Thèse présentée pour l'obtention du grade de Docteur de l'UTC

225 Pages·2017·9.41 MB·French

Par Sofia NESTORA

Molecularly imprinted polymers as selective sorbents

for recognition in complex aqueous samples

Thèse présentée
pour l’obtention du grade
de Docteur de l’UTC

Sou tenue le 13 avril 2017
Spécialité : Biotechnologie : Unité de recherche Génie
Enzymatique et Cellulaire - GEC (FRE-3580)
D2346
Dissertation
submitted for the obtention of the degree of

DOCTEUR DE L’UNIVERSITÉ DE TECHNOLOGIE DE COMPIÈGNE

Spécialité : Biotechnologie

By

Sofia Nestora
MOLECULARLY IMPRINTED POLYMERS
AS SELECTIVE SORBENTS
FOR RECOGNITION IN COMPLEX AQUEOUS SAMPLES

Thesis supervised by Prof. Karsten Haupt, Prof. Claire Rossi and Dr. Bernadette Tse Sum Bui

Disputation on April, 13th 2017, in front of a jury composed of:

Oliver BRÜGGEMANN, Prof. Johannes Kepler University of Linz, Austria Reviewer
Dietmar KNOPP, Prof. Technical University of Munich, Germany Reviewer
Andrew GREAVES, Dr. L’Oréal-Paris, France Examiner
Valerie PICHON, Prof. École Supérieure de Physique et de Chimie Industrielles, France Examiner
Karsten HAUPT, Prof. Université de Technologie de Compiègne, France Supervisor
Claire ROSSI, Prof. Université de Technologie de Compiègne, France Supervisor
Bernadette TSE SUM BUI, Dr. Université de Technologie de Compiègne, France Supervisor

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Thèse
En vue de l’obtention du grade de

DOCTEUR DE L’UNIVERSITÉ DE TECHNOLOGIE DE COMPIÈGNE

Spécialité: Biotechnologie

Par

Sofia Nestora
POLYMERES A EMPREINTE MOLECULAIRES
EN TANT QU’ADSORBANTS SELECTIFS POUR LA RECONNAISSANCE
DANS DES MILIEUX AQUEUX COMPLEXES

Thèse dirigée par Prof. Karsten Haupt, Prof. Claire Rossi et Dr. Bernadette Tse Sum Bui

Souten(cid:88)(cid:72) le Jeudi 13 Avril 2017 devant le jury composé :

Oliver BRÜGGEMANN, Prof. Johannes Kepler University of Linz, Austria Rapporteur
Dietmar KNOPP, Prof. Technical University of Munich, Germany Rapporteur
Andrew GREAVES, Dr. L’Oréal-Paris, France Examinateur
Valerie PICHON, Prof. École Supérieure de Physique et de Chimie Industrielles, France Examinatrice
Karsten HAUPT, Prof. Université de Technologie de Compiègne, France Directeur
Claire ROSSI, Prof. Université de Technologie de Compiègne, France Directrice
Bernadette TSE SUM BUI, Dr. Université de Technologie de Compiègne, France Directrice
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To my parents for all their love and support

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Acknowledgements
First, I would like to express my gratitude to Prof. Dr. Oliver Bruggemann and Prof. Dr. Dietmar
Knopp for taking the time to review my thesis as well as to Prof. Dr. Valérie Pichon, Dr. Andrew Greaves
and for being the examiners of this work.
I am sincerely thankful to my three supervisors Prof. Dr. Karsten Haupt, Dr. Bernadette Tse Sum Bui
and Prof. Dr. Claire Rossi for their constant help and advice all these years. Karsten, thank you for giving
me the opportunity to join the MIP group, it was a whole new world for me. I am deeply greatful for your
ideas and our fruitful discussions. Bernadette, thank you for your guidance, your numerous advices and
your persistence to perfection. You were the first to believe in me and taught me so much these three years.
It wasn’t always easy, but in the end it was a life lesson for me. Claire, warm thanks to you for your positive
attitude, your help and your constant availability.
There are so many people that I am grateful, from all these years in Compiegne, old and new, people
who left and people that are still here. I sincerely thank all the members of the Enzyme and Cell Engineering
Laboratory for their help and nice working environment. Aude, thank you for all your advice, your
“chemistry classes” and your delicious tartiflette. Lumi, I am deeply grateful for your help and kindness
and for the critical reading of this thesis. Elise, thank you for teaching me a bit of NMR, for improving my
French and for your friendship. Franck, thank you for your help and patience with the endless experiments
for Chapter 2. Sylvie, I am thankful for your kindness and your support. Special thanks to you Maria,
Paulina and Yannick for contributing to this work.
I would like to thank all the people from my first years in Compiegne, Serena, Zeynep, Jacqui, Li
Bin, Jean, Doudou, Laetitia, Melody, Paolo, Mira, Xuan-Anh, Selim, Carlo, Yi, Marcelina etc. You each
have a place in my heart and I have so many moments to cherish: the crazy party at the old place of Melody
in the pool with a watermelon, Paolo smoking and talking NMR, Jacqui dancing, Sere swearing in Italian,
Zeynep cooking these delicious salmon rolls. Thank you guys for giving me such happy memories. Doudou,
special thanks to you for being always there for me.
What can I say for all the people that we started the PhD life together? My office mates Paulina and
Jing Jing and the rest of the gang Maria, Nadie, Frank, Nicolas and Hassan. Jing Jing, you are my Chinese
soul mate and sister, I will waini you always. Pau, thank you so much for your friendship, your help and
positiveness. Maria, we shared so many things together, our sorrows and our happy moments, life in
Compiègne would not be the same without you. Nadie, you know I will always owe you for THAT night,
thank you for all the crazy (and unique) memories and for your friendship. Frankie, you left us and went to
Bordeaux but I forgive you, because you are the best.
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Also thanks to the people that come to our lab for a short time, Martins (crazy Latvian guy), Bilo
(crazy surtuk), Leena, Ola, Yolanda and Cecilia, I thank you for all the good moments. Mariano, it was
such a pleasure to meet you, I will always remember your nice stories and T-shirts and baby Alexandro.
How can I forget the few Greek people in Compiegne?! My dear friend Maria Gad, we came to France
together as Erasmus students and we shared some of the most wonderful experiences and I thank you for
being there with me. Maria, Maria M. and Kostas, Chrysanthi and Stelios thank you for being my Greek
family in France.
I would like also to thank all the people from every place in the world that I have meet during my
thesis. My girls, Marina, Christina, Lauri and Emna as well as Diego, Felipe, Yeli, Alexandra, Heronimo,
Florian etc. and my first friends in France, Chiara, Tommaso and Mehdi.
I will also include to my acknowledgerments my Greek friends, it was such a warm feeling to see you
when back home! Aristea, Karolina, Vicky, Maria, Georgia, Nena, Natalia and Eleutheria thank you for
making my life full of nice moments, for your support and compassion.
Finally, I would like to thank my parents for their endless love, support and patience and for their
constant encouragement. Thank you for all you have given me and for the person that I have become. Colin,
I cannot thank you enough being there for me during the last months of my thesis, for your motivation,
your care and your love. You kept me going when I had lost hope and I love you so much for that.

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Abstract in English: Molecularly imprinted polymers as selective sorbents for
recognition in complex aqueous samples

Keywords: molecularly imprinted polymer, synthetic receptor, plastic antibodies, stoichiometric monomer,
deodorant, body odor, cosmetics, solid phase extraction, betanin, beetroot

In this thesis, we have demonstrated the feasibility of preparing highly selective molecularly imprinted
polymers (MIPs) for recognition in complex aqueous matrices with applications in cosmetics and food
technology.
MIPs are synthetic tailor-made receptors, with binding affinities and specificities comparable to those of
natural antibodies. Their molecular recognition properties, combined with their high stability, mechanical
robustness, low cost and easy synthesis make them extremely attractive as selective capture materials with
applications in analytical and preparative separations, sensing and drug delivery, among others. However, their
selective recognition in aqueous samples still remains problematic and is one of the reasons for their so far lilited
commercial expansion.
In the first part, we developed a water compatible MIP for its application as an active ingredient in a
deodorant. Body odors are mainly due to volatile fatty acids generated from their glutamine conjugate precursors
by hydrolytic enzymes from bacteria present on the skin. Most currently marketed anti-perspirants and
deodorants contain, respectively aluminum salts and unspecific antibacterials. However, the extremely wide use
of these products requires alternative solutions with regard to various problems (environmental, respect of skin
ecosystem, toxicity, etc.). For this reason, a MIP was developed to capture the glutamine conjugate precursors
so that they are no longer availaible to the bacteria, thus preventing their transformation to malodorous
compounds. In order to generate binding selectivity in aqueous environments, an amidinium-based monomer
which can form a strong stoichiometric electrostatic interaction with the carboxyl groups on the template, was
synthesized. The MIP, blended in a dermo-cosmetic formulation, could capture selectively the glutamine
precursors, amidst a multitude of other molecules present in human sweat. Furthermore, the MIP did not affect
the skin bacteria, paving the way to an innovative and ‘safer’ future-generation deodorant.
In the second part, we developed a fast and efficient procedure based on molecularly imprinted solid-
phase extraction (MISPE) for the selective clean-up of betanin and its stereoisomer isobetanin from red beetroot
extracts. Betanin is a natural pigment with significant antioxidant and biological activities currently used as food
colorant. Dipicolinic acid was used as template for the MIP synthesis, because of its structural similarity to the
chromophore group of betanin The MISPE procedures were optimized allowing the almost complete removal
of carbohydrates and the majority of proteins, resulting in high extraction recovery of betanin/isobetanin in a
single step. Moreover, the whole extraction procedure was performed in environmentally friendly solvents with
either ethanol or water.
To conclude, we believe that this study paves the way towards the development of a new generation of
water compatible MIPs with improved recognition properties in highly complex aqueous environments, and
should be applicable to other biotechnological and biomedical areas as well.
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Résumé en français : Polymères à empreinte moléculaires en tant qu’adsorbants sélectifs
pour la reconnaissance dans des milieux aqueux complexes

Mots-Clés : polymères à empreinte moléculaires, récepteur synthétique, anticorps plastiques, monomer
stoechiométrique, déodorant, odeur corporelle, extraction sur phase solide, bétanine, betterave

Dans cette thèse, nous avons démontré la faisabilité de la préparation de polymères à empreinte
moléculaires (MIP) hautement sélectifs pour la reconnaissance dans des matrices aqueuses complexes avec des
applications dans les cosmétiques et en technologie alimentaire.
Les MIP (de l’anglais molecularly imprinted polymers) sont des récepteurs synthétiques comparables aux
anticorps, qui sont synthétisés par co-polymérisation de monomères fonctionnels et réticulants en présence d'un
gabarit moléculaire. Leurs propriétés de reconnaissance moléculaire, associées à leur grande stabilité, robustesse
mécanique, faible coût et leur synthèse facile les rendent extrêmement intéressants comme matériaux de capture
sélective, avec des applications dans les séparations analytiques, la détection et la vectorisation des
médicaments. Cependant, leur reconnaissance sélective dans des milieux aqueux reste toujours problématique
et c’est l'une des raisons de leur expansion commerciale restreinte.
Dans une première partie, nous avons développé un MIP fonctionnant en milieu aqueux pour son
application comme ingrédient actif dans un déodorant. Les odeurs corporelles sont principalement dues à des
acides gras volatils générés à partir de leurs précurseurs, des conjugués de glutamine par des enzymes
hydrolytiques produites à partir de bactéries présentes sur la peau. La plupart des anti-transpirants et des
déodorants actuellement commercialisés contiennent des sels d'aluminium et des agents antibactériens non
spécifiques, respectivement. Cependant, l'utilisation extrêmement étendue de ces produits nécessite des
solutions alternatives en ce qui concerne divers problèmes (environnement, respect de l'écosystème de la peau,
toxicité, etc.). Pour cette raison, un MIP a été synthétisé pour capturer les précurseurs conjugués de glutamine
afin qu'ils ne soient plus disponibles aux bactéries, empêchant ainsi leur transformation en composés
malodorants. Afin de générer des liaisons selectifs dans des environnements aqueux, un monomère à base
d'amidinium qui peut former une interaction électrostatique stoechiométrique forte avec les groupes carboxyle
sur le gabarit moleculaire a été synthétisé. Le MIP, mélangé dans une formulation dermo-cosmétique, pourrait
capter sélectivement les précurseurs conjugués de glutamine, au milieu d'une multitude d'autres molécules
présentes dans la sueur humaine. En outre, le MIP n’ affecte pas les bactéries de la peau, ouvrant la voie à des
déodorants innovateurs de nouvelle génération, moins problématiques pour la santé.
Dans une deuxième partie, nous avons développé une procédure rapide et efficace basée sur l'extraction
en phase solide à empreinte moléculaire (MISPE) pour la purification sélective de la bétanine et de son
stéréoisomère l’isobétanine à partir d'extraits de betterave. La bétanine est un pigment naturel ayant un fort
pouvoir antioxydant et dont les propriétés pharmacologiques sont de plus en plus étudiées. Ce pigment est
actuellement utilisé comme simple colorant alimentaire. Dans notre étude, l'acide dipicolinique a été utilisé
comme gabarit moleculaire pour la synthèse de MIP, en raison de sa similarité structurelle avec le groupe
chromophore de la bétanine. Les procédures MISPE ont été optimisées permettant l'élimination presque
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