TOF-SIMS structural characterization of self-assembly monolayer of cytochrome b5 onto gold substrate.

International audienceOrientation and three-dimensional structure of immobilized proteins on bio-devices are very important to assure their high performance. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is able to analyze upper surface of one layer of molecules. Orientation of immobilized proteins can be evaluated based on determination of a partial structure, representing ensemble of amino acids, on the surface part. In this study, a monolayer of cytochrome b5 was reconstituted onto gold substrate and investigated by surface plasmon resonance (SPR). After freeze-drying, the resulted protein self-assembly was evaluated using TOF-SIMS with the bismuth cluster ion source, and then TOF-SIMS spectra were analyzed to select peaks specific to cytochrome b5 and identify their chemical formula and ensembles of amino acids. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the modified cytochrome b5 and three-dimensional structure of cytochrome b5 registered in the protein data bank. Finally, fragment-ion-generating parts of the immobilized-cytochrome b5 are determined based on the suggested residues and three-dimensional structure. These results suggest the actual structure and confirm the expected orientation of immobilized protein

Revisited BIA-MS combination: Entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels

International audienceWe present the results of a study in which biomolecular interaction analysis (BIA, Biacore 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS-MS of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexe

Improvement of robotic micromanipulations using chemical functionalisations.

International audienceRobotic microhandling is disturbed by the adhesion phenomenon between the micro-object and the grippers. This phenomenon is directly linked to both the object and the gripper surface chemical composition. We propose to control adhesion by using chemical self-assembly monolayer (SAM) on both surfaces. Previous distance-force measurements done with AFM have shown that the liquid pH can be used to modify the adhesion and created repulsive force between the gripper fingers and the micro-objet. This paper shows the correlation between the force distance distance measurements and the micromanipulation tasks using chemically functionalized grippers

Robotic submerged microhandling controlled by pH swithching.

International audienceRobotic microhandling is a promising way to assemble microcomponents in order to manufacture new generation of Hybrid Micro ElectroMechanical Systems (HMEMS). However, at the scale of several micrometers, adhesion phenomenon highly perturbs the micro-objects release and the positioning. This phenomenon is directly linked to both the object and the gripper surface chemical composition. We propose to control adhesion by using chemical self-assembly monolayer (SAM) on both surfaces. Different types of chemical functionalisation have been tested and this paper only focuses on the presentation of aminosilane grafted (3 (ethoxydimethylsilyl) propyl amine (APTES) and (3 aminopropyl) triethoxysilane (APDMES)). We show that the liquid pH can be used to modify the adhesion and to switch from an attractive behaviour to a repulsive behaviour. The pH control can thus be used to increase adhesion during handling and cancel adhesion during release. Experiments have shown that the pH control is able to control the release of a micro-object. This paper shows the relevance of a new type of reliable submerged robotic microhandling principle, which is based an adjusting chemical properties of liquid

Adhesion forces controlled by chemical self-assembly and pH. Application to robotic microhandling.

International audienceRobotic microhandling is a promising way to assemble microcomponents in order to manufacture new generation of Hybrid Micro ElectroMechanical Systems (HMEMS). However, at the scale of several micrometers, adhesion phenomenon highly perturbs the micro-objects release and the positioning. This phenomenon is directly linked to both the object and the gripper surface chemical composition. We propose to control adhesion by using chemical selfassembly monolayer (SAM) on both surfaces. Different types of chemical functionalisation have been tested and this paper focuses on the presentation of aminosilane grafted (3 (ethoxydimethylsilyl) propyl amine (APTES) and (3 aminopropyl) triethoxysilane (APDMES)). We show that the liquid pH can be used to modify the adhesion and to switch from an attractive behaviour to a repulsive behaviour. The pH control can thus be used to increase adhesion during handling and cancel adhesion during release. Experiments have shown that the pH control is able to control the release of a micro-object. This paper shows the relevance of a new type of reliable submerged robotic microhandling principle, which is based on adjusting chemical properties of liquid

Reduction of a micro-object's adhesion using chemical functionalisation.

International audienceThe adhesion and interaction properties of functionalised surfaces (substrate or cantilever) were investigated by means of atomic force microscope (AFM) related force measurements. The surfaces were functionalised with a polyelectrolyte: Poly(Allylamine Hydrochloride) (PAH), or with silanes: 3 (ethoxydimethylsilyl) propyl amine (APTES) or (3 aminopropyl) triethoxysilane (APDMES). Measurements of forces acting between a bare glass sphere (functionalised or not) and a functionalised surface indicated repulsive or attractive forces, depending on functionnalisation and medium (wet or dry). Adhesion forces (pull-off) can be observed in dry medium while in wet medium, this phenomenon can be cancelled. Now, the pull-off forces is an important problem in the automation of micro-object manipulations. The cancellation of this force by chemical functionnalisation is thus a promising way to improve micro-assembly in the future

Preparation of flat gold terraces for protein chip developments

International audienceA simple method to prepare flat gold terraces on mica for atomic force microscopy biomolecular characterisation is described. The procedure includes preheating of the substrate, metal deposition and an annealing step. All of these steps are at elevated temperatures (300–420°C). This approach allows one to prepare large flat gold terraces (200– 500 nm), which constitute ideal substrates for visualisation and characterisation of a self-assembly monolayer of biomolecules at the nanoscale. The authors illustrated this potential of characterisation with the reconstitution of a protein monolayer

Strategy of macromolecular grafting onto a gold substrate dedicated to protein-protein interaction measurements.

International audienceMany biotechnology applications use proteins immobilized on surface. For biosensor, the sensing layer is a key component interfacing the transducer and the sample. Strategies employed to activate the bidimensional surface act directly on the performance of the biosensor. In this paper we propose a novel strategy for engineered proteins self-assembly. Our original supramolecular structure allows a direct and fast covalent attachment of proteins onto bare gold substrate through a homobifunctional cross-linker, 1,4-di-([2'-pyridyldithio]propionamido)butane (DPDPB). In this work, engineered proteins and linker-protein complexes were synthesized and characterized by gel electrophoresis, chromatography and spectroscopy experiments. Macromolecular construction "DPDPB-GST tag-GEC1 protein" was conceived in order to guarantee a 2D architecture enhancing the capabilities of the target (tubulin) to recognize its partner (GEC1). Surface plasmon resonance measurements clearly showed potential of this particular self-assembled protein layer compared to a commercial immunosensor interface. At the concentrations tested, the recognition process occurs between tubulin and the immobilized GEC1; moreover enhanced binding was obtained with the home-made 2D sensing layer more than with 3D carboxymethyl dextran matrix

Neuropilin-2 Expression Promotes TGF-β1-Mediated Epithelial to Mesenchymal Transition in Colorectal Cancer Cells

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors