Cancer biomarkers detection using microstructured protein chip: implementation of customized multiplex immunoassay

Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins, and immobilization conditions (buffer, pH, concentration). We have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions was evaluated on the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that each antibody displays variable biological activities depending on the surface chemistry and on the immobilization procedure. Under optimized conditions, we can reach a limit of detection in tumor antigen as low as 10 pM. Our microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody on the same chip

New concepts of integrated photonic biosensors based on porous silicon

International audienceno abstrac

Développement d'un biocapteur couplant la résonance des plasmons de surface et la microcalorimétrie pour le suivi des interactions moléculaires à l'interface liquide/solide

Dans un avenir proche, les dispositifs de détection médicaux miniaturisés en temps réels (lab-on-chip) seront au centre de la révolution des méthodes de diagnostics médicaux et d identification des processus biologiques et cela, autant au niveau clinique qu au niveau de la recherche. Pour y arriver, il est important de développer des chimies de surface stables et spécifiques, ce qui demande une compréhension des interactions intermoléculaires à l interface liquide/solide. Pour bien comprendre ces interactions, il est important de développer des instruments adaptés à la mesure près de l interface liquide/solide des différentes caractéristiques à identifier. Ce projet de recherche présente la conception, la fabrication et les expériences tests d un capteur multimodal pour l identification de processus biologiques à l interface basés sur des technologies de résonance des plasmons de surface (SPR) et de microcalorimérie. Ces deux technologies mises ensemble vont permettre d effectuer des mesures de la cinétique des interactions ainsi que des caractéristiques thermodynamiques. En premier lieu, les caractéristiques d une interaction intermoléculaire à l interface d une réaction d hybridation d ADN furent définies afin d en déduire un cahier des charges pour les transducteurs. Suite à cela, la conception des transducteurs microcalorimétrique et SPR furent réalisés en tenant compte des contraintes de chacun des transducteurs. Suite à la conception théorique des différentes parties du capteur, un procédé de fabrication compatible avec les méthodes de fabrication standard de la microélectronique fut défini et testé. Afin de s assurer de la fonctionnalité des dispositifs ainsi fabriqués, des tests de fonctionnalisation de surface furent appliqués sur les échantillons afin de tester la compatibilité du procédé de fonctionnalisation avec les méthodes de fabrication et avec une chimie de surface type. Pour terminer, un système de mélange actif fut testé et caractérisé avec le dispositif de microcalorimétrie afin de s assurer qu il était possible de mélanger les fluides avec les produits biologiques pour s assurer de la qualité de la réaction de surface. Le système développé pourra être utilisé pour effectuer la mesure d hybridation d ADN à l interface. Le système intègre deux modalités permettant la caractérisation en temps réel des interactions intermoléculaires à l interface liquide/solide. Ce type de système permet la mesure de la cinétique de différents modèles biologiques tels que les puces à sucre encore certains récepteurs cellulaires ou la mesure de conformation moléculaire à l interface. Des mesures d oxydation du glucose catalysée par la glucose oxydase sont montrées.To begin with, the characteristics of a DNA hybridization intermolecular interaction at the interface were defined in order to deduce the specifications for our transducers. Following this, the SPR and microcalorimetric transducer will be design by taking into account the constraints of each one. Following the theoretical design of the sensor, a manufacturing process compatible with standard methods of microelectronics manufacturing was tested and identified. To ensure the functionality of the devices, a test of surface functionalization on the sensor was applied to test the compatibility of the manufacturing process with the surface functionalization methods. Finally, a system of active mixing was tested and characterized with a microcalorimetric device to ensure it was possible to mix fluids with organic products to be sure of the quality of the surface reaction. The system developed can be used to mesure DNA hybridization at the interface. This system incorporates two modalities for real-time characterization of intermolecular interactions near the solid/liquid interface. This type of system allows the kinetic measurement of different biological models such as cellular receptors or it is possible to have some molecular measure conformation near the interface. Oxidation of glucose with the enzyme glucose oxidase was shown to present the possibility to measure a biological interaction.LYON-Ecole Centrale (690812301) / SudocSudocFranceF

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

Background: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation

Chemical Architecture and Applications of Nucleic Acid Derivatives Containing 1,2,3-Triazole Functionalities Synthesized via Click Chemistry

There is considerable attention directed at chemically modifying nucleic acids with robust functional groups in order to alter their properties. Since the breakthrough of copper-assisted azide-alkyne cycloadditions (CuAAC), there have been several reports describing the synthesis and properties of novel triazole-modified nucleic acid derivatives for potential downstream DNA- and RNA-based applications. This review will focus on highlighting representative novel nucleic acid molecular structures that have been synthesized via the “click” azide-alkyne cycloaddition. Many of these derivatives show compatibility for various applications that involve enzymatic transformation, nucleic acid hybridization, molecular tagging and purification, and gene silencing. The details of these applications are discussed. In conclusion, the future of nucleic acid analogues functionalized with triazoles is promising

The key role of sulfation and branching on fucoidan antitumor activity

There is an urgent need for antitumor bioactive agents with minimal or no side effects over normal adjacent cells. Fucoidan is a marine-origin polymer with known antitumor activity. However, there are still some concerns about its application due to the inconsistent experimental results, specifically its toxicity over normal cells and the mechanism behind its action. Herein, three fucoidan extracts (FEs) have been tested over normal and breast cancer cell lines. From cytotoxicity results, only one of the extracts shows selective antitumor behavior (at 0.2 mg mLâ 1), despite similarities in sulfation degree and carbohydrates composition. Although the three FEs present different molecular weights, depolymerization of selected samples discarded Mw as the key factor in the antitumor activity. Significant differences in sulfates position and branching are observed, presenting FE 2 the higher branching degree. Based on all these experimental data, it is believed that these last two properties are the ones that influence the cytotoxic effects of fucoidan extracts.The authors would like to thank the funding from projects 0687_NOVOMAR_1_P, cofunded by INTERREG 2007-2013/POCTEP, CarbPol_u_Algae (EXPL/MAR-BIO/0165/2013), and IF/00376/2014/CP1212/CT0015, funded by the Portuguese Foundation for Science and Technology, FCT, and ComplexiTE (ERC-2012-ADG 20120216-321266), funded by the European Research Council under the European Union's Seventh Framework Programme for Research and Development. The authors would also like to thank FCT, Portugal, for the scholarship of A.S.F. (SFRH/BD/102471/2014), fellowship of C.N. (SFRH/BPD/100627/2014), Investigator grants of A.M. (IF/00376/2014), R.N.-C. (IF/00373/2014), and I.P. (IF/00032/2013) and the financial support to CICECO-Aveiro Institute of Materials (POCI-01-0145-FEDER-007679, FCT UID/CTM/50011/2013) and OOPNA (UID/OUI/00062/2013), through national founds and cofinanced by the FEDER, within the PT2020 Partnership Agreement

Fucanomics and Galactanomics: Marine Distribution, Medicinal Impact, Conceptions, and Challenges

Glycomics turned out to be a very extensive project where its subdivision is consequently emerging. This is seen by the growing number of terminologies used to define subprojects concerning particular classes of bioactive carbohydrates. Sulfated fucans (SFs) and sulfated galactans (SGs) are relatively new classes of sulfated polysaccharides (SPs) that occur mostly in marine organisms, and exhibit a broad range of medicinal effects. Their structures are taxonomically dependent, and their therapeutic actions include benefits in inflammation, coagulation, thrombosis, angiogenesis, cancer, oxidation, and infections. Some red algae, marine angiosperm and invertebrates express SPs of unique structures composed of regular repeating oligomeric units of well-defined sulfation patterns. This fine pattern of structural regularity is quite rare among any naturally occurring long SPs, and enables accurate structure-biofunction correlations. Seeing that, fucanomics and galactanomics may comprise distinguished glycomics subprojects. We hereby discuss the relevance that justifies the international recognition of these subprojects in the current glycomics age associated with the beneficial outcomes that these glycans may offer in drug development

Rising from the Sea: Correlations between Sulfated Polysaccharides and Salinity in Plants

High salinity soils inhibit crop production worldwide and represent a serious agricultural problem. To meet our ever-increasing demand for food, it is essential to understand and engineer salt-resistant crops. In this study, we evaluated the occurrence and function of sulfated polysaccharides in plants. Although ubiquitously present in marine algae, the presence of sulfated polysaccharides among the species tested was restricted to halophytes, suggesting a possible correlation with salt stress or resistance. To test this hypothesis, sulfated polysaccharides from plants artificially and naturally exposed to different salinities were analyzed. Our results revealed that the sulfated polysaccharide concentration, as well as the degree to which these compounds were sulfated in halophytic species, were positively correlated with salinity. We found that sulfated polysaccharides produced by Ruppia maritima Loisel disappeared when the plant was cultivated in the absence of salt. However, subjecting the glycophyte Oryza sativa Linnaeus to salt stress did not induce the biosynthesis of sulfated polysaccharides but increased the concentration of the carboxylated polysaccharides; this finding suggests that negatively charged cell wall polysaccharides might play a role in coping with salt stress. These data suggest that the presence of sulfated polysaccharides in plants is an adaptation to high salt environments, which may have been conserved during plant evolution from marine green algae. Our results address a practical biological concept; additionally, we suggest future strategies that may be beneficial when engineering salt-resistant crops