{"id":38680,"date":"2019-12-16T13:07:21","date_gmt":"2019-12-16T11:07:21","guid":{"rendered":"https:\/\/www.iemn.fr\/?p=38680"},"modified":"2020-03-03T11:16:50","modified_gmt":"2020-03-03T09:16:50","slug":"detection-ultrasensible-de-la-toxine-de-ricine-par-des-capteurs-innovants-a-base-de-graphene-utilisant-la-spectrometrie-de-masse","status":"publish","type":"post","link":"https:\/\/www.iemn.fr\/en\/actualites\/detection-ultrasensible-de-la-toxine-de-ricine-par-des-capteurs-innovants-a-base-de-graphene-utilisant-la-spectrometrie-de-masse.html","title":{"rendered":"Soutenance de th\u00e8se : Ioana Silvia HOSU \u2013 D\u00e9tection ultrasensible de la toxine de ricine par des capteurs innovants \u00e0 base de graph\u00e8ne, utilisant la spectrom\u00e9trie de masse"},"content":{"rendered":"<div id='layer_slider_1'  class='avia-layerslider main_color avia-shadow  avia-builder-el-0  el_before_av_heading  avia-builder-el-first  container_wrap sidebar_right'  style='height: 261px;'  ><div id=\"layerslider_58_w487pfk34mk4\" data-ls-slug=\"homepageslider\" class=\"ls-wp-container fitvidsignore ls-selectable\" style=\"width:1140px;height:260px;margin:0 auto;margin-bottom: 0px;\"><div class=\"ls-slide\" data-ls=\"duration:6000;transition2d:5;\"><img loading=\"lazy\" decoding=\"async\" width=\"2600\" height=\"270\" src=\"https:\/\/www.iemn.fr\/wp-content\/uploads\/2019\/01\/sliders_news1.jpg\" class=\"ls-bg\" alt=\"\" srcset=\"https:\/\/www.iemn.fr\/wp-content\/uploads\/2019\/01\/sliders_news1.jpg 2600w, https:\/\/www.iemn.fr\/wp-content\/uploads\/2019\/01\/sliders_news1-300x31.jpg 300w, https:\/\/www.iemn.fr\/wp-content\/uploads\/2019\/01\/sliders_news1-768x80.jpg 768w, https:\/\/www.iemn.fr\/wp-content\/uploads\/2019\/01\/sliders_news1-1030x107.jpg 1030w, https:\/\/www.iemn.fr\/wp-content\/uploads\/2019\/01\/sliders_news1-1500x156.jpg 1500w, https:\/\/www.iemn.fr\/wp-content\/uploads\/2019\/01\/sliders_news1-705x73.jpg 705w\" sizes=\"auto, (max-width: 2600px) 100vw, 2600px\" \/><ls-layer style=\"font-size:14px;text-align:left;font-style:normal;text-decoration:none;text-transform:none;font-weight:700;letter-spacing:0px;border-style:solid;border-color:#000;background-position:0% 0%;background-repeat:no-repeat;width:180px;height:30px;left:0px;top:231px;line-height:32px;color:#ffffff;border-radius:6px 6px 6px 6px;padding-left:50px;background-color:rgba(0, 0, 0, 0.57);\" class=\"ls-l ls-ib-icon ls-text-layer\" data-ls=\"minfontsize:0;minmobilefontsize:0;\"><i class=\"fa fa-quote-right\" style=\"color:#ffffff;margin-right:0.8em;font-size:1em;transform:translateY( -0.125em );\"><\/i>ACTUALITES<\/ls-layer><\/div><\/div><\/div><div id='after_layer_slider_1'  class='main_color av_default_container_wrap container_wrap sidebar_right'  ><div class='container av-section-cont-open' ><div class='template-page content  av-content-small alpha units'><div class='post-entry post-entry-type-page post-entry-38680'><div class='entry-content-wrapper clearfix'>\n\n<style type=\"text\/css\" data-created_by=\"avia_inline_auto\" id=\"style-css-av-1zck48-81e665a615c337c6bf8eff1e6daaa1cd\">\n#top .av-special-heading.av-1zck48-81e665a615c337c6bf8eff1e6daaa1cd{\nmargin:0 0 10px 0;\npadding-bottom:4px;\n}\nbody .av-special-heading.av-1zck48-81e665a615c337c6bf8eff1e6daaa1cd .av-special-heading-tag .heading-char{\nfont-size:25px;\n}\n.av-special-heading.av-1zck48-81e665a615c337c6bf8eff1e6daaa1cd .av-subheading{\nfont-size:15px;\n}\n<\/style>\n<div  class='av-special-heading av-1zck48-81e665a615c337c6bf8eff1e6daaa1cd av-special-heading-h2  avia-builder-el-1  el_after_av_layerslider  el_before_av_hr  avia-builder-el-first'><h2 class='av-special-heading-tag'  itemprop=\"headline\"  >THESE : Ioana Silvia HOSU \u2013 D\u00e9tection ultrasensible de la toxine de ricine par des capteurs innovants \u00e0 base de graph\u00e8ne, utilisant la spectrom\u00e9trie de masse<\/h2><div class=\"special-heading-border\"><div class=\"special-heading-inner-border\"><\/div><\/div><\/div>\n\n<style type=\"text\/css\" data-created_by=\"avia_inline_auto\" id=\"style-css-av-18u73nj-dad6a947580930e400fc42ba200e80f1\">\n#top .hr.av-18u73nj-dad6a947580930e400fc42ba200e80f1{\nmargin-top:5px;\nmargin-bottom:5px;\n}\n.hr.av-18u73nj-dad6a947580930e400fc42ba200e80f1 .hr-inner{\nwidth:100%;\n}\n<\/style>\n<div  class='hr av-18u73nj-dad6a947580930e400fc42ba200e80f1 hr-custom  avia-builder-el-2  el_after_av_heading  el_before_av_textblock  hr-left hr-icon-no'><span class='hr-inner inner-border-av-border-thin'><span class=\"hr-inner-style\"><\/span><\/span><\/div>\n<section  class='av_textblock_section av-jriy64i8-2f4600354c0449b610997916bbd9b6bc'   itemscope=\"itemscope\" itemtype=\"https:\/\/schema.org\/BlogPosting\" itemprop=\"blogPost\" ><div class='avia_textblock'  itemprop=\"text\" >\n<style type=\"text\/css\" data-created_by=\"avia_inline_auto\" id=\"style-css-av-13ewzjw-68e036126b913e5028f77311dc66b825\">\n.av_font_icon.av-13ewzjw-68e036126b913e5028f77311dc66b825{\ncolor:#bfbfbf;\nborder-color:#bfbfbf;\n}\n.av_font_icon.av-13ewzjw-68e036126b913e5028f77311dc66b825 .av-icon-char{\nfont-size:60px;\nline-height:60px;\n}\n<\/style>\n<span  class='av_font_icon av-13ewzjw-68e036126b913e5028f77311dc66b825 avia_animate_when_visible av-icon-style- avia-icon-pos-left avia-icon-animate'><span class='av-icon-char' aria-hidden='true' data-av_icon='\ue8c9' data-av_iconfont='entypo-fontello' ><\/span><\/span>\n<p>Ioana Silvia HOSU<\/p>\n<p>Sp\u00e9cialit\u00e9 : Electronique, micro\u00e9lectronique, nano\u00e9lectronique et micro-ondes<\/p>\n<p>Etablissement\u00a0: Universit\u00e9 de Lille<\/p>\n<p>Soutenance\u00a0: 11 f\u00e9vrier 2020<\/p>\n<p><strong>Mardi 11 f\u00e9vrier 2020 \u00e0 14h00<br \/>\n<\/strong>Amphitheatre of the IEMN-Laboratoire central - Villeneuve d'Ascq<\/p>\n<\/div><\/section>\n<section  class='av_textblock_section av-jtefqx33-628129dba2299b2ecd65ebfc92eac29d'   itemscope=\"itemscope\" itemtype=\"https:\/\/schema.org\/BlogPosting\" itemprop=\"blogPost\" ><div class='avia_textblock'  itemprop=\"text\" ><div  class='hr av-kjh3zw-4dff888f744b728a1aca9b3a0971493a hr-default  avia-builder-el-6  avia-builder-el-no-sibling'><span class='hr-inner'><span class=\"hr-inner-style\"><\/span><\/span><\/div>\n<h5><strong><span style=\"color: #800000;\">Jury :<\/span><\/strong><\/h5>\n<ul>\n<li>Yannick\u00a0COFFINIER,\u00a0Charg\u00e9 de Recherche,\u00a0Universit\u00e9 de Lille &#8211; IEMN\u00a0(Directeur de th\u00e8se)<\/li>\n<li>C\u00e9line\u00a0ELIE-CAILLE,\u00a0Ma\u00eetre de Conf\u00e9rences,\u00a0Institut FEMTO-ST\u00a0(Rapporteur)<\/li>\n<li>Yann CHEVOLOT,\u00a0Directeur de Recherche,\u00a0Institut des Nanotechnologies de Lyon (Rapporteur)<\/li>\n<li>Rabah\u00a0BOUKHERROUB,\u00a0Directeur de Recherche,\u00a0IEMN (Examinateur)<\/li>\n<li>Claudia\u00a0MURACCIOLE BICH,\u00a0Ma\u00eetre de Conf\u00e9rences,\u00a0IBMM, Universit\u00e9 de Montpellier (Examinateur)<\/li>\n<li>Tuami\u00a0LASRI,\u00a0Professeur des Universit\u00e9s,\u00a0Universit\u00e9 de Lille \u2013 IEMN (Examinateur)<\/li>\n<\/ul>\n<h5>Summary:<\/h5>\n<p>Les attaques bioterroristes sont devenues plus fr\u00e9quentes ces derni\u00e8res ann\u00e9es et le large \u00e9ventail d\u2019agents bioterroristes en fait un probl\u00e8me important \u00e0 r\u00e9soudre. La ricine appartient \u00e0 la famille des prot\u00e9ines inactivant les ribosomes (RIP). Les RIP sont des toxines biologiques, solubles dans l\u2019eau, qui peuvent \u00eatre facilement extraites de l\u00e9gumes (ricine de graines de ricin, abrin de pois rosay) ou de bact\u00e9ries (toxine de Shiga). La ricine est compos\u00e9e de deux cha\u00eenes: la cha\u00eene A de la ricine, une N-glycosidase induisant la toxicit\u00e9 par \u00e9limination de l\u2019ad\u00e9nine (action d\u00e9purination) de l\u2019ARNr 28S des sous-unit\u00e9s ribosomales 60S, puis en inhibant la synth\u00e8se prot\u00e9ique, et la cha\u00eene B de la ricine, une lectine qui se lie des fragments de sucre sp\u00e9cifiques sur la membrane extracellulaire, assurant l&rsquo;absorption de la toxine. Comme ils inhibent la synth\u00e8se des prot\u00e9ines, en fonction de la voie d&rsquo;absorption (orale, par inhalation, par voie intraveineuse) et de la dose re\u00e7ue, la mort peut survenir. En l&rsquo;absence de contre-mesures efficaces, les m\u00e9thodes de d\u00e9tection de ces toxines doivent \u00eatre rapides, fiables, s\u00e9lectives et sans aucune ambigu\u00eft\u00e9, en particulier les analyses de pr\u00e9-assimilation.<\/p>\n<p>Les m\u00e9thodes actuelles principalement bas\u00e9es sur SERS, ELISA, Colorim\u00e9trie et SPR ne r\u00e9pondent pas \u00e0 toutes ces exigences. M\u00eame si la spectrom\u00e9trie de masse a \u00e9t\u00e9 utilis\u00e9e pour la d\u00e9tection de la ricine, elle ne peut pas \u00eatre r\u00e9alis\u00e9e sans une longue et fastidieuse pr\u00e9paration d&rsquo;\u00e9chantillon, c&rsquo;est-\u00e0-dire une digestion enzymatique, une extraction \/purification et une identification de prot\u00e9ines sur la base d&rsquo;une analyse peptidique suivie d&rsquo;une comparaison avec la base de donn\u00e9es existante. Dans ce travail, nous avons montr\u00e9 comment les mat\u00e9riaux \u00e0 base de carbone (nanomurs de carbone) pourraient \u00eatre appliqu\u00e9s comme mat\u00e9riaux nanostructur\u00e9s pour la d\u00e9tection de la ricine par ionisation laser\/d\u00e9sorption de surface pour la d\u00e9tection par spectrom\u00e9trie de masse (SALDI-MS) et d&rsquo;autres techniques. Tout d&rsquo;abord, l&rsquo;ad\u00e9quation des nanoparticules de carbone en tant que bonne surface SALDI a \u00e9t\u00e9 initialement \u00e9tudi\u00e9e pour des biomol\u00e9cules plus petites (saccharides, peptides jusqu\u2019\u00e0 5800 m \/ z, glucides, lipides et glucose, qui a \u00e9galement \u00e9t\u00e9 quantifi\u00e9 \u00e0 l\u2019aide de SALDI-MS). En ce qui concerne les prot\u00e9ines, elles sont difficiles \u00e0 ioniser et \u00e0 d\u00e9tecter avec SALD-MS, en raison de leur grand poids mol\u00e9culaire. La capacit\u00e9 des CNWs \u00e0 d\u00e9sorber et \u00e0 ioniser les prot\u00e9ines a n\u00e9cessit\u00e9 de nombreuses \u00e9tapes d\u2019optimisation (r\u00e9alis\u00e9e pour la d\u00e9tection de prot\u00e9ines en jouant avec la nature et la concentration de sel, le temps d\u2019incubation, les caract\u00e9ristiques physico-chimiques des nanomurs (comme la hauteur, le dopage au bore ou la morphologie). Pour ce faire, le cytochrome C a \u00e9t\u00e9 utilis\u00e9 comme prot\u00e9ine mod\u00e8le, comme il \u00e9tait habituellement d\u00e9crit pr\u00e9c\u00e9demment dans SALDI-MS, \u00e9tant facile \u00e0 d\u00e9sorber. Enfin, des nanomurs de carbone align\u00e9es verticalement ont ensuite \u00e9t\u00e9 modifi\u00e9es \u00e0 l&rsquo;aide de sucres \u00e0 lectine sp\u00e9cifiques (galactosamine), pour la d\u00e9tection directe de la cha\u00eene B de la ricine dans des \u00e9chantillons r\u00e9els, tels que des boissons sans alcool et du s\u00e9rum sanguin. Nous avons obtenu une limite de d\u00e9tection (80 ng\/0.5 \u03bcL) trois fois inf\u00e9rieure \u00e0 la dose l\u00e9tale m\u00e9diane la plus faible (DL50 = 10 \u03bcg\/kg). Cette d\u00e9tection peut \u00eatre r\u00e9alis\u00e9e dans les 10 min.<\/p>\n<p>Les surfaces multifonctionnelles sont d\u00e9crites comme des perspectives pour des outils d&rsquo;analyse bimodaux plus puissants, en combinant deux techniques, telles que: SPR (r\u00e9sonance plasmonique de surface)\/SALDI-MS, SERS (Spectroscopie Raman Exalt\u00e9e de Surface)\/SALDI-MS et EC(\u00c9lectrochimie)\/SALDI-MS. Une attention particuli\u00e8re a \u00e9t\u00e9 port\u00e9e sur la SPR \/ SALDI-MS car elle permet d\u2019obtenir des interactions quantitatives et mol\u00e9culaires en temps r\u00e9el (SPR) et une identification structurelle des analytes (MS). Diff\u00e9rentes m\u00e9thodes de d\u00e9p\u00f4t de mat\u00e9riaux de type graph\u00e8ne ont \u00e9t\u00e9 \u00e9tudi\u00e9es (m\u00e9thode de surfactant \u00e0 bulle d&rsquo;oxyde de graph\u00e8ne, transfert par voie humide de graph\u00e8ne CVD, d\u00e9p\u00f4t \u00e9lectrophor\u00e9tique de graph\u00e8ne, couche par couche en utilisant un polycation et un oxyde de graph\u00e8ne, coul\u00e9e en goutte \u00e0 goutte de graph\u00e8ne et d&rsquo;oxyde de graph\u00e8ne r\u00e9duit) . Le cytochrome C (en tant que prot\u00e9ine mod\u00e8le) a \u00e9t\u00e9 d\u00e9tect\u00e9 en utilisant les trois premi\u00e8res m\u00e9thodes et seuls les peptides ont \u00e9t\u00e9 d\u00e9tect\u00e9s pour les deux derni\u00e8res m\u00e9thodes, l&rsquo;ordre de mention \u00e9tant en corr\u00e9lation avec la diminution de l&rsquo;efficacit\u00e9 de SALDI-MS vis-\u00e0-vis des prot\u00e9ines.<br \/>\nComme ceci, cette th\u00e8se d\u00e9crit le premier capteur direct \u00e0 base de ricine SALDI-MS, capable de d\u00e9tecter une dose inf\u00e9rieure \u00e0 la dose mortelle chez l&rsquo;homme et d&rsquo;apporter une contribution importante \u00e0 la lutte contre d&rsquo;\u00e9ventuelles attaques terroristes. L&rsquo;\u00e9tude syst\u00e9matique de diff\u00e9rents param\u00e8tres qui influencent ce processus LDI-MS est \u00e9galement pr\u00e9sent\u00e9. Les surfaces doubles \u00e9tudi\u00e9es, en particulier les techniques bimodales SPR\/MS, ont pr\u00e9sent\u00e9 une coh\u00e9rence fiable pour les approches ult\u00e9rieures permettant de cr\u00e9er des outils analytiques plus puissants.<\/p>\n<h5>Abstract:<\/h5>\n<p>Bio-terroristic attacks have become more frequent in the past years and the wide range of bio-terroristic agents makes this an important issue to overcome. Ricin is part of the ribosome-inactivating proteins (RIP). RIPs are vegetable toxins, water soluble, which can be easily extracted from plants (ricin from castor beams, abrin from rosary pea) or from bacteria (Shiga toxin). These proteins are composed of two chains: ricin A chain, a glycosidase that insures the toxicity by removal of adenine (depurination) from the RNAr 28S from the 60S ribosomal subunits, followed by the inhibition of protein synthesis, and ricin B chain, a lectin that binds to specific sugar moieties on the surface of the cells, assuring transportation the cell uptake. As they inhibit protein synthesis, depending of the administration take-up (oral, inhalation, intravenously) and the dose received, cell death also occurs. In the absence of efficient counter measurements, detection methods of these toxins have to be fast, reliable, selective and suitable, especially pre-assimilation analysis. The current methods (based on SERS, ELISA, Colorimetric, SPR and MS) do not overcome all these requirements.<br \/>\nEven though mass spectrometry was used for ricin detection, it cannot be performed without long and tedious sample preparation, meaning through enzymatic digestion, extraction\/purification and identification of the proteins based on the peptide analysis, followed by comparison with an existent database (mass fingerprint). In this work, we describe how carbon-based materials (carbon nanowalls and others) can be used as nanostructured materials for specific ricin-like proteins sensors, using surface assisted laser\/desorption ionization mass spectrometry (SALDI-MS) and other techniques. The suitability of the carbon nanowalls (CNWS) was proven initially for other smaller bio-molecules (saccharides, peptides up to 5800 m\/z, carbohydrates, lipids and glucose, which was also quantified using SALDI-MS).<br \/>\nWhen it comes to proteins, they are hard to ionize and detect using SALD-MS, due in part to their big molecular weight. The ability of CNWs to desorb and ionize proteins required a lot of optimization steps of the SALDI-MS method (salt nature, concentration, and pH, incubation time, physicochemical characteristics of the nanowalls, such as height, boron doping and morphology). A systematic optimization was done using a model protein, the cytochrome C). From this, we were able, for the first time, to detect Ricin B chain without the use of organic matrix. To go further in improving Ricin detection performances, carbon nanowalls were then covalently modified using specific lectin sugars (galactosamine) and the ability to detect Ricin B chain in real samples such as soft drinks and blood serum was demonstrated within10 minutes. We obtained a limit of detection (80 ng\/0.5 \u03bcL) that is 3 times lower than the lowest median lethal dose (LD50 = 10 \u03bcg\/kg)<br \/>\nMultifunctional surfaces are described as perspectives for more powerful bimodal analytical tools, by combining two techniques, such as: SPR(Surface Plasmon Resonance)\/SALDI-MS, SERS(Surface Enhanced Raman Spectroscopy)\/SALDI-MS and EC(Electrochemistry)\/SALDI-MS. Special attention was focused on SPR\/SALDI-MS as it can achieve both quantitative and molecular interactions in real-time (SPR) and precise identification of the analytes (MS). Different depositions methods of graphene-like materials were studied to ensure a good surface coverage of the substrate : 1)bubble surfactant method of graphene oxide, 2) wet transfer of CVD pristine graphene, 3) electrophoretic deposition of graphene, 4) layer by layer using a polycation and graphene oxide, 5) drop casting of both graphene and reduced graphene oxide). Cytochrome C (as model protein) was detected using the first three methods and only peptides were detected for the last two methods, as the mentioning order is in correlation with decreasing SALDI-MS efficiency towards proteins.<br \/>\nIn this thesis, we described the first world wide ricin-like proteins SALDI-MS sensor, which is able to detect below the lethal dose in humans and bring an important contribution to the fight against eventual terroristic attacks. The systematic study of different parameters that influence this LDI-MS process is also presented. The dual surfaces studied, in particular the SPR\/MS bimodal techniques, presented reliable consistency for further approaches in creating more powerful analytical tools.<\/p>\n<\/div><\/section>","protected":false},"excerpt":{"rendered":"","protected":false},"author":2,"featured_media":38687,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[8],"tags":[],"class_list":["post-38680","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-actualites"],"_links":{"self":[{"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/posts\/38680","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/comments?post=38680"}],"version-history":[{"count":0,"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/posts\/38680\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/media\/38687"}],"wp:attachment":[{"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/media?parent=38680"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/categories?post=38680"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.iemn.fr\/en\/wp-json\/wp\/v2\/tags?post=38680"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}