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

Diagnostic du cancer par puces à protéines 3D

Un système est dit robuste s'il est possible de garantir son bon comportement Le cancer est en passe de devenir la première cause de décès dans le monde avec un nombre de cas de cancer qui a pratiquement doublé sur les trente dernières années. Le diagnostic du cancer est d autant plus important qu il est maintenant reconnu que, plus la prise en charge du patient est rapide, plus les traitements thérapeutiques sont efficaces. Ce diagnostic doit être précis, fiable, et établi dans les premiers stades de la maladie afin d augmenter significativement les chances de succès du/des traitements. Les techniques conventionnelles pour le diagnostic du cancer sont essentiellement basées sur des techniques d imagerie (radiographies, IRM ) associés à des tests cytologiques et biochimiques. Avec le développement récent des technologies de biologie moléculaire (et notamment en protéomique), de nombreux marqueurs tumoraux ont été identifiés et sont utilisés dans des tests d immunoassay pour le diagnostic voire pronostic du cancer en oncologie clinique. Cependant, le faible taux de marqueurs tumoraux dans le sérum de patient, ainsi que leur grande diversité, sont un challenge important pour l établissement d un diagnostic d autant plus que les techniques de détection souffrent souvent d un manque de sensibilité et de sélectivité. De plus, du fait de la diversité et de la variabilité des cancers, aucun marqueur tumoral n est suffisamment spécifique pour permettre un diagnostic précis. Aussi, afin d augmenter la fiabilité et la précision du diagnostic, il est nécessaire d utiliser plusieurs marqueurs tumoraux. Dans ce contexte, grâce à leur capacité d analyse haut débit en parallèle et le faible volume d échantillon nécessaire, les technologies de puces à protéines (protein microarray)présentent de nombreux avantages pour l identification de marqueurs tumoraux associés à la réponse humorale. Comme les marqueurs tumoraux sont souvent présents dans les échantillons en très faible quantité (à l échelle sub micro-molaire), il y a un besoin urgent de développer des puces à protéines avec une détection ultrasensible de marqueurs tumoraux. La spécificité du diagnostic sera fortement liée au choix des protéines que l on veut détecter(notées protéines cibles) et par conséquent au choix des protéines sondes que l on va immobiliser sur le support. Un des paramètres critiques dans le développement de puces à protéines sensibles est la chimie de surface qui détermine le mode d immobilisation de la protéine sonde sur le support et influence son activité biologique et donc sa capacité à reconnaitre et interagir avec la protéine cible que l on cherche à détecter. Comme de nombreuses études suggèrent qu un seul biomarqueur n est pas suffisamment spécifique et sensible, la recherche d une combinaison pertinente de biomarqueurs est un axe important pour l amélioration d un tel diagnostic. L objectif de ce travail de thèse est donc le développement d un outil original basé sur la technologie de puces à protéines fonctionnalisées avec différentes chimies de surface pour la détection sensible et spécifique de biomarqueurs tumoraux afin d améliorer le diagnostic du cancer. Deux types de puces à protéines seront développés pour des applications différentes. Une première puce, avec comme protéines sondes des anticorps, sera développée pour la détection de biomarqueurs tumoraux impliqués dans le cancer colorectal. Une deuxième puce, où les protéines sondes seront des antigènes, sera étudiée en vue de l identification de réponses autoimmunes de patientes atteintes d un cancer du sein. [...]Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients and 28 healthy donors sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.LYON-Ecole Centrale (690812301) / SudocSudocFranceF

Autoantibodies against heat shock proteins as biomarkers for the diagnosis and prognosis of cancer

International audienceThanks to their specificity and stability in the sera, autoantibodies (AAbs) against tumor-associated antigens (TAAs) are very attractive biomarkers for the development of less invasive serological tests for the diagnosis and prognosis of cancer. Heat shock proteins (HSP) belong to TAAs and they are over-expressed in various human cancers. Elevated HSP can stimulate the immune system to produce anti-HSP antibodies. So far, AAbs against HSP have been identified in the circulation of various cancer patients. Here we will review current literature on the use of anti-HSP antibodies for cancer diagnosis and prognosis. The challenges as well as future directions of AAbs identification in oncology are also discussed

Friction of 316L stainless steel on soft-tissue-like poly(vinyl alcohol) hydrogel in physiological liquid

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Tumor Antigen Microarray for the Diagnosis and individual therapy of Breast Cancer

2-6 juin 2014International audienceBreast cancer is one of the most challenging diseases, endangering the health of women worldwide. In 2012, there is 1.7 million new breast cancer cases and 520 000 women were dead. The five year survival of patients can be greatly improved if they are diagnosed at an early stage. However, conventional methods of diagnosis are based on imaging techniques (mammography, magnetic resonance imaging (MRI), computed tomography scans (CT), ultrasounds, positron emission tomography (PET)) which required high technology equipments, and are not available to most women. So there is a need to develop cheaper, more reliable and specific screening tools. Protein microarray has a great potential as biomarkers screening tool because it can fulfill high throughput using tiny volume of sample [1]. Protein microarray is a solid surface (typically glass) on which many proteins (up to thousands) are immobilized as probes and dedicated to catch their target in a given sample. This specific interaction can be detected by various methods such as fluorescent detection method. In many cancers, the tumor produces modified proteins (called tumor antigens) which are recognized by the immune system. Antibodies against these tumor antigens circulate in the body and represent good indicators of tumor development. In this study, we focused on the detection of these antibodies in the serum of breast cancer patients to identify relevant signature of the disease. However, protein microarray performances are influenced by various factors including surface chemistry, spotting buffer, concentration of immobilized proteins, etc [2]. So firstly, we immobilized 8 tumor antigens on 6 different surfaces in order to determine the best immobilization condition for each protein. Results turned out that each protein has its own optimal condition but 3 surfaces displayed good performances for all proteins. Then secondly, we immobilized the 8 tumor antigens on these 3 surfaces and screened 76 sera including 50 breast cancer sera and 26 healthy donor sera for the presence of antibodies against the tumor antigens. Results showed that if we consider only a single antibody, few breast cancer sera could be indentified. However, if we combine the detection of the 8 antibodies on the 3 surfaces, the sensitivity of breast cancer identification can be greatly increased from 26% up to 60%

Effect of counter body material and lubricant on the frictional properties of biomimetic PVA hydrogel

May 11th-14th, 2014International audienceno abstrac

Friction of 316L stainless steel on soft-tissue-like poly(vinyl alcohol) hydrogel in physiological liquid

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Mise en oeuvre de chitosan ou d'alginate en tant que masque de transfert dans des procédés de lithographie et de transfert

Brevet : N° de publication FR 15 53072 / Déposant cnrs / Date de dépot 09/04/2015 /no abstrac

Multiplex detection of anti-heat shock protein autoantibodies in breast cancer sera

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