by the QCM setup in situ in real time. The resulting responses were rapid, and the effects were fully reversible. Low pH resulted in full contractions of the films, whereas higher pH yielded maximal expansion in order to minimize repulsion around the charged acrylate centers. The surfaces also proved to be very robust because the responsiveness was reproducible over many cycles of repeated expansion and contraction. Using ellipsometry, copolymer layers were estimated to be approximately 220 nm in a collapsed state and approximately 340 nm in the expanded state, effectively increasing the thickness of the film by 55%.

The influence of flow cell geometry on sample dispersion in a quartz crystal microbalance (QCM) biosensor system was investigated. A circular and a rectangular flow cell and corresponding sensor ...

This paper investigates a novel quartz crystal microbalance (QCM) biosensor with a small and rectangular flow cell along with a correspondingly shaped crystal electrode. The sensor was evaluated ...

Electrochemical quartz crystal microbalance has been used to measure changes in the composition of the capacitive electrical double layer for 1-ethyl-3-methylimidazolium tris(pentafluoroethyl)-trifluorophosphate, an ionic liquid, in contact with a gold electrode surface as a function of potential. The mass difference between the cation and anion means that the technique can effectively "weigh" the surface charge accurately with high temporal resolution. This reveals quantitatively how changing the potential alters the ratio of cations and anions associated with the electrode surface, and thus the charge per unit area, as well as the kinetics associated with these interfacial processes. The measurements reveal that it is diffusion of co-ions into the interfacial region rather than expulsion of counterions that controls the relaxation. The measured potential dependent double layer capacitance experimentally validates recent theoretical predictions for counterion overscreening (low potentials) and crowding (high potentials) at electrode surfaces. This new capacity to quantitatively measure ion composition is critical for ionic liquid applications ranging from batteries, capacitors and electrodeposition through to boundary layer structure in tribology, and more broadly provides new insight into interfacial processes in concentrated electrolyte solutions.

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery.

Atomic force microscopy (AFM) and infrared reflection absorption spectroscopy (IRAS) were used for in situ investigations of the initial atmospheric corrosion of bronze. In addition ex situ XPS inv ...

Background: The interaction between biotin and avidin is utilized in a wide range of assay and diagnostic systems. A robust material capable of binding biotin should offer scope in the development of reusable assay materials and biosensor recognition elements. Results: Biotin-selective thin (3-5 nm) films have been fabricated on hexadecanethiol self assembled monolayer (SAM) coated Au/quartz resonators. The films were prepared based upon a molecular imprinting strategy where N, N'-methylenebisacrylamide and 2-acrylamido-2-methylpropanesulfonic acid were copolymerized and grafted to the SAM-coated surface in the presence of biotin methyl ester using photoinitiation with physisorbed benzophenone. The biotinyl moiety selectivity of the resonators efficiently differentiated biotinylated peptidic or carbohydrate structures from their native counterparts. Conclusions: Molecularly imprinted ultra thin films can be used for the selective recognition of biotinylated structures in a quartz crystal microbalance sensing platform. These films are stable for periods of at least a month. This strategy should prove of interest for use in other sensing and assay systems.

Three complementary experimental techniques for in situ surface analysis have been combined for the first time in order to explore the chemistry and physics of a copper surface exposed to humidified air. Infrared reflection absorption spectroscopy, quartz crystal microbalance and atomic force microscopy provide a congruent picture of the processes occurring at the surface. At a given relative humidity, cuprous oxide forms according to an approximately logarithmic rate law. In addition, an aqueous adlayer of constant mass physisorbs on the surface. Increased relative humidity stimulates the physisorption of water and enhances the nucleation rate of oxide grains, thereby increasing the formation rate of cuprous oxide.

Scanning force microscopy (SFM) and infrared reflection absorption spectroscopy (IRAS) coupled with quartz crystal microbalance (QCM) were used for in-situ investigations of the atmospheric corrosion of pure copper. Based on chemical and kinetic studies obtained by IRAS/QCM, tapping mode atomic force microscopy (TM-AFM) and phase detection imaging (PDI) were applied to gain information about the topography changes of the sample surface with emphasis on the shape and lateral distribution of the corrosion products. Investigations were carried out in synthetic air with 60 and 80% relative humidity (RH). At 60% RH, small features partly covering the surface could be observed with TM-AFM. These features were identified as cuprous oxide with IRAS. Contrary to these results, a fast formation of a layer of cuprous oxide entirely covering the sample surface was observed at 80% RH with TM-AFM. QCM investigations showed a higher formation rate of cuprous oxide at 80% than at 60% RH but in both cases, a high corrosion rate at the beginning of the exposure, which decreases with progressing time.

Scanning force microscopy (SFM) studies of atmospheric corrosion of pure copper require a clean, homogeneous and smooth metal surface in order to image small changes. In this study various preparation methods for samples of copper sheets were tested: chemical etching, mechanical polishing as well as electrochemical polishing. In addition to copper sheet samples also specimen of sputtered copper obtained by physical vapour deposition on quartz substrates were chemically etched. In conclusion, mechanical polishing with monocrystalline diamond paste and electrochemical polishing of copper sheet yielded the most suitable surface condition for in situ investigations by means of SFM. Tapping mode atomic force microscopy (TM-AFM) was applied to gain information about the topography changes of the copper surface. Furthermore, the root mean square (rms) roughness of the sample surfaces was determined and delivered additional arguments for the applicability of the various surface treatments for in situ studies.

This work describes in situ infrared reflection absorption spectroscopy (IRAS) studies of the initial interaction of platinum and palladium to humidified air into which SO 2 has been introduced. The formation of surface species could be followed in situ from 9 min to 20 h of exposure. Supported by X-ray photoelectron spectroscopy and glancing incidence X-ray diffraction analysis after exposure, the IRAS bands from surface species formed in the aqueous adlayer on both metals has been assigned to sulfuric acid, H 2 SO 4 , with no evidence of chemical reaction with the metal. When changing from humid to dry exposure conditions, the aqueous adlayer with sulfuric acid transforms into one or more phases of hydrated crystalline sulfuric acid, H 2 SO 4 .nH 2 O. On platinum there is observed an initially fast formation rate of sulfuric acid followed by a slower formation rate with prolonged exposure. The initial formation rate on palladium, however, is much slower than on platinum. After about 7 h of exposure, a sudden but well-reproduced increase occurs, whereafter the formation rate levels off.

A new experimental setup for in situ studies of the metal/atmosphere interface has been developed based on simultaneous infrared reflection absorption spectroscopy (IRAS) and quartz crystal microbalance (QCM) measurements of a metal surface. It consists of an in situ chamber in which the metal can be exposed to a well-controlled atmosphere. Four external devices are connected to the in situ chamber; a Fourier transform infrared spectrometer with external optical compartments, a QCM sensor probe with a frequency counter, a corrosive air generator, and a corrosive air analyzing system. In order to demonstrate the capability of the IRAS/QCM setup, copper was exposed to purified air at 80% relative humidity and 25 C. Under these exposure conditions, the interface between copper and air consists of cuprous oxide and water physisorbed on the oxide. The kinetics of the cuprous oxide formation could be followed in situ with both techniques. The combined IRAS/QCM results show excellent agreement with previous combined IRAS and cathodic reduction measurements and with optical calculations of the IRAS response. Under these conditions, the detection limit in terms of an equivalent Cu{sub 2}O film thickness is 10 {angstrom} for IRAS in situ analysis and 2 {angstrom} for QCM in situ analysis,more » respectively.« less

A novel hyperbranched polyethyleneimine (PEI) modified gold surface has been designed, fabricated, and investigated with respect to its ability to resist non-specific adsorption of proteins. The facile synthesis strategy, based on self-assembly, involves immobilization of polyethyleneimine to gold surfaces modified with 11-mercaptoundecanoic acid (MuDA) monolayers using traditional carbodiimide chemistry. The hyperbranched polymer brushes were characterized by X-ray photoelectron spectroscopy (XPS). Reflection absorption infrared spectroscopy (RAIRS) and ellipsometry measurements showed the thickness of the PEI brushes increases with adsorption solution ionic strength. Polymer brush surface concentrations can be improved from 2560 to 3880chains/μm(2) by changing the ionic strength of the adsorption solution (PBS) by varying NaCl concentration from 0 to 650mM. Protein adsorption (pH 7.4) was evaluated under flow injection analysis (FIA) conditions using a quartz crystal microbalance (QCM). The PEI brushes suppress protein adsorption, for example, cytochrome C, bovine serum albumin (BSA), and ribonuclease A, to less than 0.08μg/cm(2) and the protein resistance increases with increasing ionic strength of the carrier solution, performance comparable to that achieved with comparable PEG-coated surfaces. The PEI brushes were exceptionally stable, with adsorption characteristics maintained after 6months storage in aqueous conditions (pH 7.4, 25°C, PBS). The potential of hyperbranched PEI structures as protein-resistant surfaces is discussed.

Jean-Marie Lehn Constitutional Dynamic Chemistry: Bridge from Supramolecular Chemistry to Adaptive Chemistry.- Mihail Barboiu Multistate and Phase Change Selection in Constitutional Multivalent Systems.- Morakot Sakulsombat Yan Zhang Olof Ramstrom Dynamic Systemic Resolution.- Emilie Moulin Nicolas Giuseppone Dynamic Combinatorial Self-Replicating Systems.- Benjamin L. Miller DCC in the Development of Nucleic Acid Targeted and Nucleic Acid Inspired Structures.- Eugene Mahon Teodor Aastrup Mihail Barboiu Dynamic Nanoplatforms in Biosensor and Membrane Constitutional Systems.- D. Quemener A. Deratani S. Lecommandoux Dynamic Assembly of Block-Copolymers.- Radu Custelcean Dynamic Chemistry of Anion Recognition.- Nandhini Ponnuswamy Artur R. Stefankiewicz Jeremy K. M. Sanders G. Dan Pantos Supramolecular Naphthalenediimide Nanotubes.- Adrian-Mihail Stadler Juan Ramirez Synthetic Molecular Machines and Polymer/Monomer Size Switches that Operate Through Dynamic and Non-Dynamic Covalent Changes Kamel Meguellati Sylvain Ladame Reversible Covalent Chemistries Compatible with the Principles of Constitutional Dynamic Chemistry: New Reactions to Create More Diversity.

Molecular recognition in biological systems occurs mainly at interfacial environments such as membrane surfaces, enzyme active sites, or the interior of the DNA double helix. At the cell membrane surface, carbohydrate-protein recognition principles apply to a range of specific non-covalent interactions including immune response, cell proliferation, adhesion and death, cell-cell interaction and communication. Protein-protein recognition meanwhile accounts for signalling processes and ion channel structure. In this chapter we aim to describe such constitutional dynamic interfaces for biosensing and membrane transport applications. Constitutionally adaptive interfaces may mimic the recognition capabilities intrinsic to natural recognition processes. We present some recent examples of 2D and 3D constructed sensors and membranes of this type and describe their sensing and transport capabilities.

A series of streptavidin-mimicking molecularly imprinted polymers has been developed and evaluated for their biotin binding characteristics. A combination of molecular dynamics and NMR spectroscopy was used to examine potential polymer systems, in particular with the functional monomers methacrylic acid and 2-acrylamidopyridine. The synthesis of copolymers of ethylene dimethacrylate and one or both of these functional monomers was performed. A combination of radioligand binding studies and surface area analyses demonstrated the presence of selectivity in polymers prepared using methacrylic acid as the functional monomer. This was predicted by the molecular dynamics studies showing the power of this methodology as a prognostic tool for predicting the behavior of molecularly imprinted polymers.

Molecular recognition in biological systems occurs mainly at interfacial environments such as membrane surfaces, enzyme active sites, or the interior of the DNA double helix. At the cell membrane surface, carbohydrate–protein recognition principles apply to a range of specific non-covalent interactions including immune response, cell proliferation, adhesion and death, cell–cell interaction and communication. Protein–protein recognition meanwhile accounts for signalling processes and ion channel structure. In this chapter we aim to describe such constitutional dynamic interfaces for biosensing and membrane transport applications. Constitutionally adaptive interfaces may mimic the recognition capabilities intrinsic to natural recognition processes. We present some recent examples of 2D and 3D constructed sensors and membranes of this type and describe their sensing and transport capabilities.

A photoclick method based on azide photoligation and Cu-catalyzed azide−alkyne cycloaddition has been evaluated for the immobilization of carbohydrates to polymeric materials. The biomolecular recognition properties of the materials have been investigated with regard to applicable polymeric substrates and selectivity of protein binding. The method was used to functionalize a range of polymeric surfaces (polystyrene, polyacrylamide, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and polypropene) with various carbohydrate structures (based on α-d-mannose, β-d-galactose, and N-acetyl-β-d-glucosamine). The functionalized surfaces were evaluated in real-time studies of protein−carbohydrate interactions using a quartz crystal microbalance flow-through system with a series of different carbohydrate-binding proteins (lectins). The method proved to be robust and versatile, resulting in a range of efficient sensors showing high and predictable protein selectivities.