d’ordre: 2007-12 Année 2007

THESE EN COCO----TUTELLETUTELLE

entre l’Ecole Centrale de Lyon, France (Ecole doctorale Electronique, Electrotechnique, Automatique )

et l’Institut de Biologie Moléculaire et Génétique, Kyiv, Ukraine (Discipline : Biotechnologie)

présentée devant

Ecole Centrale de Lyon

pour obtenir le grade de

DOCTEUR d’ECOLE CENTRALE DE LYON

soutenue publiquement le 15 juin 2007

par

Iryna BENILOVA née SKSKRYSHEVSKARYSHEVSKA

AppApprocheroche « biocapteur » pour sonder la bioaffinité et les interactions biocatalytiques de petits xénobiotiques

Jury:

Dr. Nicole JAFFREZIC-RENAULT Présidente Pr. Roland SALESSE Rapporteur Dr. Alexandr KUKLA Rapporteur Pr. Claude MARTELET Directeur de thèse (France) Pr. Alexey SOLDATKIN Directeur de thèse (Ukraine) Dr. Sergey DZYADEVYCH Examinateur

AcknowledAcknowledggggmentsments

The present work was carried out in the former laboratory CEGELY (now AMPERE) of Ecole Centrale de Lyon (ECL) in collaboration with the Laboratory of Biomolecular Electronics (LBME) in the Institute of Molecular Biology and Genetics (IMBG) of National Academy of Sciences of Ukraine (NASU). These institutions are gratefully acknowledged. My work in French laboratories was financially supported by EGIDE (France).

I wish to thank my French supervisors, Professor Claude Martelet and Professor Nicole Jaffrezic-Renault for accepting me in CEGELY for my “co- tutelle” thesis. I am infinitely grateful for their confidence in me, their priceless help and generosity and, of course, for our fruitful scientific discussions during these years. I warmly thank Academician Anna V. El’skaya, the Director of IMBG NASU, and my Ukrainian supervisor, Professor Alexey P. Soldatkin, the Head of LBME, for giving me the chance to start my research work in the LBME and to continue it in the Ecole Centrale de Lyon. Their constant scientific and moral support has been very precious to me. I am also very grateful to Pr. Roland Salesse, Dr. Alexandr Kukla and Dr. Sergey Dzyadevych for accepting to judge this work and for their opinions.

Warm thanks to Pr. Didier Leonard and Dr. Francois Bessueille from Laboratoire de Sciences Analytiques (Université Claude Bernard – Lyon 1) for their gentleness and kind help with some manipulations. I would like to acknowledge Drs. Valentina Arkhypova, Alexandr Rachkov and Yaroslav Korpan (LBE IMBG) for all their priceless help, support and sympathy. I thank Drs. Vladimir Chegel and Yuri Ushenin from the Institute of Semiconductor Physics NASU, for initiating me in optical biosensors, for their professional advices and especially for our fruitful “New-Year collaboration” in December 2006-January 2007 which gave rise to the Part 6 of this thesis.

I would like to take the opportunity to thank Dr. Edith Pajot from INRA de Jouy-en-Josas, for her kindness, patience and all priceless help given me during all the time of my work with olfactory receptors.

My special thanks to Mmes Monique Lacroix, Anne Zucco, Josiane Chabert, Maryline Bonnefoi and Mr. Philippe Billoux for their precious help in my démarches administratives . I am also very grateful to Mme Nina R. Polischuk for editing my first article in English.

I want to warmly thank all my colleagues and friends for the unforgettable time we have had together: i) Ali, Basma, Chaker, Houcine, Imen, Mauricio, Mouna, Rita, Rodicka, Walid-1 (Hassen), Zhichang and all others from AMPERE and UCBL-1; ii) Bedja, Chaiyan, Jean-Hubert, Lamine, Lazar, Le Ha, Lucas, Siméon, Thanh, Walid-2 and all others from H9 of ECL; iii) Alena, Andrusha, Lyuda, Nadia and Dima, Oleg, Pasha, Sanya, Sasha Nikolaich and Yulia, Sasha Yashkin, Vitalik and all others from Taras Shevtchenko University (Kyiv) and IMBG NASU. Special thanks to Sveta for her help in navigating the French capital, and to Anya for our pleasant colocation (April-May 2007).

Finally, I would like to thank my parents, my parents-in-law and my husband Arthur for their constant and unconditional support: nothing would have been possible without it.

TTTableTable of contents

INTRODUCTION GENERALE 1 GENERAL INTRODUCTION 3 BIBLIOGRAPHIC REVIEW 5 1. Definition of a biosensor. 5 2. Transducer vs biorecognition event. Proper choice of detection mode. 6 3. Potent macromolecular targets of xenobiotics. 9 3.1.Biocatalytic recognition elements. 9 3.1.1. Enzymes. 9 3.1.2. Ribozymes. 10 3.2. Bioaffinity recognition elements. 11 3.2.1. Antibodies. 11 3.2.2. Lipocalins and anticalins. 12 3.2.3. Aptamers. 13 3.2.4. G protein-coupled receptors. 14 4. Immobilization of biorecognition element. 15 4.1. Covalent attachment. 16 4.2. Electrochemical attachment. 20 4.3. Physical attachment. 20 4.4. Entrapment. 22 5. Bulk properties of biofilms. 22 6. “Apparent” and “true” affinity. 24 7. Concluding remarks. 29

CHAPTER 1. Glycoalkaloids and cholinesterases 31 PART 1. Potato glycoalkaloids: true safety or false sense of security? 35 1. Introduction. 36 2. Potato glycoalkaloids and health. 37 3. Detection of potato GAs in foodstuffs and biological fluids. 39 4. Biotechnological aspects of potato breeding. 40 5. Concluding remarks. 43 PART 2. Cholinesterases inhibition by glycoalkaloidsglycoalkaloids:::: probing with potentiometric biosensorbiosensor.... 45 1. Introduction. 46 2. Experimental. 48 2.1. Materials. 48 2.2. pH-FETs. 49 2.3. Enzyme immobilization. 49 2.4. Measurements. 49 3. Results and discussion. 50 3.1. Optimal pH. 50 3.2. Specificity towards substrates. 51 3.3. Specificity towards inhibitors. 54 4. Conclusion and perspectives. 60 PART 333.3... Impedimetric study of glycoalkaloids binding to cholinesterase. 61 1. Introduction. 62 2. Experimental. 63 2.1. Materials. 63 2.2. Working electrodes. 64 2.2.1. Construction. 64 2.2.2. Surface cleaning. 64 2.2.3. Biofunctionalization. 64 2.3. Impedance measurements. 65 3. Results and discussion. 65 3.1. Biofilm stability. 65 3.2. Electrochemical characteristics of protein layers. 66 3.3. Impedimetry of glycoalkaloids. 67 3.4. Calibration curves. 70 4. Conclusion and perspectives. 72

CHAPTER 2. Odorants and olfactory receptors 73 PART 4. Natural, electronic and bioelectronic olfaction. 77 1. Introduction. 78 2. Detection of odorants. 79 3. From electronic to bioelectronic noses. 80 4. Concluding remarks. 83 PART 5. Electrochemical studstudyy of human olfactory receptor OR 1717----4040 stimulation by some odorantsodorants.... 85 1. Introduction. 86 2. Experimental. 87 2.1. Biomaterials and chemicals. 87 2.2. Single channel SPR spectrometer and gold coated substrates. 88 2.3. Pretreatment of sensor surface. 88 2.4. Self-assembly of the mixed layer on gold. 88 2.5. Blocking step and formation of the upper supporting layers. 89 2.6. Preparation and immobilization of OR 17-40. 89 2.7. Electrochemical probing. 90 2.8. Preparation of odorant solutions. 91 2.9. Monte-Carlo simulation. 91 3. Results and discussion. 92 3.1. Optical and electrochemical monitoring of biofilm assembly. 92 3.1.1. SPR monitoring. 92 3.1.2. Impedance monitoring. 95 3.2. Impedance study of already biofunctionalized SPR chips. 96 3.3. Impedance measurements in the presence of odorants at 20ºC. 98 3.4. Impedance measurements at 4ºC in the presence of odorants and GTP-K-S. 100 3.5. Impedimetric screening unrelated odorants. 104 3.6. Modeling the impact of nanosomes size on their anchoring. 105 4. Conclusion and perspectives. 107 PART 66.. Response pattern of human olfactory rreceptoreceptor OR 1717----4040 ppprobedprobed by surface plasmon resonanceresonance.... 109 1. Introduction. 110 2. Experimental. 110 2.1. Biomaterials and chemicals. 110 2.2. Sensor substrates and SPR spectrometer. 111 2.3. Pretreatment of sensor surface. 111 2.4. Two architectures of biofilms. 111 2.5. Preparation and immobilization of OR 17-40. 111 2.6. Preparation of odorants. 112 2.7. Cyclic voltammetry. 112 2.8. AFM. 113 3. Results. 113 3.1. Orientated and random immobilization of Ab. 113 3.2. Electrochemical properties of biofilms. 117 3.3. AFM study of biofilms. 118 3.4. Detection of odorant molecules. 120 4. Discussion. 123 5. Conclusion and perspectives. 125

CONCLUSION GENERALE ET PERSPECTIVES 127 GENERAL CONCLUSION AND PERSPECTIVES 129

ANNEX A. Potentiometry based on ISFETs (pH-FETs). 131 ANNEX B. Electrochemical impedance spectroscopy (EIS). 133 ANNEX C. CCC-C---1.1.1.1. Single channel SPR spectrometer and gold coated chips. 137 CCC-C---2.2.2.2. Double channel SPR spectrometer and gold coated chips. 140 ANNEX D. Plasmids designed for the heterologous expression of human olfactory receptor 17-40 and protein GM olf in yeast Saccharomyces cerevisiae (strain MC 18). 141 ANNEX E. EEE-E---1111. Preparation of helional, heptanal and blank probes for screening in the double channel SPR spectrometer. 143 EEE-E---2.2.2.2. Protocol of odorant screening in the double channel SPR spectrometer in differential mode. 144 EEE-E---3.3.3.3. Preparation of octanal, nonanal and vanillin. 144

BIBLIOGRAPHY 145 Publications et communications scientifiques 167 List of abbreviations

Ab(s) antibody(-ies) AcChE acetyl cholinesterase AcChCl acetylcholine chloride AEAEAE auxiliary electrode AFAFAFMAF MMM atomic force microscopy Biotinyl PEA biotinylated phosphoethanol amine BSA bovine serum albumin BuChE butyryl cholinesterase BuChCl butyrylcholine chloride cAMP cyclic adenosine monophosphate CPE constant phase element CVCVCV cyclic voltammetry DMSO dimethylsulfoxide DNA desoxyribonucleic acid eBuChE equine butyryl cholinesterase EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EIS electrochemical impedance spectroscopy EST2 carboxyl esterase FRFRFR flow rate GA(s) glycoalkaloid(s) GC/MS gas chromatography/mass spectrometry GPCR(s) G protein-coupled receptor(s) GTP guanosine triphosphate hBuChE human butyryl cholinesterase HPLC high performance liquid chromatography ISFET ((pHpHpHpH----FET)FET) ion (pH) sensitive field-effect transistor IUPAC International Union of Pure and Applied Chemistry LAPS light addressable potentiometric sensor MAb monoclonal antibody MHDA mercaptohexadecanoic acid MOS metal-oxide sensors MOSFET metal-oxide-silicon field effect transistor MWMWMW molecular weight NHS N-hydroxysuccinimide OBP odorant binding protein OR(s) olfactory receptor(s) PAb polyclonal antibody PBS phosphate buffered saline PrPrPrAPr AAA protein A PrG protein G PWR plasmon-waveguide resonance QCM quartz crystal microbalance RNA ribonucleic acid SAM(s) self-assembled monolayer(s) SCE saturated calomel electrode SPR surface plasmon resonance STI soybean trypsin inhibitor TMTMTM transmembrane WEWEWE working electrode XPS X-ray photoelectron spectroscopy

Introduction générale

De nos jours, les industries pharmaceutique et biotechnologique consacrent beaucoup d’efforts à la découverte de nouveaux médicaments et à l’analyse de petits xénobiotiques. Ceci comprend la modélisation moléculaire [1] et le test en routine [2] d’ agonistes et d’antagonistes de biomacromolécules comme les enzymes et les récepteurs impliquées dans les voies métaboliques principales. Les bioessais de petites molécules les plus répandus se basent sur le marquage et sont menés sur des levures Saccharomyces cerevisiae génétiquement modifiées [3-5]. Récémment une approche appelée « empreintes affines» [6] a été proposée pour explorer l’affinité de couplage de petites molécules et des protéines de panel représentatif. Cette technique basée sur le marquage peut également servir à l’élucidation de méchanismes de supression des enzymes par des inhibiteurs [7].

Biocapteurs et plateformes biosensibles sans marquage ont montré être des outils très fiables dans un domaine de quantification de xénobiotiques [8- 10], mais ils n’ont jamais été pleinement engagés en tant qu’instruments à sonder la biochimie de petites molécules. Ceci est dû au fait que les techniques d’analyse en phase hétérogéne sont souvent suspectées d’altérer les «vraies» constantes soit affines soit catalytiques. Les méthodes d’immobilization de macromolécules de bioreconnaissance, les propriétés principales des biofilms, les affinité « vraie » et « apparente » sont discutés dans une revue bibliographique . La partie expérimentale de ce travail est présentée en deux chapitres chacun étant consacré à l’application des biocapteurs sans marquage aux études d’interactions soit enzyme-inhibiteur (antagoniste) soit récepteur- 1 odorant (agoniste) en phase hétérogène. Le premier chapitre traite des glycoalcaloïdes, principalement de pomme de terre et de tomates, et leur impact sur les butyryl cholinestérases de sérum humain et équin. Afin de mieux comprendre la motivation pour le contrôle des glycoalkaloïdes dans les nouvelles variétés de pomme de terre, nous présentons tout d’abord une revue sur ces petites toxines naturelles dans une première partie de ce manuscrit. La deuxième partie décrit l’inhibition des cholinestérases par l’:-solanine, l’:- chaconine et la tomatine, étudiée en employant un biocapteur potentiométrique. Nous avons montré que l’approche « biocapteur» ne fournit pas uniquement l’information sur le pourcentage d’inhibition (données de basse qualité selon le point de vue de la chimie medicale), mais aussi permet de déterminer les constantes cinétiques et le mode d’inhibition pour chaque composant étudié. La troisième partie est consacrée à l’interaction bioaffine de glycoalcaloïdes de pomme de terre avec la cholinestérase immobilisée, étudiée en l’absence de substrats enzymatiques. Dans un premier temps, les efficacités bioaffines des glycoalcaloïdes ont été determinées en utilisant la spectroscopie d’impédance électrochimique et ont été comparées à leur impact sur la biocatalyse. Le deuxième chapitre de ce mémoire de thèse traite du récepteur olfactif humain RO 17-40 couplé à la protéine G, employé dans deux biocapteurs, électrochimique et optique en tant qu’unité complexe capable de bioreconaissance des molécules odorantes. La quatrième partie décrit l’exceptionnelle diversité chimique des molécules odorantes et les techniques actuelles de leur analyse en routine. Un concept de « nez bioélectronique » est introduit en liaison avec l’olfaction artificielle. Afin d’obtenir une bonne sensibilité du RO 17-40 entouré de sa fraction membranaire, sur la surface d’une électrode, des architectures biomoléculaires de types différents ont été construites et characterisées dans la cinquième et la sixième parties de manuscrit. Une attention spéciale a été portée au contrôle de caractéristiques des nanosomes – vésicules membranaires portant des récepteurs. Enfin, le modèle de réponse de RO 17-40 à son odorant agoniste hélional a été montré être similaire au profil pharmacologique, en forme de cloche, établi dans les nombreux bioessais utilisant des lignées cellulaires.

2

General introduction

Nowadays, great efforts in pharmaceutical and biotechnology industry are devoted to the discovery of new drugs and small xenobiotics analysis. This involves both molecular modeling [1] and routine screening [2] agonists and antagonists of biomacromolecules, namely, enzymes and receptors responsible for the crucial metabolic pathways. The most widespread bioassays of small compounds are label-based and carried out on the genetically modified budding yeast Saccharomyces cerevisiae [3-5]. Recently, the “affinity fingerprints” approach [6] was proposed to probe the affinity of small molecules binding with a representative panel of proteins. Such label-based screens may also serve for elucidation of mechanisms of inhibitors action on biocatalysts [7].

Label-free biosensors and biosensing platforms have proven to be very potent tools for xenobiotics quantification [8-10], but they have never been fully exploited as tools for probing biochemistry of small molecules. It is mainly due to the fact that surface-based analytical methods are often suspected to alter the “true” binding/catalytic constants of systems under investigation. Variety of conventional and new biorecognition macromolecules which can be implied in xenobiotics screening, methods of their immobilization onto solid transducers, key bulk properties of sensitive biofilms, as well as the concept of “apparent” and “true” affinity will be discussed in the bibliographic review . The experimental work presented in this thesis is mainly focused on label-free biosensor application for studying enzyme-inhibitor (antagonist) and receptor-odorant (agonist) interactions in heterogeneous phase. It consists of two chapters . The first chapter deals with main potato and tomato glycoalkaloids and their impact on human and equine sera butyryl

3 cholinesterase. In order to better explain the need in the routine analysis of glycoalkaloids species, a review dealing with security problems connected with potent toxicity of potato varieties is given in the first part of the manuscript. In the second part , we investigate inhibition of cholinesterases by :-solanine, :- chaconine and tomatine probed by means of potentiometric biosensor. It has been clearly shown that biosensor approach could provide not only percent inhibition information (“low-quality data” from the medicinal chemistry point of view), but also kinetic constants and inhibition characteristics for each tested compound. The third part is devoted to the bioaffinity interaction of immobilized cholinesterase with potato glycoalkaloids in the absence of enzyme substrates. For the first time, bioaffinity potencies of alkaloids have been screened by electrochemical impedance spectroscopy and then compared to their previously estimated impact on biocatalysis. The second chapter of this thesis deals with G protein coupled human olfactory receptor OR 17-40 employed as a complex biorecognition unit in electrochemical and optical biosensing of some odorant molecules in heterophase. The fourth part gives an introduction on exceptional chemical diversity of odorants and modern techniques of their routine analysis. A concept of “bioelectronic nose” has been presented in connection with the artificial olfaction. To achieve a good sensitivity of the membrane-bound OR 17-40, different types of biosensitive molecular architecture have been designed and characterized in fifth and sixth parts. Special attention has been paid to controlling the characteristics of receptor-bearing membrane vesicles – nanosomes. Finally, the pattern of response of immobilized OR 17-40 to its agonist helional has been revealed and compared to the pharmacological profiles established in numerous cell-based bioassays.

4

BibliograBibliographicphic review

1.1.1. Definition of a biosensor

The definition of a “biosensor” given in Encyclopedia of Sensors (2006) is as follows: “Biosensor is a device that converts the modification of physical or chemical properties of a biorecognition element occurring as a result of biochemical interactions in an electrical signal with an amplitude depending on the concentration of the defined analyte in the solution” [11]. A biosensor consists of two basic components connected in series: a biorecognition element ( biofilm ) and a physicochemical transducer . According to the IUPAC recommendations, a biosensor should be considered as a chemical sensor in which the recognition system utilizes a biochemical mechanism [12]. The biorecognition element of sensor translates information on analyte concentration into a biochemical output signal with a defined sensitivity and selectivity. The transducer part also called detector , sensor or electrode , converts the biochemical signal from an output domain of biorecognition system into the electrical signal [12]. There are some limitations in the use of the term “biosensor” to be underlined. As a biosensor is a self-contained integrated device, it should be distinguished from the analytical systems which require additional separation steps, hardware and/or sample processing. The two principal types of biorecognition elements which can be used in biosensor design are biocatalytic and bioaffinity ones. The first one is based on biocatalysts (mostly, enzymes and whole cells) coupled to the transducer. Transient (kinetic) or steady-state (stationary) response to substrate injection can be detected by such a system. Monitoring modulation of biocatalysis by

5 effectors (activators and inhibitors) can provide information about biochemical potencies of tested analytes. The second type of biorecognition element is based on bioaffinity interactions of macromolecules like antibodies, oligonucleotides, receptors etc. For analytical applications, the equilibrium responses are useful since they provide data about saturation of “binding capacity” of biofilm. On the other hand, kinetics of association and dissociation of biocomplex can be also successfully monitored by means of biosensors. The biocatalytical sensors are the best developed, while the bioaffinity ones have not yet reached such advanced stage due to some intrinsic properties which complicate the analysis of real samples [12].

2.2.2. TraTransdnsdnsducerucer vs biorbiorecognitionecognition event. Proper choice of detection mode

It is worth noting that a label-free biosensor becomes a powerful screening tool in the case when a chosen biosensitive element maintains all indispensable recognition properties and a chosen transducer is really able of sensing a desired molecular event. Here we will briefly overview the principal detection techniques in respect to the biochemical event to be measured. Small xenobiotics of biochemical interest include volatile and soluble compounds with MW <1000 [13]. Drugs, foreign odorants and pheromones, pesticides and natural toxins normally correspond to the xenobiotics definition [14]. Most in vivo effects of small xenobiotics are represented by affinity binding with target biomacromolecules followed by formation of biorecognition complex. The latter usually comprises conformational changes in target molecule followed by: i) catalytic transformations (in case of enzymes and rybozymes) or ii) triggering a signal cascade (in case of receptors). Different direct measurement techniques have been elaborated to detect reliably these biochemical events when occurring in heterophase. Three types of effects may be taken as more or less specific “reporters” of bioaffinity binding:

6 i) alterations in the intrinsic properties of immobilized macromolecule, e.g. changes in conformation of one or even several functionally coupled biomolecules; ii) interfacial effects which occur in biorecognition layer near the solid electrode surface, e.g. changes in mass, refractive index/dielectric permittivity, pH, electron transfer kinetics etc; iii) bulk phenomena which can be detected as alteration of bulk solution physical chemistry, e.g. ionic conductivity, pH, refractive index, temperature etc.

Changes occuring at the electrochemical interface (i.e. between metal and electrolyte) are widely exploited in biosensor elaboration, while changes in the intrinsic properties of macromolecules have attracted scientific interest comparatively recently [15, 16]. For example, plasmon-waveguide resonance (PWR) spectroscopy has been proposed as a new optical label-free tool for studying conformational changes of immobilized proteins, especially membrane-bound ones [15].

The most developed and commonly reported in the literature biosensors are based on the electrochemical detection methods [12], namely, amperometry, AC conductimetry and potentiometry. During the last 2 decades, these methods have been developed in cooperation in the Laboratory of Biomolecular Electronics (Kyiv) and in the research laboratories of Ecole Centrale de Lyon and Claude Bernard – Lyon 1 University, where the present thesis work was carried out. Recent achievements in the field of amperometric and conductimetric catalytic biosensors are well illustrated in PhD “co-tutelle” thesis defended in the UCBL by O. Schuvailo ([17], 2006), and PhD thesis defended in the ECL by M. Marrakchi ([18], 2006). The key theoretical principles of the work on potentiometric transducers related to their application in bioanalytical practice, technological aspects of their production, and measurement schemes with set-ups are discussed in details in the recent review of S. Dzyadevych [19]. In the Chapter 1 of this thesis, we will deal with potentiometry based on the pH-sensitive field-effect transistors (pH-FETs).

7 Electrochemical impedance spectroscopy (EIS) coupled to data fitting software is a multipotent electrochemical platform able to investigate the bulk and interfacial phenomena separately thus providing precise information on broad spectra of heterophase phenomena [20]. Now, this method is one of the principal analytical tools in the Laboratoire de Sciences Analytiques (UCBL). In the Chapters 1 and 2, the outstanding potencies of EIS in the monitoring of interfacial biochemical events will be shown.

Interestingly, modern electrochemical detection of small molecules consists of nothing but monitoring of biocatalytic transformations of substrates and impact of effectors on this catalysis. Therefore, the range of biorecognition events which could be detected and characterized electrochemically (except by the EIS) is rather narrow. The possibility of direct monitoring affinity binding has been successfully achieved in the optical and gravimetric sensor platforms, namely, surface plasmon resonance (SPR) spectroscopy, surface acoustic waves (SAW) and quartz-cristal microbalance (QCM) techniques.

As one can see from the increasing number of papers (especially in Analytical Biochemistry, Sensors and Actuators etc.) devoted to the SPR spectroscopy, the latter has become the one of the most advanced and reliable methods of studying bioaffinity in heterophase. SPR sensors principle and main technological achievements are outlined in works of J. Homola et al. [21- 23]. Actually the leading commercial device based on the SPR effect is Biacore (Uppsala, Sweden), nevertheless, a few similar but less expensive devices have also been developed and recognized in scientific world. A prominent example is SPR spectrometers with single and double optical channel elaborated by Prof. Yu. Shirshov’s team in the Institute of Semiconductor Physics (Kyiv) [10, 24- 27]. Single channel spectrometer and recently approved double channel device will be introduced in Chapter 2.

Table 1 summarizes some well-developed transducers and detection techniques capable of small molecules label-free sensing, in respect to the target macromolecules (see in the next section) and biochemical events taking place.

8 Table 111.1... Transduction modes in small molecules analysis

Biorecognition Biochemical Transduction AAAnalyteAnalyte element events mode Binding, Olfactory receptors; EIS, QCM, SPR, Odorants conformation lipocalins PWR, SAW etc. changes Substrates Enzyme Catalysis and its Electrochemical (+/- effectors) as catalysts modulation Binding, Enzyme as biocomplexing EIS, QCM, SPR, Pesticides conformation agents; lipocalins; aptamers PWR, SAW etc. changes

3. Potent macromolecular targets ooofof xenobiotics

3.1. Biocatalytic recognition eleeleelementsele ments

3.1.1. Enzymes An interaction of mammalian enzymes with artificial and natural pesticides is of great interest in view of investigating intoxication mechanisms and figuring out enzymatic kinetics. On the other hand, the enzymes sensitive to effector action of xenobiotics are widely used as sensitive elements in biocatalytic sensors based on different transducers [8, 9, 28]. Mono- and multi enzyme biofilms are considered as the most common and well-developed biorecognition part of biosensors [12]. As proteins endowed with catalytic functions, enzymes are capable of accelerating chemical reactions by up to 10 23 fold [29]. Amino acids residues from the active sites of enzymes participate in the chemical transformation of substrates. The main functions of these residues are to modulate the electrostatic environment and chemical catalysis, including facilitation of proton-transfer reactions and covalent chemistry at the reaction center [29]. In order to make a proper choice of target enzyme (in respect to its source, stability, type of kinetics, affinity to the analyte of interest etc.) for biosensing of small compounds, either substrates or effectors, it is useful to search within enzyme electronic databases like BRENDA (BRaunschweig ENzyme Database, http://www.brenda.uni-koeln.de ), founded in 1987 by D. Schomburgis [30].

9 In recent years, the intense development of methods for altering protein properties has allowed sorting out interesting enzyme variants. One route in enzyme engineering is molecular modelling, the second one – random mutagenesis and recombination techniques allowing directed evolution of biocatalysts [31]. It should be noted that enzymes can be used in biosensor screening of drugs not only as drug-modulated catalysts but as affinity “receptors” as well. Binding of small analytes to immobilized enzymes in the absence of enzyme substrates can be effectively monitored by mass-sensitive platforms (SPR, QCM) [32, 33] but also by means of the EIS as it will be experimentally shown in Chapter 1.

3.1.2. RiRibozymesbozymes Ribozyme ( ribo nucleic acid en zyme ) is a ribonucleic acid (RNA) possessing a catalytic activity [34]. Natural ribozymes are involved in RNA maturation through the catalysis of either their own cleavage or the cleavage of other RNAs via phosphoryl-transfer reactions. The only natural ribozyme endowed with a synthetic activity (i.e. peptide chain formation) is a rybosome [29]. Compared with protein enzymes, which are chemically much more diverse, ribozymes possess a limited repertoire of groups that take part in catalysis. Nevertheless, ribozymes act in ways that are similar to protein enzymes [29] . Recently, several groups have succeeded in creating allosterically controllable artificial ribozymes (aptazymes ) whose activities can be regulated by allosteric effector molecules [35, 36]. In allosterically controlled catalyst, the effector molecule binds to a site that is located in some distance from the active (catalytic) site. The known allosteric effectors of aptazymes are: nucleic acids, proteins, small chemical compounds including ATP, flavin mononucleotide, theophylline etc. The effectors can activate an appropriate aptazyme up to 100,000-fold [35]. Some small compound like aminoglycosides are known to allosterically modulate both ribozymes and enzymes (namely, phospholipases) [13]. Allosteric ribozymes can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals; they are

10 easily engineered, can detect diverse classes of biologically relevant molecules with high signal-to-noise ratios [35]. These features make aptazymes useful candidates for incorporation into arrays of biosensors. [35]

3.2. Bioaffinity recognition elements

3.2.13.2.1. . Antibodies Antibodies (Ab) are proteins that are produced by the mammalian immune system to bind antigens with high specificity and affinity [37]. In order to evoke immune response in a host organism, an antigen must be a foreign and quite large molecule, usually > 10 kDa. Low molecular weight xenobiotics (or haptens) are too small to produce immune response and therefore must be conjugated with any large protein e.g. bovine serum albumin. By raising Ab against hapten-protein complex, Ab can be produced which will also recognize free hapten [38]. For example, in Fig. 1, A it is depicted a structure of solanidine-protein conjugate used to generate Ab in mice for immunoassays of solanaceous glycoalkaloids [39].

AAA BBB

FigFigFig.Fig . 1. Structure of solanidine immunogen [39] (A) and IgG molecule [40] (B) .

An antibody can tightly bind an antigen (small molecule, peptide or protein) from micro to pico and even sub-picomolar affinity [37]. Natural and recombinant, poly- and monoclonal (PAb and MAb) antibodies, as well as

11 antigen-binding fragments F(ab), selected in vitro by library screening techniques, are widely used in affinity purifications and in biosensing. Usually, the production of PAb is quicker and cheaper than that of MAb, however, the source of PAb (animals) is necessarily finite and the quality of PAb can widely vary. The hybridoma cell culture has allowed the standardized long- term production of theoretically unlimited quantity of MAb against a particular antigen epitope. In the industrial purifications related to therapeutics and drugs production, the preferred choice is MAb [40]. The absolute majority of biosensor applications deal with either poly- or monoclonal IgG (Fig. 1, B), however, other isotypes (mainly, IgM) may be used in immunoaffinity purifications. Usually, direct analysis, either in homogeneous or heterogeneous phase, of small antigen binding to Abs is difficult without radioactive, fluorescent, enzymatic or high-mass nanoparticles labelling [41]. “Sandwich”, competitive and other modes of immunochemical analysis may also be applied to enhance the sensitivity of detection. Thus, SPR-based biosensing using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules has been recently developed using progesterone as a model compound [41].

3.2.2. Lipocalins and anticalins

Representing a promising alternative to recombinant Ab fragments, lipocalins belong to the family of proteins whose function is associated with storage or transport of hydrophobic organic compounds like retinol, pheromones, odorants. Some lipocalins, e.g. odorant binding protein (OBP), may also be involved in complex molecular recognition processes [42]. Lipocalins have a stable and compact core structure of Y-barrel type, and four variable loops, which create a binding site for a specific ligand. Because lipocalins are monomeric polypeptides and do not require additional factors for correct folding, they can be produced in yeast and Escherichia coli , which are less expensive to grow than mammalian cells [43].

Lipocalin scaffold can be employed for the design of so-called anticalins via targeted random mutagenesis. Discovery of a potent antibody-like affinity of

12 such lipocalins by working group of A. Skerra allowed construction of libraries of anticalins specifically directed against various targets [44].

Very recently, some lipocalins (namely, OBP) have been attempted as a biorecognition element of biosensor for small molecules [45].

3.2.3. Aptamers Aptamers (“aptus” – fitting) are RNA or DNA single-strand oligomers (15- 60 nucleotides) which can bind to a given ligand with high (in low nanomolar range) affinity and specificity due to the particularly flexible 3D structure. Synthesis and selection of aptamers against any compound (antibiotics, neurotransmitters, pesticides, drugs etc.) is provided by several companies, i.e. by Archemix (USA) and NascaCell Technologies (Germany). Aptamers have advantages over the MAbs, namely: they can be isolated from the vast synthetic libraries for virtually any target, even those that are toxic or have low immunogenicity; production of aptamer takes about 8 weeks, antibody production – about 6 month; aptamers are stable under a wide range of buffer conditions and resistant to physical or chemical denaturation etc. The method used to identify an aptamer to a chosen molecular target was first developed by Gold et al. at the University of Colorado and is called SELEX (Fig. 2) [46]. This iterative procedure comprises four steps: (1) pool preparation (~1015 unique molecules), (2) selection, (3) amplification and (4) aptamers isolation.

FigFigFig.Fig . 2. Schematic outline of aptamers selection process [47] .

13 3.2.4. G proteinprotein----coupledcoupled receptors G protein-coupled receptors (GPCRs) are characterized by 7 transmembrane (TM) spanning domains (Fig. 3) requiring a lipid environment to maintain receptor’s native conformation. In human, about 340 of 865 genes predicted to encode 7TM polypeptides are defined as olfactory receptors (ORs) [48]. Others are presented by rhodopsin, receptors for biogenic amines (acetylcholine, serotonin), opioids, cytokine, trombin, gustatory receptors etc. [49]. Binding of olfactory receptors to odorants is characterized by the affinity

constants Kd within the pico- to millimolar range [50].

The GPCRs are involved in almost all aspects of human physiology (e.g. hormonal regulation, neurotransmission), and the modulation of GPCRs is a major mechanism for modern therapeutic intervention [51, 52]. The GPCRs have proven to be the most productive area for small molecule drug discovery [51]. Allosteric modulators of GPCR are also known and present a pharmacological interest [53], Fig. 4. There are 3 categories of such allosteric modulators (M): (1) M that affect only the binding affinity of orthosteric agonists; (2) M that can impact the efficacy of an orthosteric ligand either in addition to or independently of effects on the affinity binding of orthosteric ligands; (3) M that have detectable efficiency independently of effects on the orthosteric agonists affinity [53].

During the last few years, there has been a growing interest in the application of GPCRs in label-free heterophase analysis of ligands [51, 54-57]. Ligand binding to a GPCR triggers a series of spatial and temporal biochemical events, leading to an ordered, regulated, and dynamic redistribution of cellular contents [51] which can be detected by the different kinds of biosensors.

The current GPCR-based drugs target 25-50% of the approximately 200 known GPCRs. Therefore, screening drugs against this class of proteins is still a major effort in pharmaceutical and biotech industries [51]. Biosensors and

14 biosensing platforms based on GPCRs could be twice more useful as analytical tools for receptor pharmacology probing and drug quantification as well.

FiFiFigFi ggg.... 3 3.... Schema of 7TM olfactory receptor according to Buck and Axel [58]. The protein traverses the plasma membrane 7 times, its N-terminus located extracellularly and its C-terminus – intracellularly. The vertical cylinders delineate the seven putative :-helices spanning the membrane. Variable residues are shown as black balls. The high degree of variability encountered in TM domains III, IV, V [58] .

Fig. 44.... Types of allosteric modulators. (1) Allosteric modulation of orthosteric-ligand binding affinity. (2) Allosteric modulation of orthosteric ligand efficacy. (3) Direct allosteric agonism. Adapted from Langmead et Christopoulos [53].

4.4.4. Immobilization of biorecobiorecognitiongnition element

Strategy of biorecognition element immobilization can affect biochemical events occurring on the electrode. The attachment procedure can be considered as correct if biofilm is reproducibly sensitive to the analyte. Optimization of the established immobilization protocol can include its simplification (i.e. from

15 multilayer to monolayer), reducing the amount of biomaterials and procedure duration. Nevertheless, introduction of additional steps and/or reagents in the immobilization protocol sometimes may help to enhance the sensitivity, selectivity and/or stability of biosensor.

The mode of attachment of biomolecules is generally driven by characteristics of transducer surface (hydrophobicity, roughness, biological non-inertion etc.) and by the desired properties of the future biosensor (low interface capacitance for capacitive affinity biosensors; high electrocatalytic activity for amperometric biosensors; low biofilm thickness for the SPR based sensors etc.).

4.4.4.1.4. 1. Covalent attachment

Chemical immobilization consists in the covalent or pseudo-covalent binding of biomolecule to the surface, either bare or previously modified. These strategies allow assembling of quite stable layers with a planed density of biorecognition centers. The most popular method of biomacromolecules immobilization on noble metal (Pt, Ag, Au) surface consists in the formation of thiol-Au self-assembled monolayer (SAM) based on the mercaptide bond [59]. There are 2 general immobilization strategies: i) direct coupling of SH-groups rich biomolecules to metal and ii) coupling through the SH-bearing linker.

Self-assembled alkanethiols HS-(CH2) n-X can be functionalized with different terminal groups (X): hydroxyl, amino, carboxylic acid, ester, nitrile etc. The end group functionalities change the surface wetting properties, and generally determine the chemistry of covalent coupling with macromolecules (Table 2). Additionally, there are some interesting data on the strong impact of the end COOH group on the spatial organization of mercaptohexadecanoic acid monolayer on Au [60].

Pure thiol monolayer with one type of solution-oriented headgroup may be functionalized with other thiols or phospholipides, forming so-called “mixed SAM” (Fig. 5). Mixed SAM, especially blocked with IgG or BSA (see Chapter 2),

16 provides a limited amount of functional groups for the high-affinity anchoring of an upper layer, and thus reduces non-specific interactions of analyte with first layer of thiols.

FigFigFig.Fig ... 5 . Schema of pure and mixed SAMs. Sulfur (black) is attached to a gold surface. Functional groups (shaded) are elevated into the solution. Adapted from [61] .

The site-directed attachment of antibody fragments onto gold can be performed via the chemisorption of F(ab’) fragments through a single reactive thiol group [62]. Cysteine lacking proteins like PrG can be modified via substitution of their free NH 2 groups into SH groups using 2-iminothiolane [63]. Otherwise, a direct attachment is used for the formation of intermediate linker layer. Methods of indirect covalent coupling are briefly outlined in Table 2. Since most proteins contain randomly distributed lysine residues, covalent attachment sites will be numerous resulting in random orientation (like shown in Fig. 6) and reduced biological activity of biorecognition element [62].

Fig. 6. Potential positions of Ab randomly orientated in heterophase. The antigen recognition sites may be totally exposed in solution (a), non-exposed (b), partially exposed (c) [40].

Method of the indirect attachment allows rather simple anchoring of polysaccharide matrixes (cellulose, dextrans etc.) to the thiolated gold. However, as it can be seen from Table 4, the thickness of such supporting

17 polylayers is essentially large. Recently Petri et al. have proposed synthesis of thiosulfate derivative of cellulose for ultrathin (4 ± 1 nm) supporting amorphous polylayers fabrication [64].

Polysaccharide matrix is widely used in the non-covalent high affinity immobilisation of proteins tagged with carbohydrate binding domains [65]. Hydrazine-activated polysaccharide can also be effectively used for the site- directed covalent attachment of IgG via the periodate-oxidized carbohydrate moieties of the F(c) region [40] (Fig. 1, B). Activated cellulose can be applied for covalent cross-linking of enzyme molecules on a solid surface.

Recently, thiol layer covalently attached to gold was used in the direct electrochemical biosensing of odorant molecules [66]. This original approach is based on the adsorption and penetration of hydrophobic analyte molecules (e.g., odorants, Fig. 7) into the hydrophobic thiol layer, which alters the electrical capacitance and resistance of the latter. It has been proposed to use the correlation of electrical changes to the odorants physico-chemical properties in the creation of so-called pattern recognition [66] widely applied in processing of data obtained from the “electronic noses” (see Chapter 2).

FigFigFig.Fig ... 777. Changes of interfacial resistance of gold electrode modified with dodecanthiol (1 mM) upon additions of odorants dissolved in ethanol [66] .

18 Table 222.2... Strategies of covalent attachment of target biomolecules to thiolated gold.

Target chemical groups of Soluble Substrate Mechanism of coupling macromolecule activator of coupling to be attached 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide ( EDC ) Conjugation through the accessible Lys residues of Au-S- N-hydroxy-succinimide protein (peptide bonds) (CH ) - 2 n (NHS ) [67] COOH

p-Nitrophenol (1-Hydroxy- 4-nitrobenzene) COOH,

NH 2 Thionyl chloride (SOCl 2)

(Proteins etc.) Conjugation through the Cyanuric chloride COOH groups of protein

(bonds differ from the Phenylene diisocyanate peptide –type and “Schiff

Au-S- base” type) [67]

(CH 2)n- NH 2 Conjugation through the NH 2 groups of protein Glutaric aldehyde (bonds of type “Schiff base”) [67]

OH groups are periodate- oxidized to COH groups, then aldehyde groups Au-S- easily couple to the (CH ) - 2 n {NaIO }, then hydrazine hydrazide derivative of COOH 4 OH thiol-COOH [25]

(bonds of type “Schiff (Polysaccharide base”) s etc.) OH groups are periodate- Au-S- oxidized to COH groups, (CH ) - {NaIO } 2 n 4 then Schiff bases connect NH 2 COH and NH 2 groups

19 4.2. Electrochemical attachment

This strategy includes electrochemical methods: i) embedding of biomolecules in the electropolymerized matrix like polypyrrole [68]; ii) chemical attachment of biomolecules onto such a matrix [69], [68], iii) voltage-dependent precipitation of biomolecules bearing electrochemical tag [70]. The latter method seems to be quite promising in view of reduction of sensing layer thickness.

Modulation of the protein molecule via insertion of special tags, i.e., hexahistidine sequence (6His) may allow avoiding covalent binding chemistry. The N of the imidazole residue of His coordinates divalent metal ions (Co 2+ , Ni 2+ , Zn 2+ , Cu 2+ , Ce 2+ ). Protein tagged with 6His can be immobilized onto conductive electrode surface via electrochemical reduction of the 6His- coordinated metal ions at cathodic potential (-200 mV) at the neutral pH. An electrochemical oxidation and release of biomatrix occurs at +100 mV [70].

4.3. Physical attachment

A direct adsorption of biomolecules on a solid surface from aqueous solutions is usually called “physical” attachment due to the forces responsible of this process: ionic, Van der Waals, hydrophobic. The adhesion of proteins on metal surfaces is usually stable thus allowing the fabrication of pseudo-mono and even polylayer structures. For example, physical adsorption can be applied in the immobilization of cheap and stable “bulk” proteins (BSA, STI, bacterial PrA and PrG etc.) PrG and PrA layer are used for the oriented immobilization of IgG via non-covalent bioaffinity interaction with F(c) fragments [63], [40]. Stability of such layers can be enhanced by cross-linking protein molecules by the glutaric aldehyde. Immobilization of cysteine-rich proteins like STI is improved by Au-S bonds formation [71]. Any pseudo-monolayer of above-described bulk protein (1 st component) can be used for electrostatic “anchoring” of oppositely charged molecules (2 nd component), following the scheme [72]:

20 pI of 1 st component < pH of working buffer < p I of 2 nd component

Assemblies mainly based on the electrostatic interactions are quite sensitive to the changes of pH and t º of working buffer solutions and can suffer from high potentials applied; therefore, these biofilms are often not suitable for the electrochemical studies. However, a physical attachment may be a useful strategy to immobilize proteins whose affinity sites are poorly studied [73], [72]. Noncovalent interactions also allow reliable anchoring of protein/amphiphile Langmuir-Blodgett films [74], Langmuir-Schaefer films [75], artificial lipid and natural protein/lipid monolayers [76, 77] to thiolated hydrophobic substrates. Chemistry and techniques (1-5) of such a transfer are presented in Fig. 8.

FigFigFig.Fig . 8. Building up lipid bilayers on gold. Adapted from Steinem et al. [75]

21 4.4. EntrapEntrapmentment

Since there is no need of “direct” contact of biocatalytic element with the sample, enzymes can be entrapped into polymers: polyacrylamide, agar gel, polyurethane, poly(vinyl)alcohol, sol-gels, redox hydrogels etc. [12, 17, 78]. Accessibility and activity of enzymes immobilized in this way depends on the perculiarities of local microenvironment: pH, ionic strength, pores dimensions etc. [78] Covalent attachment and glutaraldehyde-mediated reticulation are usually more complicated than entrapment but much more feasible when the biorecognition element must be fabricated directly on the transducer. Additionally, more stable and reproducible activities are reachable using covalent immobilization [12].

5.5.5. Bulk properpropertiesties of biofilms

The most important parameters of biofilms and methods for their evaluation are briefly listed in Table 3 and Table 4. Control of “bulk” properties provide additional information about state of a sensitive surface, its stability, physicochemical and mechanical properties, either initial or altered with biorecognition events, and generally could help in the improvement of biofilm design.

Table 333.3... Estimation of bulk parameters of biosensitive films.

Bulk parameter Routine technique Thickness Ellipsometry, AFM, XPS etc. Roughness AFM Wettability Contact angle measurement Refractive index (n) SPR, ellipsometry Rate of electron transfer Cyclic voltammetry through the film with redox probe Nanomechanical properties AFM-based “force spectroscopy”[79]

22 Table 444.4... Bulk properties of some thiol, protein and polysaccharide films formed on the gold substrates.

CCConcentrationConcentration Effective Electron Film of the reagent Thickness, refractive transfer composition used for the film nmnmnm index (n) rate formation Hexadecanthiol m, Au 1.89 [60]

16-Mercapto- Well- hexadecanoic acid m, Au 1.94 [60] organized 0.4 – 1 mM n/a layers are 22-Mercapto- fully docosanoic acid m, Au 2.57 [60] insulating [80], [81], taken as [*] etc. 11 –Mercapto- 1 mM 1.9-2.0 [82] 1.45 [82] undecanoic acid m, Au

Quite 1.367 BSA Au 0.2 mg/ml 6.87 [24] high [*] [24]

Restricted taken as due to BSA Au-SAM 1 mg/ml 0.83 [82] 1.45 [82] the SAM

Probably BSA Au-Abs 0.5 mg/ml 0.9 [*] high

taken as Restricted 1.36 [*] due to Neutravidin Au-SAM 0.03 mg/ml 15 [*] the SAM [80], [*]

1.343- Probably IgG Au 0.2 mg/ml 10.5-13.8 [24] 1.363 high [24] Restricted taken as due to IgGAu-SAM 0.1 mg/ml 1.9-5.7 [82] 1.45 [82] the SAM

Probably Fibrinogen Au >0.01 mg/ml 6-7 [27] n/a high

Aldehyde- ethylcellulose Au-SAM Probably 20-50 [25] restricted ~1.335 Dextran Au-SAM 10 mg/ml by large [25] thickness Aldehyde- 80-140 [25] dextransulfonate Au-SAM

m – film is a monolayer; Au – substrate is a bare gold surface; Au-SAM – gold + thiols, Au-Abs – gold + antibodies; n/a – data not available, *– experimental section of the present work.

23 6. “Apparent” and “true” affinity

The affinity, either “true” or “apparent”, is determinated both by the ligand and the macromolecule. That is why the modern computational chemistry is still not able to evaluate affinity with high reliability [7]: too many parameters related mainly to macromolecules metastable conformations, tautomeric forms, ionization states etc. have to be taken into account. Certainly, the knowledge of full 3D structure of a macromolecule is required for such a modelling. Historically, affinity is assumed as the force that causes (bio)chemical reactions/formation of biorecognition complex (C).

k→ass P + L C ; (1) ← kdiss Affinity, or how tightly a ligand (L) binds to a protein (P), is usually determinated by the value of dissociation constant Kd having dimension of molar concentration: P ⋅ L K = [ ] [ ]; (2) d []C

The Kd correspond to the concentration of ligand [L] at which 50% of the binding sites on a particular protein (antibody, receptor etc) is occupied, i.e. the concentration of ligand, at which the concentration of [C], equals the concentration of protein with no ligand bound, [P]. The smaller the dissociation constant, the more tightly bound the ligand is, or the higher the affinity between ligand and protein.

It is worth noting that inhibition constant Ki is nothing but Kd of enzyme-inhibitor complex (see also Chapter 1, Part 1), and thus a Ki of X mol/l estimated for the enzymatic reaction suggests that a binding event with affinity X might be expected [32]. Affinity recognition process is governed by the interplay of non-covalent interactions of comparable strength: electrostatic (ionic) attraction, the Van der Waals interactions, formation of hydrogen bonds and hydrophobicity [83]. In an aqueous medium non-covalent bonds are typically of the order of 1-2 kcal/mol [83] (for comparison: the energy associated to the thiols chemisorption on gold is 40-45 kcal/mol [74]), the high specificity of

24 biomolecular recognition can be achieved if a large number of non-covalent bonds can be formed. The energy ∆E associated with ligand binding is defined as: ∆ = − ⋅ ⋅ E k T ln K d ; (3) where the thermal energy k ⋅T =0.58 kcal/mol at room temperature [50]. In

-4 case of weak affinity, e.g. Kd of the order of 10 M, ∆E =5.34 kcal/mol.

From the kinetical point of view, affinity is: k = diss K d ; (4) kass

−1 where kdiss is the dissociation rate constant having dimensions of s ; 1 and kass is the association rate constant having dimensions of . (mol l) ⋅ s

The classical method of measuring Kd with affinity biosensors involves probing a few concentrations of ligand [L] with the same surface and regenerating this surface between binding cycles [32]. For this purpose, e.g. in Biacore-based analysis, the kinetic rate constants are estimated by fitting the entire binding profile (curve) to an appropriate interaction model. Usually it is the integrated first-order rate equation [84] as follows:

R L k − + R = max [ ] ass 1− e ()kass []L kdiss t ; (5) t + () kass []L kdiss where Rt is the sensor response at time t, R max reflects the binding capacity of the immobilized biorecognition element, [L] is the concentration of ligand, kass and kdiss are above-described rate constants,. The term (kass [L] + kdiss ) is the apparent pseudo-first order rate constant ( k) with dimensions of s -1 [84]. As it may be expected, a high affinity of immobilized biorecognition molecules leads to large errors in kass and kdiss determined by pseudo-first-order analysis due to the significant re-binding phenomenon during dissociation etc. On the basis of such a pseudo-first-order approximation, ligand desorption from the surface is determined as follows:

− R = R e kdisst ; (6) t 0 ( ) where R0 is the sensor response at the beginning of dissociation. Equation (6) provides good description of systems endowed with Kd within a micromolar

25 range, but is unsuitable for high affinity systems when Kd lays in a nanomolar range or even lower. Second-order equations for biosensor analysis of experimental kinetic data on the high-affinity systems are proposed by Edwards et al. [84]. Initial association rate analysis based on the linear regression of the initial part of binding curve obtained by means of optical biosensor has been also proposed for high affinity interactions analysis [85]; and in this case the entire binding profile is not required for rate constant estimation.

Interestingly, two compounds having similar Kd, can vary by several orders of magnitude in their kinetic rate constants. From a drug creation perspective, a rapidly dissociating drug will be more susceptible to excretion and thus must be administered more frequently. Lower affinity (but better stability) seems to be preferable since may be compensated by increased concentration of drug [32]. Recently, Karlsson et al. has proposed a robust protocol for Biacore- based drug screening allowing “kinetic titration” of the affinity surface by soluble ligand. It allows collecting kinetic data from the same surface sequentially even without regeneration steps [86].

However, let us return to the discussion on “true” and “apparent”

(untrue?) affinity. The Kd (as well as any other parameter of affinity reaction) is considered as “true” if it has been measured in homogeneous phase (i.e. in solution); and Kd obtained in any “unusual” conditions, i.e. in heterophase assay must be called “apparent”.

Does the apparent Kd estimated by means of biosensor-based assay for any potent drug or toxin, present any practical value apart from the scientific interest? Otherwise, when the Kd absolute values measured in homo-and heterophase are similar, can we expect the same process of biorecognition and similar stability of affinity complex in both cases? There are several lines of evidence that the answer will be “yes” if the experiment in heterophase is designed correctly, however, some below-described “intrinsic” challenges should also be taken into consideration. It is well known that three processes, diffusion, convection and migration may affect mass transport of analyte thus masking the “true” rate constants.

26 Migration of analyte molecules in solution in applied electric field is usually thought to be of low importance for steady-state (but not for kinetic) measurements. The major challenges of heterophase analysis, however, are related to convection and diffusion. According to the first Fick’s low, ∂C(x,t) j(x,t) = −D ; (7) ∂x where j(x,t) is the diffusion flux having dimensions of m −2 ⋅ s −1 or, with respect to substance amount, mol ⋅ m −2 ⋅ s −1 ; C(x,t) is a concentration, m −3 or mol ⋅ m −3 being function of time t and position x; D is a diffusion coefficient, or diffusivity, m 2 ⋅ s −1 , proportional to the velocity of diffusing molecules. The latter depends on the temperature, viscosity and particles size. For biomolecules D is usually near 10 -11 -10 -10 m 2 ⋅ s −1 . Now let us consider diffusion processes near the polarized electrode surface, Fig. 9. In well mixed solutions, in the bulk (convective) region the concentration C of analyte is constant with respect to distance, and C=C0.

Fig. 99.... Fictious profile of Nernst, or Nernst diffusion layer [87]. (www-biol.paisley.ac.uk/marco/Enzyme_Electrode/Chapter2/Chapter2_page3.htm. )

Then there is a fictious region where the concentration falls to zero, i.e. substance instantaneously undergoes reduction/oxidation. This region is called Nernst diffusion layer with thickness e, and for well stirred aqueous

27 media e is 1-10 fm [87, 88]. The diffusion flux j of analyte is related to e by the formula [88]: DC j = 0 ; (8) δ For high molecular mass molecules, such as proteins, the mass transport to the surface through a Nernst diffusion layer significantly limits the rate of reaction [88]. These mass transport limitations lower the quality of apparent kinetic data measured for analytes with MW>10,000-20,000. In case of small molecules, mass transport of analyte from solution to affinity surface less alters the rate of binding, but it still may impact the systems endowed with fast k ass [33]. Nevertheless, a routine analysis of small molecules (even with very fast association rate) can be carried out by biosensors; e.g. experimental and data processing protocols for mass transport-limited reactions are available for Biacore sensor platform [33]. As to heterophase biocatalysis , it is well known that it occurs in all dimensions of enzyme-containing biofilm (Fig. 10). Thickness and porosity of enzyme polylayer will dictate its diffusion properties.

Fig. 10 10.... Direct (label-free) biorecognition and biocatalysis in heterogeneous phase.

Usually, the thicker the biofilm, the stronger the impact of inner mass transport of analyte and product on apparent catalytic parameters, and the lower the biocatalytic sensor sensitivity [11]. Another key point in surface-based analysis of biochemical potencies is immobilized state of one of the recognition partner, which is suspected to alter its affinity or catalytic properties and thus is the principal reason of scepticism concerning the validity of biosensor-measured affinity [89]. Thus, strong reticulation of biorecognition molecules may result in better stability of biofilm,

28 but in biochemical studies, gentle immobilization techniques are preferable. Thus, in the recent comparative study carried out by 30 independent users of Biacore it was clearly shown that the immobilization of an enzyme does not necessarily affect its binding constants [90]. The main advantages and drawbacks of biosensor-based screening ligands for immobilized macromolecules are outlined in Table 5.

Table 555.5... Advantages and drawbacks of label-free biosensor-based profiling of ligands for macromolecules in heterogeneous phase.

Drawbacks Advantages • Proper choice of immobilization • Opportunity of obtaining a full strategy is crucial for probing kinetic profile of drug binding drug biochemistry • Possibility of surface regeneration • Control of biofilm thickness is (especially for enzyme-containing needed biofilms)

• Optimization of analysis • Several assays can be performed protocol (buffer capacity, ionic on the same biofilm: important strength of working solution, its when the quantity of recognition pH, temperature, intensity of macromolecule is limited stirring etc.) is required for every type of transducer or • Mass transport effect is minimal platform employed for small molecules

• Mass transport usually alters • Biosensing may be the unique the rate of high molecular possibility to probe some weight ligand binding to the substrates: i.e., acetyl choline affinity surface instead of acetyl thiocholine required for chromophore production in spectrophotometry

7. Concluding remarks

An analysis of the literature proves that biosensors in fact can serve as highly valuable tools for investigating biochemical interactions of small agonists and antagonists with natural and synthetic biomolecules. The majority of the latter has been designed specially for incorporation in biosensor arrays. A large number of diverse strategies of coupling biopolymers to appropriate transducers are available to design accurately a highly sensitive biofilms. Experimental protocols as well as models of data analysis are

29 currently available for several affinity systems mainly those probed by means of Biacore technique. In the next chapter devoted to glycoalkaloids and cholinesterases we will present two experimental approaches to evaluation of kinetic and affinity parameters by means of label-free electrochemical biosensing.

30

CCChChhhaaaapppptttteeeerrrr 111

GGGlGlllyyyyccccooooaaaallllkkkkaaaallllooooiiiiddddssss aaanannndddd ccchchhhoooolllliiiinnnneeeesssstttteeeerrrraaaasssseeeessss

Summary

PART 1. Potato glycoalkaloids: true safety or false sense of security? 353535 PART 2. Cholinesterase inhibition by glycoalkaloids: probing with potentiometric biosensor 454545 PART 3. Impedimetric study of glycoalkaloids binding to cholinesterase 616161

31

32

CCChChhhaaaappppiiiittttrrrreeee 111

GGGlGlllyyyyccccooooaaaallllccccaaaallllooooïïïïddddeeeessss eeetettt ccchchhhoooolllliiiinnnneeeessssttttéééérrrraaaasssseeeessss

Cette partie de travail concerne les glycoalcaloïdes stéroïdiques, leurs effets toxiques sur l’organisme humain et notamment leur activité contre la cholinestérase qui va nous intéresser dans le travail expérimental. Ce dernier est consacré à l’exploitation de l’action inhibitrice des glycoalcaloïdes sur les cholinestérases par un biocapteur potentiométrique et pour l’étude impédimétrique des couplages affins des glycoalcaloïdes et de la cholinestérase en absence de substrat enzymatique. D’abord, la cinétique d’inhibition des butyryl cholinestérases humaine et équine par l’:-solanine, l’:-chaconine et la tomatine a été étudiée en employant un biocapteur basé sur des transistors à effet de champ (pH-FETs) sensibles aux changements de pH dus à l’hydrolyse enzymatique de la butyrylcholine. Tous ces glycoalcaloïdes sont des inhibiteurs réversibles ; on a montré que l’inhibition de l’enzyme équine est compétitive et celle de l’enzyme humaine est de caractère mixte. L’affinité de chaque glycoalcaloïde a été évaluée à travers le calcul de constantes d’inhibition Kiapp et des coefficients d’inhibition I50 . L’:-chaconine s’est révélé être l’inhibiteur le plus fort de deux enzymes. Puis, nous avons tenté la détection impédimétrique directe des interactions des glycoalcaloïdes et de la butyryl cholinestérase équine immobilisée sur une électrode d’or. Les interactions affines de l’:-chaconine et de l’:-solanine avec l’enzyme ont conduit à la diminution spécifique de la résistance interfaciale de polarisation. Nous avons montré que les électrodes modifiées par la butyryl cholinestérase sont plus sensibles à l’:-chaconine qu’à l’:-solanine. La confrontation des valeurs de I50 mesurées par le capteur biocatalytique basé sur les pH-FETs et par le capteur bioaffin impédimétrique a montré leur bon accord.

33

34

Part 1

Potato glycoalglycoalkaloids:kaloids: true safety or false sesensense of security?

As one of the major agricultural crops, the cultivated potato is consumed each day by millions of people from diverse cultural backgrounds. A product of global importance, the potato tuber contains low molecular weight toxic compounds – glycoalkaloids (GAs) that cause sporadic outbreaks of poisoning in humans, as well as many livestock deaths. In this part we will discuss some aspects of the potato GAs chemistry, their toxic effects and risk factors, methods of detection of GAs and biotechnological aspects of potato breeding. An attempt has been made to answer a question of vital importance – are potato GAs dangerous to humans and animals and, if so, to what extent?

Data presented in Part 1 were published in Trends in Biotechnology , 2004.

35 1. Introduction Glycoalkaloids (GAs), or alkamines, are nitrogen-containing derivatives of higher plants steroid glycosides – saponins [91]. GAs have been found in several vegetables and fruits (including sugar beets, apples, cherries and bell peppers), but mainly in the plants of the Nightshade family [92], particularly the potato (Fig. 1.1, A) – an everyday food for many people. :-Solanine and :- chaconine account for 95% of GAs present in Solanum tuberosum , and consist of a nonpolar lipophylic steroid nucleus, which is extended by two fused nitrogen-containing heterocyclic rings at one end and bound to a polar water soluble trisaccharide at the other (Fig. 1.1, B).

AAA BBB

FigFigFig.Fig ... 1.1.1. 1.1.1. (A) Tubers of some potato varieties cultivated in France, Exposition Botanique, Parc de la Tête d’Or, Lyon, 2007. (B) Chemical structures of :-solanine and :-chaconine. Steroid backbone, or aglycone of both glycoalkaloids is presented by the solanidine.

Several research studies [93-95] have shown the toxicity of steroid alkaloids to be defined not only by their concentration but also by the nature and number of sugar molecules (the carbohydrate moiety attached to the 3-OH aglycone group), as well as by their stereochemical orientation. The :-form is more toxic than the Y-form, which in turn is more toxic than the g-form. GAs are thought to protect the crop against certain pests and diseases caused by insects and fungi. Several factors associated with growth, harvest and post- harvest treatment might lead to an increment in GA content to high and toxic levels in potato tubers, especially underneath the skin. Major factors causing this increment are genetic variations, and growth and storage conditions,

36 including exposure to light and tuber injury [96]. Recently, Surjawan et al. reported that free sulfhydryl groups in some sulfur compounds act directly on natural potato toxins and substantially reduce their toxicity [97]. In recent decades, the extreme toxicity of glycoalkaloids has been a focus of scientific attention, especially concerning GAs in the potato ( Solanum tuberosum Lam.). Potatoes are an essential component of the diet of humans and animals and are, thus, a potential source of food poisoning [93]. The steroidal alkaloids are teratogenic, embryotoxic and genotoxic compounds with potent permeabilizing properties towards mitochondrial membranes [98, 99]. The issues presented here support the need to analyze and summarize published data regarding the toxicity of the potato GAs and to facilitate investigations in this field. The development of biosensors, novel analytical devices and medical diagnostics is essential. Significant efforts in developing and using more efficient breeding schemes to generate superior potato varieties are also important. Here, we review the literature concerning the safety of potato GAs and discuss future research and development.

2. Potato glglycoalkaloidsycoalkaloids and health The toxicity data from in vitro and animal studies indicate that chaconine is the most toxic alkaloid of the potato GAs [93]. It is teratogenic, exhibiting strong lytic properties and inhibits butyryl- and acetyl cholinesterase activities [100]. Due to the latter, chaconine and other GAs can prolongate an action of myorelaxants, anesthetics and analgesics [101, 102], and affect the ester prodrugs activation in blood serum [101]. At lower doses, the toxicity of GAs in humans causes mainly gastrointestinal disturbances such as vomiting, diarrhea and abdominal pain. However, at higher doses, the toxicity of GAs in humans produces more severe symptoms, including neurological disorders [103]. Several cases of lethal poisoning caused by GA exposure have been reported [104]. Many authors note that the symptoms of potato poisoning are perhaps determined by the joint action of GAs. Thus, existing data on the toxic effect of an individual GA, as well as of a specific mixture of GAs, cannot be used to predict how a human will respond to a combination of potato GAs and other ones [103], because our knowledge is based mainly on the data obtained for

37 experimental animals. Moreover, for many of the drugs that are coming onto the market and being prescribed, we really do not know anything about their interaction with potato GAs [105]. Anti-cancer activity has been reported recently for some GAs, such as :- solamargine, :-solasonine and the aglycone solasodine [106]. Solamargine, a herbal and molluscidal medicine derived from Solanum incanum , was used to study anticancer activity in human hepatoma cells (Hep3B) and to characterize changes in cell morphology, DNA content and gene expression of cells after solamargine treatment. Kuo et al. [106] suggested that the appearance in solamargine-treated cells of chromatin condensation and DNA fragmentation gave rise to cell death by apoptosis. In addition, a parallel upregulation of tumor necrosis factor receptors (TNFR)-I and -II on Hep3B cells was detected after solamargine treatment, and the solamargine-mediated cytotoxicity could be neutralized with either TNFR-I-specific or TNFR-II-specific antibodies. The authors also revealed that the actions of TNFR-I or TNFR-II on Hep3B cells might be independent and that both were involved in the mechanisms of solamargine-mediated apoptosis [106]. Solasonine isolated from the thricomes of young branches and fruits from Solanum crinitum Lam. and solasodine isolated from Solanum jabrense Agra and M. Nee have been assayed [107] against cultured murine Ehrlich carcinoma and human K562 leukemia cells. The in vitro exposure of these cancer cells to these products resulted in a dose-dependent inhibition of growth. The study revealed a low activity of the aglycone solasodine on cultured murine Ehrlich carcinoma and human K562 leukemia cells, and pointed out the essential role of the sugar moiety in the cytotoxic activity of solasonine [107]. Chaconine is the most effective compound of the potato GAs at inactivating herpes simplex virus (HSV), and the important role of the carbohydrate moiety in the interaction of GAs with membrane sugar receptors has been established. Moreover, tests carried out with a hydrophilic cream containing either a crude extract of Solanum americanum fruits or solamargine, applied topically, healed patients suffering from herpes zoster virus, HSV and herpes genitalis virus after three to ten days [95]. It can be concluded that facilitation of research and development programs in this direction will be of

38 great interest and importance, especially in investigating the anti-cancer activity of other known and newly discovered or artificially designed GAs, and in producing novel antiviral creams, which could prove to be cheaper alternatives to the antiviral drugs already on the market. This discussion suggests that potato GAs, particularly solanine and chaconine, are toxic to humans and animals, and that this problem should no longer be ignored as it could turn into a serious health threat. One important way of controlling GA content in new potato cultivars resulting from traditional breeding and genetic engineering is to develop simple tests for the detection of solanaceous alkaloids.

3. Detection of potato GAGAss in foodstuffs and biological fluids The existing methodologies for GA detection (HPLC, mass-spectroscopy, immunoassays, [38, 108, 109]) have already been reviewed and critically evaluated [93, 98], so our discussion will focus on the methods that have been developed recently in the Laboratory of Biomolecular Electronics (IMBG NASU, Ukraine) in collaboration with CEGELY (Ecole Centrale de Lyon, France). Thus, a new method for the detection of solanaceous GAs, based on pH-sensitive field-effect transistors as transducers, coupled to butyryl cholinesterase, has been proposed [110-112]. It has been shown that :-solanine, :-chaconine and solanidine can be detected over a concentration range from 0.2 to 100 fM, depending on the type of GA, source of enzyme and concentration of butyrylcholine in a measuring cell. The detection limits for ISFET-based sensors are estimated to be 0.5 fM for chaconine and 2.0 fM for solanine and solanidine. High reproducibility (the relative standard deviation (RSD) was ~1.5% and 5.0% for intra- and inter-sensor responses, respectively], and operational (100 measurements or 7 h) and storage stability (up to three months) of the biosensors developed have been shown. It has been revealed that all of the investigated potato alkaloids are reversible inhibitors of horse butyryl cholinesterase immobilized on the transducer surface. Protocols for the detection of GAs in model solutions and potato juices have been optimized [112, 113]. The constructed sensors could also be proposed for the detection of these alkaloids in blood serum, liver etc.

39 4. Biotechnological aspects of potato breeding Market standards either specify or restrict the selection of the variety of potatoes to be grown, although many of these varieties have limitations. Therefore, the problem of breeding and evaluating new varieties is of great importance. In addition, new lines of potato must meet or exceed the market quality standards. They should have host-plant resistance to Colorado potato beetle, late blight, verticillium wilt, viruses and storage diseases, as well as a low content of GAs and glucose. The GA content is one of the most important properties because GA concentration is strongly dependent on the variety of potato, and even on the year of harvest. Continued genetic improvements are demanded to meet the needs of a changing world [114]. Potatoes were one of the first crop plants in which transgenic plants were successfully regenerated. Potato transformation has since become well developed, and now offers a real alternative approach for the improvement of cultivars. The advantage of this approach is that it theoretically permits the incorporation of a single gene into otherwise elite clones to affect their improvement. Potato transformation can be accomplished by direct uptake of DNA into protoplasts, although Agrobacterium mediated transformation using binary vectors is the preferred method and is performed routinely in many laboratories. Kanamycin (aminoglycoside antibiotic) resistance has been used as the marker for the selection of transformed cells and their regeneration into complete plants. Other selectable marker systems used for potato transformation include methotrexate (known as standard antirheumatic drug) and hygromycin (antibiotic) resistance. The integration of a gene into the potato genome can occur in a complete, truncated or rearranged manner, and can occur as single copies or tandem repeats at one or more integration sites. The preferred event is the integration (insertion) of single intact transgenes. Transgene expression is highly variable among the populations of transgenic potatoes. It is generally attributed to ‘position effects’ resulting from the random integration of the transgene into different sites of the plant genome. Potato transformation is highly unpredictable with respect to the integration and expression of transgenes and the frequency of somaclonal variation among transgenic lines. Only ~10–20%

40 of transgenic lines have the desired magnitude and required spatial and temporal pattern and/or specificity of transgene expression. Although the activation of genes involved in the biosynthesis of GAs is theoretically possible during potato transformation, such events are very rare. No such activation has been reported to date, but this does not mean that it could not occur in any specific transgenic line. Recently, potato DNA sequences encoding the enzyme solanidine-UDP- glucose glucosyltransferase have been obtained, patented and used to reduce GAs content in solanaceous plants [115]. The resistance to some potato diseases has been conferred through the expression of the gene encoding peroxide-generating glucose oxidase [116]. It is easy to assume that the combination of these two approaches could be an elegant way to obtain a new potato variety, with the desired low GA levels and disease resistance. Transgenic potato ( S. tuberosum cv. Désirée) plants overexpressing a soybean ( Glycine max ) type1 sterol methyltransferase (GmSMT) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic GAs [117]. Transgenic plants displayed an increased total sterol level in both leaves and tubers mainly because of increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a non-alkylated sterol, decreased. Associated with this was a decreased GA level in leaves and tubers, down to 41% and 63% wild-type levels, respectively. The results show that GA biosynthesis can be downregulated in transgenic potato plants by reducing the content of free non- alkylated sterols. Arnqvist et al. [117] consider cholesterol to be a precursor in GA biosynthesis. Esposito et al. [118] reported the results of chemical analyses performed on two groups of new potato genotypes. The first group contained five clones transformed with the gene ech42 , which encodes an endochitinase – an enzyme that breaks down chitin of the outer shell of arthropods and the cell walls of fungi. The second included 21 interspecific hybrids between the cultivated potato, S. tuberosum , and the wild species, S. commersonii , obtained by either somatic fusion or sexual hybridization. For transgenic tubers, the results indicated a substantial equivalence between the transgenic genotypes

41 and the cultivated control for the considered traits, and suggested that the insertion of a gene encoding chitinase does not alter other metabolic pathways of potato tubers and does not cause unintentional pleiotropic effects. For interspecific hybrids, wide variability for all of the parameters analyzed was found. For some useful traits (e.g. soluble solids and proteins, and dry matter content), the interspecific hybrids performed better than both the cultivated control and the wild species. In several genotypes, GA levels were close to or lower than those of the control varieties, suggesting that selection for low GAs content is possible . The results also indicated that GAs from S. commersonii might be lost rapidly. Indeed, some hybrids were found to have the same GA profile as S. tuberosum . Finally, the results showed that, among the parameters considered, GAs content is the most sensitive parameter to variation. The authors concluded that GAs determination should be used for routine control of genotypes produced by interspecific hybridization . Another problem concerning potato production is attributed to resistance to pests and diseases. Betz et al. [119] transferred a single gene encoding an insecticide protein from Bacillus thuringiensis (Bt) into corn. The gene confers resistance to the European corn borer, a devastating insect pest. The same gene can be transferred into other vegetables and confer resistance to the same, or related, insect pests. It has, in fact, been transferred into potatoes to confer resistance to the Colorado potato beetle, a devastating pest of potatoes. The influence of alterations in genome constitution on the relative proportions of GA aglycones in a range of interspecific somatic hybrids between wild Solanum species ( Solanum brevidens Phil.) and cultivated potato has been defined [120]. The S. brevidens parental species produces tomatidine aglycone, whereas the S. tuberosum line produces solanidine aglycone. It has been demonstrated that specific undesirable traits derived from wild Solanum species, such as alien GAs expression, could be reduced by the production of ‘second generation’ somatic hybrids of potato, in addition to improved tuberization and without the elimination of virus and bacterial disease characteristics. Transgenic resistance offers a simple and effective basis for nematode control in potato crops. Such control recently realized in Bolivia is an example of the potential benefits of genetically modified food [121]. If the crop is grown

42 each year, nematode losses caused by potato cyst nematodes are ~40%. Elimination of nematode losses and production of 12 tonnes of potatoes per hectare is a target for agriculturists.

5. Concluding remarks The concentration of GAs in potatoes destined for human consumption in many countries, 200 mg/kg of fresh weight – which is generally accepted as a ‘total alkaloid taste standard’ – has a ‘zero’ safety threshold. One reason for this conclusion is best stated by the words of Parnell et al. [122], in a paper published 23 years ago: “Many authors have assumed without further evidence that levels below 200 mg/kg are safe. They ignore the fact that the 200 mg/kg level only relates to acute and/or subacute effects and not to possible chronic effects.” It is obvious that the existing total alkaloid taste standard should be revised and new guidelines for potato consumers and breeders should be formulated. Future research must focus on: i) Identifying the enzymes involved in the biosynthesis of potato GAs, because the exact pathway for the conversion of cholesterol and cholestanol to GAs has not yet been ascertained, as well as investigating the mechanisms of the combined action of GAs with other chemicals on humans and animals. ii) Regular investigation of potato lines currently on the market throughout the world, as well as novel varieties resulting from breeding programs, to accurately assess whether or not they contain high GA levels. iii) Choice of cultivation and storage conditions and postharvest treatment, providing a maximal decrease in the alkaloid levels in potato. iv) Systematic research concerning embryotoxicity, genotoxicity and teratogenicity of GAs, aimed at establishing a GA ‘toxicologically proved concentration standard’ safe for human consumption. v) Development of smart analytical devices able to control the GA level in foodstuffs, biological fluids and tissues both selectively and specifically. vi) Evaluation of the possible anticancer activity of natural and artificial GAs, and development and production of novel drugs and antiviral creams containing GAs.

43

44

Part 2

Cholinesterase inhiinhibitionbition by glycoalkaloidsglycoalkaloids:: probing witwithhhh potentiometric biosensor

The kinetics of inhibition of human and horse sera butyryl cholinesterases by solanaceous glycoalkaloids :-solanine, :-chaconine and tomatine has been studied by means of a potentiometric biosensor based on pH-sensitive field-effect transistors (pH-FETs). Using acetyl- and butyryl choline as substrates, the optimal pH and the apparent kinetic parameters

(Kmapp , Vmaxapp ) of immobilized cholinesterases have been estimated in the absence of inhibitors. All studied glycoalkaloids have been shown to be reversible inhibitors of both cholinesterases and to inhibit the horse and human immobilized enzymes in competitive and mixed modes, respectively. The affinity of each enzyme towards :-solanine, :-chaconine and tomatine has been estimated through calculation of apparent inhibition constants Kiapp and inhibition coefficients I50. An application of the butyryl cholinesterases studied in the biosensors for glycoalkaloids detection within concentration range from 10 -7 to 10 -4 M will be discussed.

Results presented in Part 2 were published in Pesticide Biochemistry and Physiology , 2006, Ukrainian Biochemical Journal , 2006.

45 1.1.1. Introduction Mechanisms of mammalian enzymes interaction with agrochemicals and natural toxins is one of the major current topics in the research on biochemistry and toxicology. On the other hand, the enzymes sensitive to effector action of xenobiotics are widely used as sensitive elements in the biocatalytic sensors based on different types of transducers [8, 9, 28]. An example of such analytic device is a recently elaborated potentiometric biosensor based on pH-sensitive field-effect transistors (pH-FETs) and immobilized horse serum cholinesterase for direct quantitative detection of glycoalkaloids :-solanine and :-chaconine in potatoes [111-113, 123] and tomatine in tomatoes [124]. Usually, an express inhibitory analysis by enzymatic biosensors is routinely used for rapid calculation of toxin concentration in real samples. In order to determine the analyte amount, the level of enzyme inhibition/activity is evaluated from comparison of the biosensor responses to the substrate addition before and after contact of biocatalytic film with toxin solution and then plotted versus inhibitor concentrations [110, 112]. For characterizing inhibitor efficiency it is very popular to determine the inhibition coefficient or I 50 (other commonly used names: IC50 , i 0.5 ). This parameter is borrowed from routine usage in pharmacology and reveals the effector concentration at which the enzymatic reaction is inhibited by 50%

[125]. However, I50 is insufficient for adequate evaluation of both inhibition type and affinity of reversible inhibitor towards target enzyme, because I50 depends on the inhibition mechanism. Thus, I50 cannot be accepted as a complete characteristic of affinity in the case of competitive inhibition because of its strong dependence on the initial substrate concentration S0, chosen at investigator’s own discretion, and the equilibrium constant of the dissociation of the enzyme-substrate complex, Ks:  S  =  + 0  ⋅ I 50 1  K i ; (2.1)  K S 

where Ki is an inhibition constant, i.e. constant of dissociation of enzyme-inhibitor complex [EI] having dimensions of concentration [126]: [E]⋅[I] K = (2.2) i [EI]

46 where [I ] and [E] are the concentrations of free inhibitor and enzyme.

Ki serves as a measure of inhibitor potency, i.e. the affinity towards enzyme, and Ki = I 50 only in case of full non-competitive inhibition [125] which is thought to have a quite low occurrence in nature [126].

Cholinesterase is one of the crucial enzymes responsible for the nervous system functioning. In fact, vertebrates possess two types of cholinesterases: acetyl cholinesterase (EC 3.1.1.7) and butyryl cholinesterase (EC 3.1.1.8) with different kinetic properties and specificity towards various substrates and inhibitors [127, 128]. In human beings, acetyl cholinesterase (AcChE) is localized in neurons and erythrocytes; butyryl cholinesterase (BuChE) – in neurons, glia and blood serum [128]. Some low molecular weight compounds e.g. natural and synthetic drugs and pesticides are known to be reversible and irreversible inhibitors of both cholinesterases. For example, drugs inhibiting brain AcChE and BuChE are commonly prescribed for the symptomatic treatment of Alzheimer’s disease (AD) [128]. Anti-AD drugs of the first generation were physostigmine and tacrine . The second generation elaborated in nineties is currently represented in the market by the dual AcChE and BuChE inhibitor rivastigmine and AcChE inhibitors donepezil and galantamine [128, 129]. The latter chemical is a phenanthrene alkaloid [129]. In this paragraph we will deal with anticholinesterase activity of potato glycoalkaloids (GAs). As it has been mentioned in Part 1, main potato GAs :- solanine and :-chaconine in certain concentrations can provoke a serious food poisoning [130-132], meanwhile the principal tomato alkaloid :-tomatine seems to be much safer for humans [39]. Commercially available glycoalkaloid known as tomatine is a mixture of dehydrotomatine and :-tomatine (Fig. 2.1, A, B). Both are present in all parts of tomato plant. Green tomatoes can contain up to 500 mg of :-tomatine per kg of fresh fruit weight [39]. Importantly, microwaving and frying do not affect GAs level in foodstuff. Solanaceous GAs consumed with food enter in the bloodstream [131, 132], where like some other xenobiotics they can interact with soluble BuChE. The latter constitutes about 0.1% of serum proteins [133] and is often considered as a bioscavenger enzyme [101, 134]. High affinity of serum BuChE towards solanaceous GAs can be an example of xenobiotic inactivation through

47 binding with the target protein. Serum cholinesterase inhibition by GAs seems to be reversible [135].

AAA BBB

FFFigFigigig.... 222.2...1.1.1.1. Chemical structure of dehydrotomatine (A) and :-tomatine (B). Steroid backbone of tomatine molecule is presented by tomatidine.

Kinetics of cholinesterase inhibition by possible pharmacophores has been intensively studied during last years [2, 134, 136]. Although the anticholinesterase activity of GAs was revealed almost fifty years ago [137], the mechanisms of cholinesterase inhibition by these natural toxins are still poorly known. The main goal of this work was a comparative investigation of kinetic properties of immobilized butyryl cholinesterases from human (hBuChE) and from equine (eBuChE) sera and their affinity towards main potato glycoalkaloids – :-solanine and :-chaconine, and main tomato glycoalkaloid – tomatine.

2. Experimental

2.1. Materials Butyryl cholinesterases from horse serum (activity 11.4 U/mg solid) and human serum (6.3 U/mg solid), bovine serum albumin (BSA) with 98% purity, acetyl choline chloride (AcChCl) with 99% purity, butyryl choline chloride (BuChCl) with 98% purity, crystal glycoalkaloids :-chaconine, :-solanine and

48 tomatine (a mixture of :-tomatine and dehydrotomatine in ratio about 10:1) with purity 95%, 95% and 98%, respectively, were purchased from Sigma. For enzyme immobilization a saturated vapor of 25% aqueous solution of glutaraldehyde obtained from Serva was used. All reagents for buffers preparation were of analytical grade. All solutions were prepared with high- purity water with resistance18.2 Mi. Glycoalkaloids were dissolved in 5 mM acetic acid to final concentration 2 mM. The substrates were prepared as 0.2 M stock solutions in water.

2.2. pHpH----FETsFETs Design and operation mode of potentiometric transducers based on the ion sensitive field effect transistors (ISFETs, or, in our case, pH-FETs) is presented in Annex A.

2.3. Enzyme immobilization Biorecognition films on the transducer surface were formed by a method of protein cross-linking in saturated vapors of glutaraldehyde [138]. Enzyme and BSA were dissolved in 20 mM K-phosphate buffer pH 7.4 to final concentration 5% (w/v) of each protein, after that 10% (v/v) glycerol was added as plasticizer and stabilizer to avoid protein conformation changes during immobilization and to improve proteins adhesiveness to solid surface [11]. A drop of this mixture was deposited on the sensitive surface of one transducer, while the same mixture, but containing 10% (w/v) BSA instead of enzyme, was deposited on “reference” transducer surface. For biosensitive layer formation, the sensor chip was placed in a saturated glutaraldehyde vapor for 20-30 min, and then dried at room temperature and during 10-20 min washed in 5 mM K-phosphate buffer pH 7.5 in order to remove unbound molecules.

2.4. Measurements Measurements were performed in daylight at room temperature using a stirred working buffer solution in an open glass cell (V=2 ml). To equilibrate biosensitive layer and achieve a stable signal, biosensors were preincubated during 20 min in a working buffer.

49 The differential output signal between working and reference ISFET was registered using an experimental device presented in Annex A, Fig. A-2. For enzyme kinetics estimation the following procedure was applied:

i) a baseline (“0” signal) was registered in a working buffer solution;

ii) an aliquot of stock substrate solution was added into the vessel with working buffer and kinetics of the signal change was immediately recorded;

iii) biosensitive layer was washed from substrate with working buffer;

iv) sensor was again immersed into buffer, an aliquot of inhibitor solution was added into the vessel and the reaction was started by adding substrate; kinetics of signal change was immediately recorded;

v) biosensitive layer was washed with a working buffer down to “0” signal.

For percent evaluation of level of enzyme inhibition, the measurements were performed in a steady-state mode [112, 113].

3. Results and discussion

3.1. Optimal pH The enzyme activity as well as the pH-sensitive biosensor response can vary significantly depending on different pH of the medium. So, first of all, optimal pH of immobilized BuChEs was determined (Fig.2.2). Measurements were performed in 2.5 mM multi-component “polymix” buffer [112]. Since its capacity is stable over a wide range of pH (5-9), this buffer was chosen to study pH influence on the biosensor response. As a substrate, 1 mM BuChCl solution was used.

It was shown (Fig.2.2) that the optimal pH of eBuChE was in the range of 7.3-8.1, which is in excellent agreement with the literature data on optimal pH

50 values obtained for dissolved eBuChE (7.6 at 25 ºC) and immobilized eBuChE (from 8.0 to 8.7) found in the literature [139, 140]. Optimal pH value for hBuChE was found to be 7.2-7.7 – slightly more acidic than the optimal pH value (about 8.0) found by Masson et al. [141] for dissolved human BuChE using benzoyl choline as a substrate.

FigFigFig.Fig ...2.22.22.22.2.... Effect of pH on the response of biosensor with immobilized hBuChE (1) and eBuChE (2) at room temperature in 2.5 mM “polymix” buffer, [BuChCl]=1 mM.

As it can be seen in Fig.2.2, the responses obtained in “polymix” buffer are quite small, that is why for further investigations another solution was taken as the working buffer, 5 mM K-phosphate buffer pH 7.5, providing 10 fold higher sensor signals.

3.2. SSpecificitypecificity to towardswards substrates Both immobilized BuChEs were found to follow the Michaelis-Menten kinetics of hydrolysis of butyryl- and acetyl choline in the concentration range of 0.25-2.5 mM (Fig.2.3). For eBuChE a decrease in the maximal reaction rate at excess of BuChCl (>15 mM) was shown, meanwhile for AcChCl this phenomenon was not observed.

51 The soluble BuChE is well known not to be inhibited by substrate excess [128], so the signal decrease in case of the immobilized enzyme can be explained as a consequence of the restricted diffusion of the product of this reaction, butyrate anions, out of the biosensing layer. The butyrate anions are more hydrophobic than acetate ones, thus, their mobility in the protein layer is presumably worse than that of the acetate ones. The excess of butyrate anions in the biosensing layer may act as a buffer accepting protons generated during enzymatic transformation of butyryl choline, therefore, the sensor signal decreases.

According to Michaelis-Menten equation, rate of enzymatic catalysis V and constant of Michaelis Km are related as follows: V S V = max [ ] ; (2.3) + K m []S

The apparent Michaelis constants Kmapp and maximal rates Vmaxapp values were calculated by linearization of data in Lineweaver-Burk (1/ V vs 1/ [S], Fig.2.3, A, B), Eadie-Hofstee ( V vs V/[S]) and Hanes-Woolf ([S]/V vs [S]) coordinates derived from the equation 2.3. Then the average values of kinetic parameters obtained by three graphic methods were calculated (Table 2.1). It was found that immobilized eBuChE had almost the same affinity to BuCh and AcCh, as hBuChE. However, the hydrolysis of butyryl choline by eBuChE is 2.5 fold and by hBuChE – 3.2 fold faster than that of acetyl choline.

Table 2.2.2.1.2. 1.1.1. Apparent kinetic parameters of immobilized hBuChE and eBuChE measured in the absence of inhibitors. Each value presents an average of calculated by three different graphic methods, mean±SD.

Apparent eBuChE, eBuChE, hBuChE, hBuChE, parameter BuChCl AcChCl BuChCl AcChCl

Kmapp , mM 1.05±0.25 0.87±0.39 0.33±0.04 0.39±0.14

Vmaxapp ,mV/min 102.25±5.70 40.43±2.20 25.38±1.68 7.75±0.72

52

Fig. 2.32.32.3.2.3 ... (A) Responses of biosensor with immobilized eBuChE (inset) or hBuChE versus concentrations of BuChCl (1, 1’) and AcChCl (2, 2’). (B) Responses of biosensor with immobilized eBuChE versus concentrations of BuChCl (1) and AcChCl (2).

Measurements were conducted in the kinetic mode (A) or steady-state mode (B).

53

Fig. 2 2....4444.... Lineweaver-Burk plots corresponding to Fig.2.3 A and Fig.2.3 inset. Biofilm: hBuChE (A) or eBuChE (B), Enzyme substrates: 1 – BuChCl, 2 – AcChCl.

The obtained apparent Michaelis constant (Table 2.1) of immobilized horse BuChE is in good agreement with Km=1.52 mM, presented in literature for dissolved equine serum BuChE (substrate – butyryl choline [142]) but essentially larger in comparison with 0.3 mM (the same substrate) found by Main et al. [143]. As for human BuChE, the main part of investigations has been performed with butyryl thiocholine, not butyryl choline, as a substrate; so, we had no opportunity to compare properly the affinities obtained by means of biosensor with the literature data on dissolved enzyme.

Determination of kinetic parameters of the enzymatic reaction is of great practical importance in applied biochemistry and biotechnology. For example, when the substrate concentration exceeds Km several times and thus the enzyme works at almost maximal rate, the measured Vmax value can be used for estimation of the active enzyme amount in different media.

3.3. Specificity towards inhibitors The dependence of eBuChE-modified sensor responses on different BuChCl concentrations in the presence of 20 fM :-chaconine in working buffer is shown in Fig.2.5. The best sensitivity to inhibitor is achieved at low substrate concentrations (0.2-1.2 mM), and this phenomenon could be considered as one of the attributes of competitive inhibition.

54 For very low substrate concentrations (<0.1 mM) the enzyme in the membrane is in excess, hence, it is involved in the substrate hydrolysis only partly and its binding with inhibitor can be compensated by involving free enzyme molecules in the reaction. Due to this compensatory mechanism, a percent inhibition measured by biosensor at very low substrate concentration will be lower than the actual decrease in enzymatic activity after inhibition. This effect is typical for immobilized enzymes irrespective of both the inhibition mechanism and the method used for the detection of enzymatic activity [144].

Fig. 2.52.52.5.2.5 ... Level of inhibition of the immobilised eBuChE with 20 fM :-chaconine versus BuChCl concentrations in working buffer.

At the BuChCl concentration equal to Kmapp the immobilized eBuChE presented almost maximal sensitivity towards inhibitor; meanwhile the detectable biosensor response to BuChCl addition was quite large. The similar results (not shown) were obtained for :-solanine and tomatine. It was also shown that the BuChE inhibition by glycoalkaloids was time- independent, which indicated its reversibility. It was established that :- solanine, :-chaconine and tomatine inhibited reversibly both human and horse immobilized BuChEs, and this phenomenon does not contradict the known data [131, 135].

55 To assess the type of inhibition by GAs, the data on kinetics were analyzed according to classical methods of Dixon [145] and Cornish-Bowden [146]. The eBuChE inhibition by :-solanine, :-chaconine and tomatine was fully competitive which was confirmed by means of the three lines intersecting in the second quadrant above x-axis in Dixon’s coordinates, Fig. 2.6, and three parallel lines in the Cornish-Bowden coordinates ( [S]/V vs [I ], where [I ] is free inhibitor concentration), Fig. 2.6 inset. The data presented in Fig. 2.6 for :-chaconine were typical for eBuChE inhibition by :-solanine and tomatine as well.

Fig. 2.62.62.6.2.6 ... Kinetics of inhibition of immobilized eBuChE by :-chaconine presented in the Dixon and Cornish-Bowden (inset) plots. 1 - 0.5 mM, 2 - 1 mM, 3 - 2 mM BuChCl.

Since the investigated GAs inhibited eBuChE in a competitive mode, we could not estimate their affinity to immobilized eBuChE from I50 values only. So, the inhibition constants were evaluated from the Dixon plots in the concentration range 0-10 µM (:-chaconine) or 0-14 µM (:-solanine and

56 tomatine) at the BuChCl concentrations corresponding to 0.5 Kmapp , 1 K mapp and

2 K mapp (Table 2.2).

Table 2.2.2. 2.2.2. Apparent inhibition parameters of immobilized hBuChE and eBuChE measured in 5 mM K-phosphate buffer pH 7.5.

Inhibitor eBuChE hBuChE eBuChE hBuChE hBuChE Kiapp, µM Kiapp, µM (1 mM (0.2 mM (1 mM BuChCl) BuChCl) BuChCl) I50, µM I50, µM I50, µM :-Chaconine 1.39 0.17 11.20 0.18 0.28 :-Solanine 3.33 0.22 27.90 0.34 0.52 Tomatine 1.69 1.30 11.90 0.88 1.24

The inhibition of human BuChE by glycoalkaloids seemed to be more complex than that of equine serum BuChE. While the Dixon plot revealed a competitive inhibition of hBuChE by :-solanine (Fig. 2.7, A), tomatine (Fig. 2.7 B) and :-chaconine (data not shown because the plot was similar to Fig. 2.7, A) the Cornish-Bowden plot showed non-parallel curves intersecting in the third quadrant, Fig. 2.7 insets, i.e. a mixed inhibition by each alkaloid.

The apparent inhibition constants ( Kiapp ) for hBuChE were calculated from the Dixon plots in the concentration range 0-0.4 µM :-chaconine, 0-1.6 µM :-solanine and 0-2.4 µM tomatine at the substrate concentrations 0.152 mM and 0.3 mM which corresponded to 0.5 Kmapp and 1 Kmapp . It was shown that the affinity of immobilized hBuChE to tomatine was similar to that of eBuChE since the corresponding Kiapp values did not vary significantly; however, I50 values obviously differed probably due to another mechanism of inhibition.

57

Fig. 2.72.7.... (A) Kinetics of inhibition of immobilized hBuChE by :-solanine presented in the Dixon and Cornish-Bowden (inset) plots. 1 - 0.152 mM, 2 - 0.3 mM, 3 - 0.6 mM BuChCl. (B) Kinetics of inhibition of immobilized hBuChE with tomatine presented in the Dixon and Cornish-Bowden (inset) plots. 1 – 0.152 mM, 2 – 0.3 mM, 3 – 0.6 mM BuChCl.

58 From Table 2.2 it can be seen that the I50 values of hBuChE measured at

[BuChCl]=0.2 mM (about 0.6 Kmapp ) and 1 mM (about 3 K mapp ) increase at a high substrate concentration confirming a competitive caractere of inhibition. But it is also known that the effect of competitive inhibitor at a concentration equal to its inhibition constant, on the enzyme operating at half of maximal rate Vmax can be totally nullified by doubling [S] from Km to 2 Km [147].

For eBuChE at [S]=Kmapp and [I ]=Kiapp the inhibition level does not exceed 15% for all GAs (Fig. 2.8). As for hBuChE, at [S]=3 K mapp and [I ]=Kiapp the inhibition level is about 40% in all cases (Fig. 2.8) which more likely indicates the inhibition not to be pure competitive. From Fig. 2.8 it can be seen that human BuChE is one-order more sensitive to glycoalkaloids :-solanine and :-chaconine than equine enzyme; and immobilized hBuChE is more sensitive in comparison to soluble hBuChE [131, 148, 149].

FigFigFig.Fig ... 2.82.82.8.2.8 ... Calibration curves of biosensor based on hBuChE (1, 2, 3) and eBuChE (1’, 2’, 3’) for the determination of α-chaconine (1’, 1), α-solanine (2’, 2) and tomatine (3’, 3). [BuChCl]=1 mM.

The choice of proper – whether human (hBuChE) or horse (eBuChE) – immobilized butyryl cholinesterase as a sensitive component of the 59 potentiometric biosensor for glycoalkaloids depends on the GAs concentration to be analyzed. Naturally, eBuChE is preferable for biosensor production since it is cheaper. However, application of less sensitive eBuChE is restricted to the samples with high glycoalkaloids concentration – e.g., tomatoes [124] or potatoes [113] (more than 150 mg/kg). On the contrary, preference is given to highly sensitive hBuChE to probe biological samples with rather low GAs content, such as blood serum or plasma (about 1-10 fg/l [132]).

4. Conclusion and perspectives So, human and horse sera butyryl cholinesterases immobilized on pH- sensitive surface of potentiometric transducer at optimal pH have followed the Michaelis kinetics of hydrolysis of their substrates: acetyl choline and butyryl choline in the concentration range of 0.25-2.5 mM. Potato glycoalkaloids :-solanine and :-chaconine as well as tomato glycoalkaloid tomatine have inhibited immobilized eBuChE reversibly and competitively while for hBuChE the mixed inhibition was more probable.

Considering Kiapp as a characteristic of inhibition, one can conclude that :- chaconine is the most potent inhibitor for both BuChEs; :-solanine is the most feeble inhibitor for eBuChE while tomatine – for hBuChE. The application of studied butyryl cholinesterases in the potentiometric biosensors allows detection of glycoalkaloids within the concentration range of 10 -7-10 -4 M which corresponds to practical needs. Since one of the basic advantages of ISFETs is their compatibility with technologies of microelectronics, i.e. opportunity to integrate biosensor with its processing and amplifying circuits on the same chip, this technique coupled to any enzyme(s) of interest open the way to elaboration of reagentless high throughput multisensor platforms able to screen xenobiotics and drugs e.g. the anti-cholinesterase ones.

60

Part 3

Impedimetric study of glycoalglycoalkaloidskaloids binding to cholinesterase

In this part of work, a direct impedimetric analysis of potato glycoalkaloids binding to butyryl cholinesterase (BuChE) was performed in the absence of any enzyme substrate. The molecules of BuChE from equine serum were cross-linked onto gold surface of working electrode. Affinity interaction of :-chaconine and :-solanine with immobilized enzyme caused a significant decrease in the interfacial polarization resistance of modified electrode. No effect was observed after :-chaconine contact with bovine serum albumin and bacterial carboxylesterase immobilized onto gold. Electrodes modified with

BuChE were more sensitive to :-chaconine, and found I50 values were in quite good agreement with those measured by biocatalytic ISFET-based sensors reported in Part 2.

Results presented in Part 3 were published in Electroanalysis , 2006.

61 1.1.1. Introduction Glycoalkaloids (GAs) are natural toxins of Solanaceae plant family which includes widely consumed potatoes. The principal potato GAs are :-solanine and :-chaconine. Due to their strong lytic properties [91, 150] and anti cholinesterase activity, potato GAs can provoke a serious food poisoning [132] in humans and animals. Solanaceous GAs consumed with potato meal enter the bloodstream [132], where theoretically can bind with several detoxicant proteins. Thus, non-specific cholinesterase, or butyryl cholinesterase (EC 3.1.1.8) is a high-affinity target of GAs. It is known that neutralization of toxins through hydrolysis or binding in the bloodstream involves the participation of some other enzymes, such as carboxyl esterase (EC 3.1.1.1) found in some mammalian but not human plasma [101], paraoxonase, and the most abundant blood protein, drug carrier also proven to be an esterase – serum albumin [151]. Thus the possibility of glycoalkaloids binding with these proteins cannot be excluded.

Detection of synthetic and natural toxins in aqueous solutions is one of the most important directions in the field of biocatalytic and bioaffinity sensors development. As a biorecognition element it is theoretically possible to use antibodies, aptamers, receptors, enzymes (see Bibliographic review). High- affinity aptamers [152], membrane-anchored receptors [74] or monoclonal antibodies [153] against a small xenobiotic firstly must be prepared using quite complex procedures, meanwhile, many mammalian enzymes known to be natural analyte’s targets are commercially available. Study of affinity interactions of small molecules and biopolymers attached to a solid surface is often difficult without labeling binding partners. On the other hand, an influence of low molecular weight effector on a target enzyme is usually estimated from changes in enzymatic activity and kinetic properties of enzyme (see Part 2). However, several investigations of direct enzyme interactions with small effector molecules using neither labels nor enzyme substrate have been reported recently [33, 89, 154]. An example is a study of binding between immobilized bovine carbonic anhydrase (MW 30,000) and its inhibitor – acetazolamide (MW 222.2) in concentration range of 4-1000 nM by means of surface plasmon resonance spectroscopy [33]. An application

62 of non-faradaic electrochemical impedance spectroscopy (EIS) for label-free and substrate-free detection of affinity interaction between enzyme and small effector molecule has never been yet reported. EIS is a powerful tool for the study of high-affinity interactions. From fitting impedance data to an appropriate (equivalent) electrical circuit, one can get specific characteristics of working electrode (WE), in particular, properties of electrode/electrolyte interface that can be changed due to the nature of biorecognition events under scrutiny (see Annex B).

The main goal of this work was to investigate a possibility of label-free detection of affinity interaction between potato glycoalkaloids, :-chaconine and :-solanine, and horse serum butyryl cholinesterase (MW~440,000) immobilized on gold electrode. In order to control the binding specificity of cholinesterase, :-chaconine was incubated with immobilized bovine serum albumin (MW 66,000) and bacterial carboxyl esterase (MW 34,000). Neither redox species nor enzyme substrates were used in the present study.

2.2.2. Experimental

2.1. Materials Butyryl cholinesterase (BuChE) from horse serum (11.4 U/mg), bovine serum albumin (BSA), crystal :-chaconine and :-solanine (95% purity) were purchased from Sigma. Each glycoalkaloid was dissolved in 5 mM acetic acid in order to obtain the final concentration of 2 mM. Lyophilized carboxyl esterase (esterase EST2) from Alicyclobacillus acidocaldarius with purity more than 90% was a gift from Instituto di Biochimica delle Proteine (Naple, Italy). For biomaterial immobilization we have used saturated vapors of 25% aqueous solution of glutaraldehyde obtained from Serva. Acetone, ethanol and hydrogen peroxide used for electrode pretreatment were of analytical grade from Fluka. All solutions were prepared from analytical grade reagents and MilliPore MilliQ ultrapure water (resistance 18.2 Mi cm).

63 2.2. Working electrodes

2.2.1. Construction Gold electrodes were provided by Laboratoire d’Analyse et d’Architecture des Systèmes (Toulouse). They were fabricated using standard silicon technologies. <100>-oriented, P-type (3-5 i cm) silicon wafers were thermally oxidized to grow an 800 nm-thick film oxide. Then, a 30 nm-thick titanium layer and a 300 nm-thick gold top layer were deposited by evaporation under vacuum.

2.2.2. SSSurfaceSurface cleaning Before use, gold electrodes were sonicated in acetone during 10 min in order to eliminate a protective polymer film from the surface, then dried under nitrogen flow. Afterwards, gold surface was cleaned from impurities by fresh- prepared “piranha” mixture (H 2O2: H 2SO 4, 3:7, v/v) during 1 min and washed in deionized water. Then substrates were sonicated in ethanol. Electrodes rinced with water were finally dried under a nitrogen flow.

2.2.3. BiofBiofunctionalizationunctionalization Solutions of BuChE, EST2 and BSA (5 mg/ml) were prepared in 20 mM K-phosphate buffer with 0.07 M NaCl, pH 7.5. In order to stabilize proteins, glycerol was added in solutions up to 5% (v/v). For biosensitive layer formation, a drop (20 fl) of protein solution was placed on electrode surface. A coating layer was formed by spin-coating (20000 rpm) during 10-20 s. Electrodes with biomaterial were exposed to saturated vapors of glutaraldehyde for 30 min to allow a biofilm formation, then dried at +4 ºC overnight. Glutaraldehyde reacts with available amino groups leading to cross- linking of protein molecules through Schiff bases (-N=CH-). It is known that diaminomonocarbonic acids have not been found in the active site of butyryl cholinesterase [155], so one could suppose that during immobilization the biological activity of enzyme was not modified significantly. In order to remove any unbound molecules from the surface, electrodes were washing in 5 mM working buffer (KH 2PO 4 + NaOH, pH 7.5, with 0.07 M

64 NaCl) during 30 min at room temperature. The pH of working buffer was chosen taking into account the optimal pH value found for horse serum cholinesterase cross-linked onto pH-sensitive field effect transistors (see Part 2), and the optimal pH for EST2 reported by Manco et al. [156].

2.3. ImpedaImpedancence measurements Impedance measurements were carried out in three-electrode electrochemical cell, V=5 ml, placed into a Faraday cage in order to improve the signal-to-noise ratio (for details, see Annex B). The impedance analyzer Voltalab 40 controlled by the software VoltaMaster 4.0, was used to sweep impedance within the frequencies range 0.633 – 10 5 Hz, acquiring 5 points per decade. An excitation voltage of 10 mV was superimposed on a DC potential of -200 mV. As a working electrode (WE) a gold-coated Si electrode modified with biomolecules was used; WE measurement surface area was 0.28 cm 2, as a reference electrode (RE) – a saturated calomel electrode purchased from Radiometer Analytical SA. A Pt plate (surface area 1.21 cm 2) was used as an auxiliary electrode (AE). Active surfaces of AE and WE were in contact with cell content and were confined with a special O-ring. Working buffer was mixed with a magnetic stirrer. All measurements were carried out at room temperature

3. Results and discussion

3.1. BBBiofilmBiofilm stability Protein-coated electrodes were investigated for stability and repeatability of impedance spectra in contact with a working buffer at continuous stirring. In this aim, total impedance spectra were acquired during 60 min at the same conditions. The system was considered as stable if the last serie of 8-10 Nyquist curves could be superposed with a precision of ±5%. For electrodes modified with BSA and EST2 a good repeatability of measurements was achieved in less than 30 min while for BuChE a stabilization time about 60 min was necessary.

65 3.2. ElectrochemiElectrochemicalcal characteristics of protein layerslayers Formation of a complex between immobilized biopolymer and a free molecule of analyte usually impacts two principal parameters of working electrode interface: the capacitance and the resistance [157]. In order to evaluate these parameters, the impedance spectra were fitted to equivalent electrical circuit by means of ZView2 (Scribner Associates, USA) software. The data obtained from BuChE and EST2-modified electrodes were satisfactorily fitted with an equivalent circuit shown in Annex B, Fig. B-1, A over the entire frequencies range. Fitting of spectra from BSA-modified electrode was performed using the circuit shown in Annex B, Fig. B-1, B.

Table 3.1 summarizes the data obtained after fitting the spectra from all studied electrodes. As it can be seen, once an electrode is modified with proteins, the CPE value decreases 2-fold and the Rp increases 3-4-fold. All the n values are about 0.9 (except BSA-modified electrode, where n=0.84), so the CPE could be considered as a capacitive element.

Table 3.3.3.1.3. 1.1.1. The fitting values calculated in ZView2 for the gold electrodes, bare and modified with proteins. Surface area of electrodes is taken into account. All errors here represent standard deviations based on n=5.

222 Electrode RRRs, FFF CPECPE----TTT,,, nnn RRRppp, FFF HHH GF(rad/s) 111-1---nnn (((×11101000---4-444))) Bare Au 45±16 11.3±1.5 0.95±0.01 1234±227 9.8±0.02 Au-BuChE 147±17 5.5±0.7 0.89±0.02 6957±1974 8.0±1.2 Au-EST2 198 7.2 0.91 8245 5.3 Au-BSA 148 6.6 0.84 8471 9.0

Since protein solutions were deposited on electrodes manually, it was evidently impossible to obtain biofilms with the same thickness, so, the CPE and Rp absolute values of identical BuChE-modified electrodes varied at 13% and 28%, respectively (Table 3.1).

The strategy of enzyme immobilisation used in the present work was chosen in the view of the following peculiarities of glycoalkaloids:

66 i) their small size which favour a penetration into the thick and/or porous multilayer; ii) their detergent properties and thus an ability to interact with aliphatic and amphiphilic organic molecules (e.g. long-chain thiols) via hydrophobic forces, leading to appearance of non-specific responses.

Another aim was to compare the sensitivity of BuChE-based impedimetric detection of glycoalkaloids with that performed by means of catalytic biosensors based on the BuChE cross-linked with BSA [158, 159]. Since it was shown that :-chaconine and :-solanine do not inhibit an esterase activity of serum albumin [131], the last could be applied for the biocatalytic sensor elaboration in order to stabilize the working enzyme in cross-linked layer. As for the affinity biosensor development, before introducing BSA into a biorecognition element as a stabilizer, it is worth studying its ability to bind glycoalkaloids.

333.33.3.3.3.. Impedimetry of glycoalkaloids On reaching a satisfactory repeatability of impedance spectra, aliquots of :-chaconine or :-solanine bulk solutions were injected into the electrochemical cell, after that the impedance measurements were started immediately.

Results are presented as series of Nyquist spectra (Fig. 3.1, A, B). It can be seen that impedance decreases significantly after GAs contact with the BuChE-modified electrodes, while there is only a slight variation of semicircles radius in case of BSA and EST2-modified electrodes.

A possible contribution of a glycoalkaloid “solvent”, 5 mM acetic acid, to the total impedance was also studied. The blank probes of volume from 1 to 60 fl, corresponding to 0.4-24 fM glycoalkaloid (replaced with water) in 5 mM acetic acid, were added into cell with BuChE-modified electrode, and no shift of the Nyquist spectra was observed (see Table 3.2), therefore, impedance changes in the presence of glycoalkaloids could be considered as a specific response.

67 The fitting parameters of 4 electrodes, selected for the highest n value, calculated with chi-squared 62<9.8 ×10 -4 before and after contact with :- chaconine, :-solanine and blank probes, are summarized in Table 3.2.

Table 33.2..2..2..2. The fitting values calculated in ZView2 for the protein-modified electrodes before and after contact with glycoalkaloids or blank probes. Surface area of electrodes is taken into account.

Q= CPE-T, fF (rad/s) 1-n

As it can be seen, 1.2-fold increase in the CPE was observed for the 20 fM and 16 fM of :-chaconine injected over the BuChE- and the EST2- modified electrodes, respectively. The CPE values of BuChE-modified electrodes contacted with :-solanine and blank probes as well, were changed insignificantly.

Essential decrease in Rp values was showed by BuChE-modified electrodes after their contact with either :-chaconine (about 40%) or :-solanine (about 20%). A slight increase (about 1%) of polarization resistance was observed after the electrode contact with blank probes. Since both size and mass of glycoalkaloid molecule are quite small in comparison to the molecule of protein, the thickness of enzyme layer cannot be altered significantly by the binding event itself, so, a decrease of CPE (and increase of n value) caused only by mass changes in biorecognition layer is impossible. An essential diminution of Rp observed after contact of BuChE with both GAs, could be caused by charge redistribution or/and conductivity changes in enzyme layer.

68

FigFigFig.Fig ... 3.13.13.1.3.1 ... Nyquist plots of Au electrodes modified with BuChE (A), EST2 (B) and BSA (B inset) under various concentrations of :-chaconine and :-solanine: (A) a – 0 fM, b – 4 fM, c – 8 fM, d – 12 fM, e – 16 fM, f – 20 fM :-chaconine; (A inset) a – 0 fM, b – 4 fM, c – 8 fM, d – 16 fM, e – 20 fM, f – 24 fM, g – 28 fM :-solanine; (B) a – 0 fM, b – 4 fM, c – 8 fM, d – 16 fM :-chaconine; (B inset) a – 0 fM, b – 4 fM, c – 8 fM, d – 12 fM :-chaconine.

69 In the case of :-solanine, variation of Rp evidently can not arise from the removal of enzyme molecules from electrode into buffer because the CPE value remained constant. Presumably, decrease of the Rp parameter could result from conformation changes in charged enzyme molecules after binding with glycoalkaloid. Thus, when the capacitive element CPE did not vary significantly, the charge quantity in the enzyme layer might remain constant, while a charge distribution profile could be changed due to the alteration of enzyme conformation.

As to EST2-modified electrode, there were no significant variations

(about 1%) of the Rp after contact with :-chaconine, as it can be seen from Table 3.2. Increase of capacitance (CPE) here can be explained as a non- specific removal of protein molecules from biofilm. However, the absence of drastical decrease in Rp in this case reveals that the above-observed diminution of Rp may nevertheless be considered as a specific attribute of affinity interactions of glycoalkaloids and BuChE.

Surprisingly, it was found to be quite difficult to regenerate a sensing surface after contact with GAs. That is why to obtain dose-response curves it was chosen a method of successive injection of various glycoalkaloids concentrations over the same biofilm. Nevertheless, after the contact of immobilized BuChE with blank probes, a “zero” spectrum (“baseline”) was restored very quickly by changing the bulk electrolyte in the cell.

3.3. Calibration curves For practical use, direct monitoring the impedance value at a fixed value of frequency seems to be satisfactory and therefore is preferable [153]. The impedance data obtained for each electrode were analyzed at the frequency 1 Hz (Fig. 3.2). Using the Origin software, the dependence of the relative variation of total impedance modulus (| Z|0-|Z|), on the concentration of GA was fitted to the sigmoidal equation .

Shifts of the fitted Rp parameter before and after enzyme contact with glycoalkaloids ( Rp0 – Rp) for BuChE-modified electrodes were plotted vs glycoalkaloid concentrations and fitted to the linear equation (Table 3.3).

70 Table 333.3...3.3.3.3. Calibration plots for impedimetric detection of glycoalkaloids. Linear range, Linear range, Parameter III-I---chaconinechaconine III-I---solaninesolanine GMGMGM GMGMGM Y=-190.9+283.2 X, Y=-235.2+93.2 X, |Z|0-|Z| 0-16 0-20 R=0.995 R=0.965 Y=-106.6+261.3 X, Y=-136.3+37.5 X, Rp0 -Rp 0-20 0-24 R=0.997 R=0.958

Fig. 333.3...2222.... Calibration curves for the Au electrodes modified with BuChE (a, b), BSA (c), EST2 (d) after contact with :-chaconine (a, c, d) or :-solanine (b), representing the dependence of total impedance modulus changes at the frequency 1 Hz from GAs concentrations. Sigmoidal fit was applied for approximation of the curves (a) and (b). Error bars represent SD based on n=2.

Data obtained from bioaffinity impedimetric sensor platform were compared to data obtained from the biocatalytic ISFET-based sensors (Table

3.4). In this purpose, the I50 values were extracted from the dose-response curves obtained in the presence of enzyme substrate (see Part 2) and in the absence of substrate.

I50 parameter reveals the analyte concentration resulting in a 50% inhibition of sensor response and is chosen to compare two effects of glycoalkaloids on immobilized BuChE: affinity binding and modulation of biocatalytic properties.

71 Table 3.3.3.4.3. 4.4.4. I50 parameters, estimated through BuChE-based catalytic and affinity biosensing in the presence of potato glycoalkaloids.

Characteristics PPPotPotototentiometrentiometrentiometryyyy I Impempempedimetrydimetry

Need of the yes no enzyme substrate

I50 for :- 11.2 11.3 chaconine, fM

I50 for :-solanine, 27.9 15.3 fM

It was shown an excellent agreement in the I50 values for :-chaconine for both types of sensor techniques. However, the sensitivity of :-solanine analysis was 2-fold better in the absence of enzymatic substrates. Since :-solanine is less potent competitive inhibitor of horse BuChE than :-chaconine (Part 2, [112]), the affinity biosensing is more sensitive mode of its label-free detection.

4. Conclusion and perspectives In this chapter, the possibility of label-free electrochemical impedimetry of affinity interactions of small effectors, :-chaconine and :-solanine, and immobilized enzyme, butyryl cholinesterase from horse serum, has been shown. Impedance results interpreted in terms of modification of charge repartition in the biosensitive layer suggest a modification of enzyme conformation, but need to be corroborated by more detailed investigations. For example, SPR spectroscopy could highlight an impact of glycoalkaloids on dielectric properties and mechanical stability of immobilized enzyme layers.

72

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Summary

PART 4. Natural, electronic and bioelectronic olfaction 777777 PART 5. Electrochemical study of human olfactory receptor OR 17-40 stimulation by some odorants 858585 PART 6. Response pattern of human olfactory receptor OR 17-40 probed by surface plasmon resonance 109

73

74

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Ce chapitre est consacré à differents aspects de l’olfaction naturelle et artificielle. Dans la partie experimentale, nous présentons deux biocapteurs basés sur un récepteur olfactif humain RO 17-40 spécifiquement sensible à hélional. RO 17-40 a été co-exprimé hétérologiquement avec la protéine G: olf par des lévures. En vue de la réalisation des biocapteurs le RO 17-40 a été immobilisé dans sa fraction membranaire, sur une électrode d’or. Une méthode de réalisation d’une multicouche auto-assemblée a permis de construire de manière répétable une nanoarchitecture complexe qui a été caractérisée par voltamètrie cyclique, microscopie à force atomique, résonance plasmonique de surface (SPR) et spectroscopie d’impédance non-faradique (EIS). Les deux dernières techniques ont également servi à évaluer l’activation spécifique de RO 17-40 par l’hélional – son odorant préféré. La décroissance d’impédance non-faradique, systématiquement observée en présence d’hélional, peut être attribuée au changement de conformation du récepteur. La sensibilité de RO 17-40 dans la gamme 10 -12 – 10 -5 M d’hélional, à température ambiante et à 4ºC en présence de GTP-g-S, a révélé des courbes en forme de cloche avec un maximum à 10 -10 -10 -11 M. Ces données ont été confirmées par la technique SPR en utilisant le GTP-g-S comme stimulateur de dissociation de la protéine G après l’activation du récepteur par l’odorant spécifique. Ces résultats ouvrent la voie à plusieurs perspectives d’application de ce type de biocapteurs pour le suivi direct des odorants d’intérêt médical, environemental, agroalimentaire (« nez bioélectronique ») et les études de pharmacologie de récepteurs « orphelins ».

75

76

Part 4

Natural, electronic and bioelectronic olfaction

In this paragraph, we review the literature concerning detection of odorants – low molecular weight xenobiotics of exceptional chemical diversity which are percepted via olfactory system producing neutral, pleasant or pungent smell sensations in human beings. It will be shown that many factors which usually affect odor perception in human testers may also present a source of artefactual responses of odorant sensitive techniques e.g. “electronic noses”. Some analytical techniques for monitoring odors will be presented with a special attention to those based on mammalian olfactory receptors. A concept of a “bioelectronic nose” will be introduced.

77 1.1.1. Introduction It is well known that smell (odor) sensation in mammalians can be evoked by a large number of small chemicals (MW <300). Some molecules are strong nasal, ocular and throat irritants, others (short-chain volatile fatty acids, phenols, amines, indoles, and sulphur-containing compounds) are responsible for unpleasant odors associated with livestock production facilities and their waste steams. Other molecules e.g. various aldehydes are widely used as ingredients in food, perfumes and cosmetics. Many compounds which are pleasant at ppm levels, are pungent at higher concentrations. Table 4.1 summarizes data on the odor and flavour features of five aldehydes, helional, heptanal, octanal, nonanal and vanillin, which were studied in the present work (see Parts 5 and 6).

Table 4.14.1. Some features of odorants [160] studied in the present work.

Odorant name Odor Flavour Application Helional® ,Mw 192.22

(:-methyl-3,4-methylene- dioxy Sweet floral, Limited use in perfume hydrocinnamic aldehyde) No data CH3 mildy compositions, available herbaceous in soap CH2 CH CHO

O

O CH 2

n-Heptanal, Mw 114.19 HC : fatty- HC : Fatty- Ingredient in a few special

rancid; rancid; fragrances; (n-heptyl aldehyde,

enanthal) LC : LC : Imitator of apple, ginger,

“fermented- Sweet, honey, melon flavours CH (CH ) –CHO 3 2 5 fruit”-like nut-like, fruity (1-5 ppm)

n-Octanal, Mw 128.22 HC : harsh- Ingredient in many fatty; HC : fatty; (n-octyl aldehyde, fragrances;

caprylic aldehyde) LC : sweet, LC : sweet, Imitator of butter, chocolate, orange- and apricot-plum- CH (CH ) –CHO apricot flavours 3 2 6 honey-like like (0.1-5 ppm)

n-Nonanal, Mw 142.24 HC : fatty- Trace amounts (<0.1%) are floral, waxy; used in many fragrance LC : refreshing, (n-nonyl aldehyde, types; citrusy, waxy pelargonic aldehyde) LC : rosy,

sweet, fresh Imitator of citrus flavour CH 3(CH 2)7–CHO (0.2-6 ppm) 78 Odorant name Odor Flavour Application Vanillin, Mw 152.15 In almost any type of (4-hydroxy-3- fragrance; methoxybenzaldehyde, HC : intensely vanillic aldehyde, Masking agent in ill-smelling sweet; Lioxin®) products (rubber, plastic Sweet, fruity etc.); CHO LC : creamy

In ice-cream, chocolate,

O CH3 candies flavor OH (from 50 to 20000 ppm)

HC – high concentration, LC – low concentration

2.2.2. DDDetectionDetection of odorants Simultaneous detection of diverse odorants in liquid and gaseous mixtures is important for quality control in food, beverage and fragrance industries. Detection of some odorants i.e. formaldehyde, acetone, heptanal etc. in human breath, blood and urine can be extremely useful in clinical diagnostics of diabetes, liver diseases, cancer of lung and prostate [18, 161]. However, accurate measurement of odorants in odors is challenging due to the usually weak solubility of these molecules in water and their extremely low concentrations ranging from high ppm (1/1,000,000) to low ppb (1/1,000,000,000) in real samples.

Nowadays, the instruments capable of a broad-band routine analysis of complex odors are mainly presented by mass-spectrometers and gas chromatographs combined to mass spectrometers (GC/MS). There are two main drawbacks related to the latter: i) requirement of an extraction, separation or preconcentration step; ii) a spectrum of odorant peaks obtained by means of GC/MS does not provide sufficient information about odor/flavour quality itself which, however, directly arises from the human odor impressions [162].

Therefore, the traditional human panels are routinely used in the testing of wine, foodstuff, coffee, perfumes. Nevertheless, attempts are being made throughout the world to create an analytical tool able to “evaluate” the quality of product aroma in “human-like” manner. A very recent and successful

79 example is a “robot-sommelier” (Fig. 4.1) developed in 2006 by NEC System Technologies and Mie University (Japan). This “wine robot” has the ability to distinguish between a few dozen types of wine. Infrared light in fired through the wine sample, and robot performs differentiation between samples by determining different wavelengths of absorbed light. Certainly, this robot is still incapable to replace human testers but it may be very useful to help in control of wine quality.

Fig. 4.1. Robot-sommelier uses infrared sensors to distinguish between varieties of wine. Photo is taken from http://news.bbc.co.uk/2/hi/technology/5312220.stm

3.3.3. From electronic to bioelectronic noses Artificial olfaction is currently presented by artificial ( electronic) noses – the arrays of semiselective sensors coupled to the pattern-recognition systems [162-164]. Such artificial “olfactory” instrumentation is able to analyze odorant mixture without its separation thus mimicking mammalian olfaction [50, 165], Fig. 4.2, A, B. As sensitive (but not selective!) elements of electronic noses, conductive organic polymers [166, 167], porphyrins [168], calixarenes [169- 171], can be used. Various physical phenomena have been employed for array- based odor sensing: changes in resistance, impedance, current, capacitance, temperature, optical properties [172]. Metal-oxide sensors (MOS), metal-oxide– silicon field-effect transistors (MOSFETs), SAW devices, quartz resonators and fiber-optic chemical sensors have proved to be suitable for analysis of complex odors [163]. Nowadays, many “electronic noses” are commercialized and are successfully used in the control of livestock wastes, quality of grain, coffee etc. In the last years, the curiosity of scientists has stimulated the development of a bioelectronic nose [162, 173] – multisensor device whose biorecognition part consists of G protein coupled olfactory receptors (ORs). In fact, the discovery of ORs multigene family [58, 174] (Buck and Axel, Nobel prize in physiology, 2004), prominent advances in heterologous expression of

80 mammalian ORs [175-177] and in the understanding of olfactory combinatorial code [178] have opened the way to creation of receptors-based high throughput biosensor platforms. They will serve: i) to study the pharmacology of ORs; ii) as tool for deorphanisation of poorly studied ORs; iii) as human nose-like biomedical tool able to monitor toxic, irritating and unpleasant odors in biological liquids e.g. saliva, blood, urine or breath of patients; iv) as tool for quality control in food industry etc. There are many recent publications concerning the elaboration of individual sensors based on the ORs expressed either in olfactory sensory neurons [173] or in heterologous cells [56, 80, 179, 180] and coupled to various transducers like QCM [179, 180], LAPS [173], substrates for SPR [56] and EIS [80] techniques. Odorant detection by means of OR-based biosensors is based on monitoring either of ligand binding or molecular events triggered by agonist-stimulated receptor [51]. The monobiosensors with the best analytical characteristics and performances will be used in real bioelectronic noses. Although there are human ORs predicted to be narrowly selective for the individual odorants (in contrast to the rodent ORs which are able to respond to a large repertoire of ligands [181]), the sensitivity of human nose does not arise from value of Kd of odorant-receptor coupling itself but rather from the signal processing in brain [163], and therefore a choice of appropriate data processing model is an extremely important step in the bioelectronic nose elaboration. Artificial neural networks are one of the most widely used pattern recognition models [182, 183] dealing with complex non-linear dose- dependence relations and therefore can be very suitable for the bioelectronic noses. Nevertheless, dealing with time-consuming complex training of such networks requires very high stability of ORs coupled to the transducers and a satisfactory low level of stochastic responses as well. Table 4.2 summarizes the factors affecting either natural or artificial sensing of odors. As it can be seen, these factors are quite similar.

81 AAA

BBB

Fig. 4.2. Human olfaction (A) vs olfactory (bio)sensor (B). Images are taken from the site of Tübingen university: http://www.ipc.uni- tuebingen.de/weimar/pictures . 10 components depicted in (A) and (B) determines the overall performance of the both natural and artificial olfactory systems [172]

82 Table 4.2 . Factors which affect natural and artificial olfaction. FactFactorsors that affect odor sensing Main ssourcesources of arteartefactualfactual responses by human testers of odor detection techniques

1. Number of olfactory receptors (ORs) 1. Low stability of sensitive elements. available to bind odorants. 2. Inappropriate immobilization technique 2. Sequence polymorphism within (especially for bioelectronic nose). human genes of ORs [181]. 3. Odorant solubility (for measurements 3. Genetically determinated anosmia for carried out in aqueous solutions). some odorants [181]. 4. Changes in the humidity and 4. Different solubility of odorants in temperature of the air or water sample aqueous phase of nasal mucus. associated with the odorant [184].

5. Temperature, humidity, influence of 5. Electrochemical reaction between presence of alcohol and CO 2. odorant and electrode itself (for bioelectronic nose). 6. Chemical interaction between individual odorants [184]. 6. Chemical interaction between individual odorants in sample [184]. 7. Mental, emotional state of tester [163].

8. Sensitivity changes owing to age [163].

4. Concluding remarks GPCRs-based bioelectronics becomes an increasingly important discipline. Nevertheless, much development work is still required before GPCRs and especially ORs based sensors can reach their full biomedical and pharmacological potential. As professor W. Göpel wrote in one of his last articles in 1999: “ By taking advantage of characteristic similarities and differences of components in technical and biological systems, high-performance hybrid systems will be developed in the future ” [172]. New achievements in ORs biotechnology, reductions in transducer size, its coupling with faster computers and more powerful neural networks will ultimately open up a lot of application areas for the bioelectronic noses.

83

84

Part 5

ElectrocElectrochemicalhemical study of human olfactory recereceptorptor OR 1717----4040 stimulstimulationation by some odorants

The human olfactory receptor OR 17-40 heterologously co-expressed with G olf protein was employed as a bio recognition part of label-free impedimetric biosensor platform. The receptor in its natural membrane environment was anchored to a gold-coated glass substrate modified with thiol-based multilayer. Formation of the biosensitive film on thiolated surface was attempted in flow cell and stationary cell, and was monitored by means of SPR or EIS technique, respectively. Then, a stimulation of immobilized OR 17- 40 with its cognate odorant (helional) and unrelated odorants (heptanal, octanal, nonanal, vanillin) in PBS was probed by means of the EIS at various conditions. Activation of OR 17-40 in the presence of GTP-g-S at 4 ºC was found to improve the sensitivity of impedimetric detection of helional, probably via the enhancement of the specific biochemical signal.

An article based on the results presented in Part 5 is accepted for publication in Material Science Engineering C, 2007.

85 1.1.1. Introduction About half of the G-protein coupled receptors family in human is constituted from the olfactory receptors (ORs) [49, 58, 185]. These transmembrane proteins are capable of discrimination between a large number of small volatile compounds from environment [185-187]. The molecular mechanism of the ORs recognition specificity to odorants is one of the current major themes in the research on olfaction [186, 187].

There is a particularly growing interest in the elaboration of biosensor platforms based on the ORs expressed either in olfactory sensory neurons [173] or in heterologous cells [56, 179, 180, 188]. Although all these tools are label-free and seem to allow a direct dose-dependent detection of odorants, a simple interpretation of signals may only be reached in the case of mass- sensitive techniques. In other cases, especially dealing with the OR-bearing whole cells, several simultaneous processes (conformational changes of OR and G proteins, variations in membrane conductance etc. [185]) contribute to the total biosensor response. Understanding the relationships between these complex processes is important for the controlled enhancement of the biosensor sensitivity and selectivity.

Previously, our group has studied the sensitivity of heterologously expressed rat OR I7 [80] and human OR 17-40 [56] towards a set of different odorants, by means of the EIS and the SPR technique, respectively. In this work, for the first time, a stimulation of SPR chip-coupled OR 17- 40 by five ligands, preferential (helional [174-176]) and unrelated (heptanal, octanal, nonanal, vanillin [175, 176]), was probed by means of nonfaradaic EIS. In order to improve sensitivity of such an impedimetric label-free sensor platform via the enhancement of the biochemical signal, ligand-receptor interactions were also investigated in the presence of the guanosine triphosphate (GTP-g-S).

86 2. Experimental

2.1. Biomaterials and chemicals

Gαolf protein and human OR 17-40 tagged with cmyc sequence on its N- terminus were heterologously co-expressed in yeast Saccharomyces cerevisiae [177] (cells from strain MC18 used in this work are shown in Fig. 5.1). Fig. 5.2 provides an evidence of successful expression of receptor.

Schema of G olf and cmyc-17-40 plasmids design is given in Annex D. The membrane fraction of yeast was prepared as described in [56]. Stock suspension of membrane fragments with protein content 3 mg/ml was aliquoted and frozen at -80 0 C. Monoclonal anti-cmyc antibody (Ab) was obtained from Roche Molecular Biochemical and biotinylated by means of DSB-XTM Biotin Protein Labeling Kit (Molecular Probes, Leiden, Netherlands). Stock solution of biotinylated Ab (2.55 mg/ml) was divided into aliquots and stored at -20 0 C.

Fig.5.1. Optical visualization of yeast from strain MC18 expressing OR 17-40 immobilized onto a solid surface coated by 0.1 % polylysine.

Fig. 5.25.2.... Evidence of successful heterologous expression of OR 17-40: Confocal visualization of cmyc-OR 1740 expression in yeast, strain MC18. Diameter of a single cell is ~4 fm. Immunolabeling was performed with anti-cmyc IgG and Alexa488-coupled secondary antibody on nonpermeabilized spheroplasts. The image was kindly provided by Dr. Edith Pajot (INRA).

87 Dimethyl sulfoxide (DMSO) for odorant dilution and heptanal were obtained from Sigma. Helional was a kind gift from Givaudan-Roure (Switzerland). 16-Mercaptohexadecanoic acid (MHDA; 90% purity) and phospholipide 1,2-dipalmitoyl-sn -glycero-3-phosphoethanolamine-N-biotinyl sodium salt (biotinyl-PEA) were purchased, respectively, from Aldrich and Avanti Polar Lipids. GTP-g-S (guanosine-5’-O-(3-thiotriphosphate); MW 563, 93% purity), bovine serum albumin (BSA; 98% purity) and goat IgG were obtained from Sigma, neutravidin – from Pierce.

Absolute ethanol, HCl (37%), HNO3 (65%), H 2O2 (30%) and NH 4OH (25%) were purchased from Fluka and used as received. As a working buffer, a phosphate-buffered saline (PBS) was used with the following composition: 8 mM Na 2HPO 4, 1.5 mM KH 2PO 4, 3 mM KCl, 150 mM NaCl, pH 7.0 [176]. All reagents for PBS preparation were of analytical grade. All aqueous solutions were prepared with ultrapure water from Milli-Q system.

2.2. Single channel SSPRPR spectrometer and gold substrates For detailed information, see Annex C-1.

2.3. Pretreatment of sensor surface

Before work, substrates were cleaned by a mixture “aqua regia” (H2O +

HCl + HNO 3, 16 + 3 + 1, v/v) during 1-1.5 min and then – with a basic mixture

(H 2O + H 2O2 + NH 4OH, 5 + 1 + 1, v/v) during 15 seconds. Thoroughly rinsed with water, every chip was then immersed into ethanol for several seconds.

2.4. SelfSelf----assemblyassembly of the mixed layer on gold To obtain mixed self-assembled monolayer (SAM) on gold surface, 1 mM MHDA and 0.1 mM biotinyl-PEA dissolved in ethanol were incubated with freshly cleaned chip during 21 h [74]. MHDA is fixed onto Au via the chemisorption of SH-groups, as to biotinyl-PEA, it can be inserted between long-chain thiols due to the numerous hydrophobic interactions. Such mixed SAM provides a good basis for the

88 further anchoring of biomolecules to the chip surface. To elute unfixed molecules, chip was rinsed with ethanol, and dried under a nitrogen flow.

2.5. Blocking step and formation of the upper supporting layers For SPR monitoring, flow cell (case 1): In order to saturate all non- specific adsorption sites on SAM surface, heterogeneous layer was treated by 1 mM solution either of IgG or BSA in PBS in the flow cell: 0.3 ml of such solution was run on the chip at the flow rate (FR) 0.04 ml/min. Neutravidin (0.5 µM, 0.3 ml solution in PBS) and biotinylated anti-cmyc Ab (0.5 µM, 0.3 ml solution in PBS) were subsequently run on the blocked SAM at the FR 0.04 ml/min. Before formation of any upper molecular layer, the previous one was rinsed with PBS during 5-15 min.

For EIS study, stationary cell (case 2): The experimental conditions were as follows: i) thiolated gold was incubated with 0.15 ml of PBS solution of molecules to be adsorbed; BSA was chosen as a blocking agent; ii) concentration of solutions were the same as the above-indicated ones; iii) time of incubation with working electrode surface: BSA – 30 min; neutravidin – 60 min; Ab – 90 min. Incubation with PBS (2 times x 0.5 ml) after each step – 15 min.

2.2.2.6.2. 6. Preparation and immobilization of OR 1717----40404040 Stock suspension of OR 17-40 in its natural membrane fraction was thawed and resuspended in PBS on ice up to the OR concentration 70 µg/ml. 0.3 ml of this suspension was treated in the ultrasonic bath Elmasonic (35 kHz, 120 W) in ice-cold water during 10 or 20 min in order to obtain more or less heterogeneous suspension of membrane nanovesicles [56], where ORs may be orientated in both directions: N-terminus outside or inside the vesicles. Nanosomes were mainly spherical, but open or closed collapsed fragments were also found [56]. 0.3 ml of such a suspension was immediately run on the chip (FR 0.04 ml/min), or, in the case 2 , 0.15 ml was loaded into the stationary

89 electrochemical cell for incubation, then the electrode surface was gently washed with PBS.

2.7. Electrochemical probing After immobilization of the OR 17-40, SPR chip was rinsed with PBS and transferred into a three-electrode electrochemical glass cell (V=5ml, see Annex B) placed into a Faradaic cage and filled with PBS ( case 1 ). Case 2 : the electrochemical investigations were continued in the same stationary cell.

The EIS measurements were carried out using the impedance spectrometer Voltalab 40. Measurements in the presence of thermally unstable GTP-g-S were conducted at 4±0.5 ºC, the cell being connected to the circulator thermostat Julabo F25 (France), Fig. 5.3. All other measurements were performed at room temperature (20±1 ºC).

As a reference a saturated calomel electrode (SCE) from Radiometer Analytical was employed, and a platinum plate was used as an auxiliary electrode. Active surface area of AE was 0.64 cm 2, and 0.25 cm 2 for the WE i.e. biofunctionalized chip.

Fig. 5.3. Cryothermostat-circulator maintaining low temperature in the cell.

Impedance was swept in the frequency range 10 5-0.1 Hz at DC potential

90 -700 mV vs SCE with superimposed excitation voltage of 10 mV. Data were visualized as Cole-Cole (Nyquist) plots via the VoltaMaster 4.0 software.

2.8. Preparation of odorant solsolutionsutions Only freshly prepared solutions of odorants were used in all experiments. Stock 0.1 M solutions were prepared by the dilution of corresponding aldehyde in DMSO (see Annexes E-1 and E-3), but further dilutions were performed only in PBS. 10 -4 M dilution was directly prepared from 0.1 M, then further dilutions were obtained by successive 1:10 dilutions. Finally, 50 fl of 100-fold odorant solution was injected into the working buffer. To take into account a possible solvent effect in the impedance shift, blank probes at the various dilutions were prepared, replacing helional by PBS.

2.9. MonteMonte----CarloCarlo simusimulationlation In order to estimate the impact of nanosomes size on their efficient immobilization, a Monte-Carlo simulation was used. A correspondent model was created using MATLAB 7.0 software.

91 3. Results and discussion

3.1. Optical and electelectrochemicalrochemical monitoring of biofilm assembly Two routes of OR-containing biofilm formation were studied and compared: i) in the flow cell of the SPR spectrometer and ii) in the stationary electrochemical cell of the VoltaLab. In the first case, biofunctionalized SPR chips must be transferred from the flow cell to the stationary one for further investigations. The electrodes biofunctionalized directly in the electrochemical cell, were applied to odorant detection without any replacement, thus presenting methodologically more convenient step.

3.1.1. SPR monitoring A typical kinetics of step-by-step formation of the OR-containing biofilm is shown in Fig. 5.4, A. At first, PBS was run on the chip surface functionalized with SAM; afterwards it was treated with IgG or BSA. Adsorption of BSA onto the surface produced a signal shift of about 0.12 arc degrees (Fig. 5.4, B), while the response to the IgG anchoring was about 2.5 times higher (data not shown), probably due to the difference in the molecular mass of BSA (66 kDa) and IgG (150 kDa).

The next step of surface modification consisted in the immobilization of neutravidin. Neutravidin (60 kDa) presents an interesting alternative to the streptavidin since it is a neutral protein without any carbohydrate moieties, thus possessing rather low capabilities of nonspecific binding at physiological pH [189]. As it is known, avidin-biotin and streptavidin-biotin binding are one of the highest affinity non-covalent bioorganic interactions occurring between large protein and small molecule (the molecular mass of biotin, also known as

-16 -14 vitamin H, is 244 Da), demonstrating Kd of 6 ×10 M and 4 ×10 M, respectively [37]. Once formed, the affinity bond between avidin and biotin is unaffected by extremes of pH, temperature and denaturing agents. All these features of avidin and streptavidin are shared by neutravidin. Neutravidin molecule possesses 4 binding sites (two on each opposite sides), presenting a very suitable linker for the consecutive immobilization of any biotinylated molecules. As it can be seen from Fig. 5.4, B, anchoring of 92 neutravidin to the BSA-blocked or IgG-blocked SAM produces a resonance angle shift of about 0.2-0.25 arc degrees. However, coupling of neutravidin to the non-blocked SAM produced a higher signal presumably due to some spatial restriction of accessibility of biotin groups after BSA/IgG blockage step.

Biotinylated anti-cmyc Ab was immobilized over the neutravidin layer. Anchoring of Ab onto the surface blocked either with IgG or BSA produced a signal of about 0.3 arc degrees being 1.7 times weaker than it could be expected from the molecular mass ratio Ab : neutravidin=2.5. Maximal SPR response to Ab anchoring (0.55 arc degrees) was achieved on the non-blocked neutravidin layer, however, the binding “capacity” of the latter was shown to be statistically the same as that of the BSA and IgG blocked structure (see Fig. 5.4, B).

Finally, a suspension of OR 17-40-containing nanosomes was run on the surface and incubated for about 20-30 min, then the surface was rinsed with PBS. The best average SPR signal for the OR 17-40 immobilization was obtained on the basis of the BSA-blocked support (Fig. 5.4, B). The latter was even more efficient than the non-blocked structure demonstrating a good accessibility and a proper orientation of the anti-cmyc binding sites of Ab. As to the IgG-blocked multilayer, it was 3 times less capable of nanosomes binding than the biofilm blocked with BSA.

.The impact of ultrasonication duration on the nanosomes adsorption on the BSA-blocked surfaces was also investigated. It was found that nanovesicles pretreated during 10 min produced the SPR signal of 0.26±0.07 arc degrees (n=8), while those sonicated during 20 min yielded 0.22±0.03 arc degrees ( n=5). So, the SPR chips modified by the BSA-blocked multilayer coated with OR-bearing nanosomes sonicated during 10 min were chosen for further electrochemical studies.

A step-by-step formation of OR-containing biofilm took about 280-330 min and was a fully real time controllable process.

93

C

Nanosome with G:- Biotinylated Ab coupled OR

BSA Neutravidin

Biotinyl-PEA Au-MHDA Fig. 5.4 (A) Typical building-up kinetics of the OR-containing SAM-based biofilm. (B) SPR responses to the step-by-step formation of OR-containing multilayer. All measurements were performed in the flow cell in PBS at pH 7.0.

(C) Scheme of the biofilm architecture.

94 3.1.2. Impedance monitoring It is also possible to successfully build up the OR-containing biofilm directly in the stationary cell of VoltaLab by means of step-by-step incubation of thiolated surface of the WE with proteins and, finally, with nanosome suspension (Fig. 5.5).

FigFigFig.Fig ... 5.55.5.... Impedance spectra of OR-containing multilayer formation in the electrochemical cell. Measurements were carried out in PBS pH 7.0 at 20 0C, without stirring in order to avoid fresh layers damaging.

However, it was quite difficult to find an optimal immobilization time for each molecular layer formation since the impedimetry in VoltaLab configuration actually does not allow monitoring adsorption in the kinetic mode.

Obtained impedance spectra were fitted with an electrical circuit composed of the ohmic resistance of bulk electrolyte ( Rs) in series with a parallel combination of the CPE (constant phase element with more or less capacitive characteristics depending on the biofilm behavior) and the Rp (interfacial polarization resistance in the absence of redox species) (as described in Part 3 and Annex B, Fig. B-1, A).

95 Interestingly, an increase in the total impedance modulus (Fig. 5.5) and, particularly, in the polarization resistance Rp (Table 5.1) was systematically observed after immobilization of each layer. This differs from the data previously reported in [80, 190], probably due to the different providers of gold electrodes, and/or different cleaning procedures applied, and/or due to the different DC potentials applied to WE. Nevertheless, an increase in electrochemical impedance during layer-by- layer functionalization of thiolated gold with large molecules of biopolymers (protein or DNA) is a well documented phenomenon [157, 191, 192] generally attributed to the step-by-step increase of the multilayer thickness leading to increase of interfacial resistance to the applied voltage. It was found that the multilayer formation did not alter significantly neither CPE nor n values, but the polarization resistance only. It was observed

1.2 fold increases in the Rp after the first 30 min of biofilm incubation in PBS without stirring. In the next section, we will compare the Rp of functionalized electrodes transferred from the flow cell with Rp described in this section.

Table 5.15.1.... The fitting values calculated in ZView2 for the SPR chips step-by-step modified with biomolecules in the stationary electrochemical cell.

Frequency range, CPECPE----T,T,T,T, HHH222 Electrode RRRs,s,s, FFF nnn RRRppp,,, F HzHzHz µF(rad/s) 111-1---nnnn (((× 10 ---4-444))) Au-SAM 63291–2 79 3.5 0.9 11431 7.1 +Neutravidin 63291–2 78 3.7 0.89 15589 7.2 +Abs 63291–1.6 76 3.3 0.9 17378 6.8 +Nanosomes 63291–1.6 77 3.6 0.89 18299 7.2 Multilayer after 30 min 63291–6.3291 76 3.7 0.9 22603 10.5 in PBS at 20 0C

3.23.23.2.3.2 . Impedance study of already biofunctionalized SSPRPR chips Once functionalized, the SPR chip was employed as a working electrode in the electrochemical cell. Multilayer revealed a high stability upon contact with PBS solution and rather high complex impedance as well (Fig. 5.6). At potential -700 mV vs SCE the OR-containing biofilm remained stable in time,

96 and crude complex impedance data (e.g., like in Figs. 5.6, 5.7, 5.10) obtained at various temperature (20 ºC or 4ºC) gave a good fit to an above-described electrical circuit shown in Fig. 5.8 (inset) over the broad frequency range.

Table 5.25.2.... The fitting values calculated in ZView2 for the SPR chips coated with gold, bare and modified with OR 17-40 via SAM-based multilayer in the flow cell of SPR spectrometer.

Frequency Temperature, CPECPE----T,T, HHH222 Electrode RRRs,s,s, F nnn RRRppp, F, F range, Hz ºººCCC µF(rad/s) 111-1---nnnn (((×101010 ---4-444))) Bare Au 10 5-1.6 20 53 13.7 0.95 9350 9 Au-biofilm 10 5-1.6 20 51 5.2 0.86 29328 11 Bare Au 4×10 3-2.5 4 57 19.9 0.91 1319 8 Au- 4×10 3-2.5 4 86 3.4 0.92 19054 11 biofilm

AAA BBB

FigFigFig.Fig . 5.6. Impedance diagrams of SAM-based OR-containing biofilm under various potentials of polarization vs SCE. Measurements were performed in PBS pH 7.0 at 20 ºC (A) and 4 ºC (B).

Table 5.2 presents the fitting values calculated in ZView2 software for impedance spectra obtained from the SPR sensor chips coated by gold, either bare or already modified with OR-based biofilm. As it can be seen, the ratio of CPE values of bare Au to CPE of modified Au is independent on temperature and remains about 4±1. The n values of the modified electrodes are close to 0.9 reflecting mainly capacitive behavior of the corresponding CPE and quite good insulating properties of the immobilized biofilms.

97 There is another interesting feature to discuss, namely, a systematic ~1.5 fold decrease in the total impedance (Fig. 5.6, A, B) and especially in the

Rp and CPE values of biofilms (Tables 5.1 and 5.2) at low temperature. It is not clear whether this “temperature effect” could be attributed to the specific changes at the biointerface, or it reflects only an intrinsic “calibration” drift in the measurement system, e.g. in the SCE.

From Tables 5.1 and 5.2 it can be seen that biofilms obtained either in flow cell or in stationary cell, demonstrate quite similar electrochemical characteristics. Estimated capacitance (CPE) of electrode modified with nanosome containing biofilms (3.6 – 5.2 fF/cm 2) correlated with the capacitance of biological membranes (2 – 7 fF/cm 2) cited by Steinem et al. in [76], but as it is mentioned in Annex B, capacitance of the working electrode also contributes to the fitted CPE .

3.3. Impedance measurements in the presence of odorants at 20 000CCC In this experiment, aliquots of helional and heptanal were injected randomly into electrochemical cell to the final concentrations 10-11 , 10 -10 , 10 -9, 10 -8 and 10 -7 M in PBS. The impedance spectrum was recorded immediately after ligand injection. Then the surface was washed with PBS, stabilized during 10-15 min and used for the further measurements of odorants. Sensor response to 10 -10 M of helional at 20 ºC is shown in Fig. 5.7, A. Injection of the same aliquot of heptanal produced only a slight shift of the impedance spectrum (Fig. 5.7, B).

In order to control the signal specificity, a SAM-based biofilm without OR 17-40 was formed on the sensor chip and then exposed to helional and heptanal (from 10 -11 to 10 -7 M in PBS) at room temperature. All applied concentrations of both odorants produced insignificant variation of total impedance (data not shown). Obtained impedance data were fitted with the above-described electrical circuit in the frequency range 10 5-31 Hz. It was found that injection of any ligand affected significantly neither CPE nor n values, but the polarization resistance only, therefore the Rp shift was taken as a “reporter” of the OR 17-

98 40 activity, i.e. as the sensor signal. Thus, the best signal was found for 10 -10

M helional, among the five concentrations tested in Fig. 5.8, where R0 is the fitting value of Rp before odorant injection, and R is the fitting value of Rp upon odorant stimulation of receptors.

Stimulation of OR 17-40 with the blank solutions corresponding to 10 -11 -

-7 -9 10 M helional resulted in Rp shift ≤0.015. Therefore, the signals to 10 and 10 -7 M helional and almost all responses to the heptanal should be considered as non-specific and/or insignificant.

FigFigFig.Fig ... 5.7. Sensor responses to the injection of 10 -10 M helional (A) and 10 -10 M heptanal (B). Measurements were performed in PBS pH 7.0 at 20 ºC.

99

FigFigFig.Fig . 5.8. Polarization resistance Rp changes under stimulation of the OR 17-40 with helional or heptanal at 20 ºC in PBS. Error bars are based on n=2.

Inset: Equivalent electrical circuit applied for the fitting of experimental spectra.

3.4. Impedance measurements at 4 ºC in the presence of odorants and GTPGTP----LLLL----SSSS It is known that the early events of olfactory signal transduction in vertebrates involve interaction of activated OR with G protein [193], which triggers the adenylate cyclase III catalyzed generation of cAMP thus leading to changes in membrane conductance [50, 194, 195]. Heterotrimeric G protein located on the cytoplasmic face of cell membrane possesses :, Y and g subunits [196], Fig. 5.9. In the presence of GTP (or GTP-g-S), odorant-activated receptor mobilizes G:, which then dissociates from GYg-dimer [196] being bound with GTP (or GTP-g-S), as it is shown in Fig. 5.9, A, B. In such a way, mobilization of G: protein represents a biochemical signal by itself [56, 177].

100 GTP-g-S is a non-hydrolysable analogue of GTP recently reported to enhance an OR 17-40-based SPR sensor response to some odorants including helional [56].

AAA BBB Fig. 55....9999. Mobilization of G: triggered by agonist-receptor binding. In cell, desorption of G: results from the GDP/GTP exchange (as it is shown in picture); in nanosome – probably from GTP binding to G: upon activation of olfactory receptor [56]. There is no GYg in nanosomes used in the present work.

So, the next step of our work was a stimulation of OR 17-40 with various concentrations of odorants in the presence of GTP-g-S in PBS at 4 ºC. Two concentrations of helional, 10 -11 and 10 -7 M were applied at 4 ºC in the absence of GTP-g-S in order to know whether application of GTP-g-S will enhance a response of the impedimetric sensor or not.

Procedure of measurements in the presence of GTP-g-S was as follows: at first, an electrochemical cell filled with PBS was cooled to 4 ºC, then an aliquot of 1 mM GTP-g-S PBS solution was injected to the final concentration 10 fM [56], and odorant was injected immediately after.

All impedance spectra obtained at 4 ºC were satisfactorily fitted with above-described model circuit in the frequency range 4 ×10 3-2.5 Hz . While

applied without GTP-g-S, helional slightly altered the Rp parameter (Fig. 5.10, A), but in the presence of GTP-g-S the response to helional increased (Fig. 5.10, B). Fig. 5.11 (inset) demonstrates OR 17-40-based sensor responses to be 4 fold increased in the presence of GTP-g-S in the working buffer thus

101 supporting a hypothesis about the enhancing role of GTP-g-S in such a mode of odorant analysis.

FigFigFig.Fig ... 5.105.10. Sensor responses to 10 -11 M helional. Measurements were conducted at 4 ºC in the absence (A) or presence (B) of 10 fM GTP-g-S in PBS pH 7.0.

In addition, a significant shift in the maximum of sensor sensitivity to helional was observed at 4 ºC in the presence of GTP-g-S. As it can be seen from Fig. 5.11, the maximal sensor response was already achieved after the injection of helional concentration 10 -11 M.

FigFigFig.Fig ... 5.115.11.... Polarization resistance Rp changes under stimulation of the OR 17-40 with helional at 4 ºC in PBS with 10 fM GTP-g-S. Error bars are based on n=2.

Inset: Rp changes under stimulation of the OR 1740 with helional in the presence or absence of GTP-g-S.

102 In order to control whether an introduction of the GTP-g-S enhances non-specific responses, immobilized OR 17-40 was stimulated with blank probes and heptanal. Responses to the latter were found to be comparable with the alteration of Rp (~0.025) caused by the blank probes in the presence of

º GTP-g-S (Fig. 5.12). This non-specific shift Rp was 1.7 times larger at 4 C instead of 20 ºC, in the presence of GTP-g-S. Therefore, the net advantage should be estimated as a 2.3 fold increase in the specific response. Since orientation of ORs in nanosomes varies, it may be necessary to introduce GTP-g-S in membrane suspension before its ultrasonication. In this way, GTP-g-S included in nanosome will allow additional mobilization of G:.

As GTP-g-S is not very stable at 20 ºC, odorant detection in the presence of GTP-g-S at 20 ºC was not attempted.

Working at low temperature was found to increase the sensor lifetime from several hours (10-15 measurements at 20 ºC) to 2 days of continuous work at 4 ºC. The most evident reason of this phenomenon is better preservation of receptor and/or G protein proper conformation which is of high importance for the intrinsic activity of these proteins.

FigFigFig.Fig ... 5.125.12.... Rp changes under stimulation of OR 17-40 with heptanal and blank probes at 4 ºC in PBS with 10 fM GTP-g-S. Error bars are based on n=2.

103 3.3.3.5.3. 5. Impedimetric screening unrelated odorants To assess the specificity of the impedimetric response observed at 4 ºC in the presence of GTP-g-S, immobilized OR 17-40 was probed with 3 unrelated odorants: octanal, nonanal and vanillin within the broad range of concentrations (10 -12 – 10 -7 M) and compared to already obtained data on helional and heptanal (Fig. 5.13). The horizontal line (Fig. 5.13) cuts off the average non-specificity of impedance alteration estimated from Fig. 5.12. However, remembering that vanillin must be absolutely ineffective ligand (according to the data reported by Wetzel et al. in his pioneer work on specificity of OR 17-40 [175]), one may suppose the “non-specific” Rp shift to be equal to 0.03-0.04.

Therefore, an array of significant data shown in Fig. 5.13 is located within the range 10 -12 – 10 -9 M, revealing a surprisingly high response to 10 -12 M of heptanal and significant response to 10 -11 M of octanal and 10 -10 M of heptanal. No essential signal on higher concentrations (up to 10 -5 M, data not shown) of the odorants including helional was observed.

Bell-shaped profile of the OR sensitivity helional differs from the response pattern observed on the thiolated gold electrodes [66] (see Bibliographic review) but corroborates some previously reported pharmacological profiles of OR 17-40 obtained in intracellular calcium and bioluminescence cell-based assays [176, 177].

104

FigFigFig.Fig ... 5.135.13.... Rp changes at stimulation of OR 17-40 with 5 various odorants at 4 ºC in the presence of GTP-g-S. Horizontal line cuts off an average non-specificity of resistance changes estimated from Fig. 5.7.

3.63.63.6.3.6 . S. SizeS ize impact on the nanosomes anchoring In section 3.1.1 we remarked a slight but evident difference of 15% in the average SPR signals to adsorption of the membrane vesicles sonicated during 10 and 20 min. Since the duration of suspension sonication apparently correlates to its homogeneity, one can suggest that the dispersity of vesicles size could affect their immobilization. In order to calculate the possible impact of nanosomes size on their surface coverage factor (SCF) we have used the Monte-Carlo simulation. Here the SCF is the ratio of an electrode surface area covered with nanosomes to a total surface accessible for specific anchoring of membrane vesicles and it evidently has to correlate with the SPR signal variation.

The following assumptions were used for the simulation: i) all nanosomes were of the same shape (spheres) which could vary in size (diameter). Two cases were considered: all

105 nanosomes had the same diameter (dispersity of sizes is equal to 0%), or various diameters (dispersity of sizes is equal to 80% like in the suspension of vesicles from 50 to 500 nm); ii) electrode surface was considered as a flat disk, whose diameter was much larger than that of a single nanosome; iii) fixed number of attempts was used to place a nanosome on the electrode surface. If no attempt was successful, the electrode was considered as optimally covered by nanosomes. The result of this simulation (Fig. 5.14) demonstrates that the wider the dispersity of the nanosomes sizes, the larger the area covered. It is obvious that the absolute value of vesicles diameter does not impact the SCF (Fig. 5.14), therefore, only varying the dispersity of the nanosomes diameters can one increase the SCF and the amount of olfactory receptor on the surface as well.

FigFigFig.Fig . 5.14. Monte-Carlo simulation of nanosomes distribution on the flat electrode surface. The present result was obtained using the following parameters values: electrode diameter: 100 a.u., nanosome diameter: 1 a.u., dispersity of nanosomes diameters: 0% or 80%, number of placement attempts: 1000, number of modeling cycles: 10000.

So, the above-described model can explain a more efficient adsorption of the nanosomes obtained after only 10 min of sonication. Nevertheless, it is not

106 evident whether the more dense coverage will enhance or restrict the biofilm sensitivity to odorant, and this has to be studied. Discussion on structural and functional properties of the adsorbed nanosomes will be continued in Part 6.

4. Conclusion and perspectives Layer-by-layer formation of the OR 17-40-based biofilm on the SPR sensor chip was an efficient and well-reproducible process. Under injections of helional resulting in submicromolar to subnanomolar concentrations, activation of OR 17-40 was electrochemically revealed. Applying GTP-g-S at 4 ºC as a specific enhancer of biochemical signal, it became possible to detect 10 -11 M helional. The low temperature improved lifetime of such an “olfactory biosensor” up to 2 days of continuous work. Already obtained results provide a basis for the usability of membrane- associated human olfactory receptors in the construction of label-free biosensors as elements of a bioelectronic nose.

107

108

Part 6

RRResponseResponse pattern of human olfactory receptor OR 1717----4040 probed by ssurfaceurface plasmon resonance

Agonist-response pattern of olfactory receptor OR 17-40 heterologously co-expressed with Gαolf in yeast was probed by means of double channel SPR sensor platform “NanoSPR-6”. Receptors in their native membrane environment were specifically captured via anti-cmyc antibody attached to the gold-coated substrate in orientated (via thiol-based architecture) or random (via physical adsorption) way. Effective thickness of each molecular layer in biofilm was estimated from experimental SPR spectra. In order to compensate the effect of solvent on the refractive index changes, detection of odorants was performed in the differential mode. Dose-dependent signals induced by helional were discussed in terms of conformational changes of OR 17-40

-10 and/or Gαolf . Bell-shaped response profile with double maximum (within 10 - 10 -8 M and near 10 -6 M) was established for helional. Unrelated odorant heptanal used as control did not evoke significant response of immobilized OR 17-40.

Results presented in Part 6 have been submitted to the Journal of Biotechnology .

109 1. Introduction Recent advances in the pharmacology of olfactory receptors (ORs) and other G protein-coupled receptors (GPCRs) result mainly from the development of in vitro screening of receptors agonists [51, 197]. The pharmacological data available on mammalian olfactory receptors include various dose-response profiles. Thus, a typical adsorption curve with a remarkably broad linear part (10 -11 -10 -3 M) was obtained by means of the quartz crystal microbalance technique for the heterologously expressed rat OR I7 exposed to its preferential ligand – octanal [180]. The increase in calcium signal of OR I7 from isolated olfactory neurons was sigmoid within the concentration range of octanal 10 -7-10 -5 M [198]. Other data obtained from intracellular calcium and bioluminescence assays revealed the response pattern of heterologously expressed OR I7 and OR 17-40 to be bell-shaped within the concentration range of agonists (helional and octanal, respectively) 10 -14 -10 -3 M [176, 177]. However, a bell-shaped curve always comprises at least two more or less narrow parts of linear sensitivity, which are of great interest for biosensor development. The goal of the present study was to probe the pattern of immobilized OR 17-40 response to its preferential odorant helional and the unrelated one – heptanal in heterogeneous phase. Two different receptor-based biofilms were designed; their thickness, relief and biosensing efficiency were compared.

2. Experimental

2.1. Biomaterials and chemicals Human OR 17-40 tagged with cmyc sequence and biotinylated anti-cmyc antibody were obtained as described in Part 5. 16-MHDA, biotinyl-PEA, GTP-g-S, BSA, neutravidin, DMSO, heptanal, organic solvents and mineral acids were similar as described in Part 5.

3-/4- Fe(CN) 6 were obtained from Sigma. Helional was a kind gift from Givaudan-Roure (Switzerland). Composition of PBS used as a working buffer was similar to that described in Part 5.

110 2.2. Sensor substrates and SPR spectrometer For detailed information, see Annex C.

2.3. Pretreatment of sensor surface The same procedure as described in Part 5 was applied.

2.4. Two architectures of biofilms Two different biofilms were designed and probed: i) Au + (MHDA+biotinyl-PEA) + neutravidin + biotinylated Ab + OR 17-40 (“A1” biofilm); ii) Au + biotinylated Ab + OR 17-40 (“A2” biofilm).

To obtain SAM layer onto gold, 1 mM MHDA and 0.1 mM biotinyl-PEA were dissolved in ethanol and incubated with freshly cleaned chip during 21 h. To wash the surface from unfixed molecules, the chip was rinsed with ethanol, and dried under a nitrogen flow. Neutravidin (0.5 µM) and/or biotinylated Ab (0.5 µM) were subsequently run through both channels from the one stock solution (0.3 ml in PBS) at the flow rate (FR) 0.02 ml/min. Before formation of any upper molecular layer the previous one was rinsed with PBS during 5-15 min. In order to saturate all non-specific adsorption sites on modified surface, Ab layer was blocked by BSA (0.5 mg/ml in PBS) which was run on a chip at the FR 0.02 ml/min.

2.52.52.5.2.5 . Preparation and immobilization of OR 1717----40404040 Stock suspension of OR 17-40 in its membrane fraction was diluted in PBS on ice down to the protein concentration 70 µg/ml. 0.3 ml of this suspension was treated in the ultrasonic bath Bandelin Sonorex Super RK 102H (35 kHz, 140 W) in ice-cold water during 20 min in order to obtain membrane vesicles called nanosomes due to their size (~50 nm) [56, 190].

111

FigFigFig.Fig . 6.1. AFM image of a single nanosome adsorbed onto a solid surface. The image was kindly provided by Dr. Edith Pajot (INRA).

Afterwards, the suspension was immediately run on the chip at FR 0.02 ml/min. As it is reported in [199], one nanosome (Fig. 6.1) of 50 nm diameter can contain up to 10 ORs. Orientation of ORs in lipid bilayer may vary. Most probably, ORs may be orientated in both directions: N-terminus located outside or inside the vesicles.

2.2.2.62. 666.. PrPreparationeparation of odorants Stock 0.1 M solutions of odorants were prepared freshly on the day of experiment in DMSO, further dilutions (from 10 -4 to 10 -12 M) were obtained by successive 1:10 dilutions in PBS. The blank probes at the various dilutions were prepared replacing the odorant by PBS. Blank solutions were run via the reference channel simultaneously with odorant probe to compensate possible effect of DMSO in odorant detection. Additionally, each odorant solution and each blank probe contained 10 fM of GTP-g-S prepared on ice from the 1 mM solution. Detailed protocols of probes preparation and screening in differential mode are given in Annexes E-1 and E-2.

2.72.72.7.2.7 ... Cyclic voltammetry SPR substrate modified with biofilm A1 or A2 was transferred into an electrochemical glass cell (V=5ml) and used as a working electrode. The measurements were conducted at a room temperature using impedance analyzer Voltalab 40 in the 4 mM potassium ferricyanide (3-/4-), dissolved in PBS. For more details on electrochemical measurements see Annex B.

112 Saturated calomel electrode (SCE) from Radiometer Analytical was employed as a reference, a platinum plate – as an auxiliary electrode. Active surface area of the latter was 0.64 cm 2, and 0.25 cm 2 – of the working electrode. Scan rate was 50 mV/s.

2.82.82.8.2.8 . AFM The substrates were imaged with Nanoscope III microscope (Digital Instrument, USA). A pyramidal silicon tip RTESP (Veeco) with a spring constant of 20-80 N/m was used to perform the experiments. The cantilever was oscillated at 300 kHz resonance frequency with free amplitude of 2 V. All experiments were performed in air at room temperature and relative humidity of 60%. Images were taken in tapping mode to avoid damaging the surface of the sample.

3. Results

3.1. Orientated and random immoimmobilizationbilization of Ab ORs carried by nanosomes were immobilized via highly specific interactions of cmyc sequence with corresponding antibodies attached to the gold in orientated (1) or random (2) way. In the first case, antibodies were attached to neutravidin specifically coupled to biotinyl-PEA (Fig. 6.2, A, B). Random immobilization involved a direct Ab attachment onto the freshly cleaned gold (Fig. 6.3, A, B).

To estimate the thickness of each molecular layer the experimental SPR spectra were fitted to the theoretical curves on the basis of five-phase Fresnel calculations using the Nelder–Mead algorithm of minimization [24]. As a basis, an effective refractive index of n=1.36 was used for protein layers [24], and of n=1.46 for a membrane vesicle [200]. The shape of biomolecules and nanosomes was considered as globular. The calculated values of thickness are presented in Table 6.1.

113 As it is shown in Table 6.1, a signal for the specific anchoring of biotinylated Ab onto SAM was about 2 times lower than a response to its direct adsorption probably due to the limited quantity of affinity sites (biotinyl-PEA) at the surface. The spatial orientation of immobilized Ab is crucial for OR anchoring since the latter is based on the highly specific interaction via cmyc tag. While the ratio Abs: nanosomes in case of biofilm A1 is close to 1, it is equal to 7 for the A2 suggesting that probably only ~11% of immobilized antibodies are properly orientated. Therefore, a random orientation of Ab layer resulted in a nanosome layer of comparatively low density (Table 6.1).

Table 6.1. SPR response to the layer-by-layer formation of 2 types of biofilms and calculated thickness of each molecular layer. Constant of filling (V) reflects a level of surface coverage with lipidic biomaterial estimated from AFM data (see Section 3.3).

A1, A2, A1, effective A2, effective Layer arc arc thickness, nm thickness, nm degrees degrees

MHDA-based SAM n/a 1.9 [60] – –

Neutravidin 0.34±0.03 15±1 – – Biotinylated Ab 0.26±0.02 12.5±0.75 0.42±0.07 18.5±1 BSA 0.01±0.00 0.5±0.1 0.02±0.01 0.9±0.2 OR17-40-bearing 11±0.75 11±0.75 0.20±0.09 0.06±0.01 nanosomes (V=1) (V=0.25) Total thickness of multilayer adsorbed ~41 ~30 on gold, nm

Table 6.2. Biorecognition efficiency of various configurations of G protein coupled olfactory receptor in a nanosome (see Figs. 6.2 B and 6.3 B).

Configuration 1 2 3 Flexibility of N-end, crucial for odorant – + + binding

Accessibility of G:olf to GTP-g-S – – + Biorecognition efficiency Low Middle High

114 A

FigFigFig.Fig . 6.2 (A) Sensogram of layer-by-layer assembly of A1 biofilm. Inset: SPR angle shift corresponds to each step of multilayer A1 formation, reflecting the state of surface washed with PBS before/after the immobilization of biomolecules.

(B) Schema of A1 multilayer. Possible orientations (1, 2, 3) of G:olf coupled receptors in a nanosome are described in Table 6.2.

115 A

FigFigFig.Fig ... 6.36.3.... Sensogram of layer-by-layer assembly of A2 biofilm. Inset: SPR angle shift corresponds to each step of multilayer A2 formation, reflecting the state of surface washed with PBS before/after the immobilization of biomolecules.

(B) Schema of A2 multilayer. Possible orientations (1, 2, 3) of G:olf coupled receptors in a nanosome are described in Table 6.2.

116 3.2. Electrochemical properties of biofilms As it was revealed by cyclic voltammetry measurements, the A1 biofilm was endowed with strong insulating properties (Fig. 6.4). The electron transfer through the A2 was 50% weaker than the redox kinetics on bare Au (Fig. 6.5). The insulating properties of A2 were found to increase after its overnight incubation in PBS at room temperature (Fig. 6.5, A). In order to clarify the nature of this phenomenon, a biofilm consisting only of randomly adsorbed Ab was probed under the same conditions. This layer of antibodies demonstrated an increased penetrability to redox couple after 12 h of contact with PBS (data not shown). Therefore, some increase of insulating properties of A2 biofilm can be attributed to the nanosomes fusion on the top of sensor surface [190]. Since redox peaks did not completely disappear, one could conclude that the membrane vesicles did not merge into a continuous layer (Fig. 6.5, B,C), therefore, the fusion of nanosomes on the electrode surface was only partial, corroborating recently reported AFM-based data [199].

FigFigFig.Fig . 6.4. Cyclic voltammogram of bare Au electrode and SAM-based biofilm A1 (inset).

117 AAA

BBB CCC

FigFigFig.Fig ... 6.56.5.... (A) Cyclic voltammogram of A2 biofilm, freshly immobilized and stored during 12 h in PBS at 20 0C. (B,C) Schema of redox probe penetration into the freshly-formed (B) A2 biofilm. After 12 h the redox probe transfer became restricted as it is shown in (C).

3.3. AFM study of biofilms AFM images of biofilms were taken after the second day of work; several interesting features were revealed. Clear difference in relief was observed between A1 (Fig. 6.6, A) and A2 (Fig. 6.6, B) biofilms. Significant porosity of A2 can be attributed to the spatial non- homogeneity of biofilm attached to the randomly oriented Ab. As it can be seen, the surface profile of A1 biofilm was rather smooth due to the proper orientation of immobilized multilayer. In the calculations of thickness (Table 6.1), the level of coverage of A1 with lipidic biomaterial was

118 taken as 1. The A2 surface coverage was estimated as 0.25 from the level of porosity observed.

AAA

119 BBB

FigFigFig.Fig . 6.6. AFM images with cross sectional profiles corresponding to A1 and A2 biofilms. Images were taken in tapping mode at the working “spots” of corresponding biofilms after using them for odorant detection.

3.4. Detection of odorant molecules It is widely accepted that an olfactory signal is transmitted into sensory neuron via an interaction of OR with heterotrimeric G protein located at the cytoplasmic face of neuron membrane [196]. Activated OR promotes GTP binding with G:olf subunit followed by its liberation from Yg dimer [56].

Surface-grafted membrane fragments bearing G: olf and ORs employed in this work present a biorecognition system where the above-described conformational changes of receptor and G protein are supposed to occur upon OR stimulation with odorant producing a detectable functional response. 120

FigFigFig.Fig ... 6.7. SPR sensor responses obtained on biofilm A2. 10 -6M helional was run via the working channel and corresponding blank probe – via the reference channel. Inset: differential signal on 10 -6M helional.

The odorants helional and heptanal were tested on A1 and A2 in the concentration range from 10 -12 to 10 -5 M. Helional is documented as a cognate odorant for the OR 17-40 [174- 176]. Measurements were conducted in the differential mode (Figs. 6.7 and 6.8) at the day of biofilm formation (“1st day” of work) and were continued the next day (“2nd day” of work). As it can be seen from Figs. 6.7 and 6.8, the observed signals showed a dose-dependent “dissociation” character. Signals obtained on thicker A1 biofilm with comparatively dense nanosomes layer were in general weaker than those from A2 (Figs. 6.8, 6.9).

121 AAA B

FigFigFig.Fig ... 6.86.8.... Typical kinetics of SPR signals to helional obtained from the A1 (A) and A2 (B) biofilm in differential mode. 50 point smoothing was applied to all curves.

AAA BBB

FigFigFig.Fig ... 6.96.9.... Responses to various concentrations of helional obtained on A1 (A) and A2 (B) biofilms.

Both types of biofilm revealed a sensitivity of immobilized OR 17-40 to helional (signals values varied dose-dependently up to 18 arc s) and were not sensitive to heptanal. Sensitivity of receptors to helional after the overnight storage of SPR substrate coated with biofilm A1 decreased essentially (Fig. 6.9, A), while A2 demonstrated only a slight decrease in response (Fig. 6.9, B) to helional.

122 The profile of OR 17-40 response to helional established in this study is presented in Fig. 6.10.

20 20 1st day 2nd day

HELIONAL 16 16 HELIONAL HEPTANAL HEPTANAL

12 12

8 8

4 4 Differentialarcsignal, s Differential signal, arc Differential s signal, 0 0 -12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5 log C, M log C, M AAA BBB

FigFigFig.Fig ... 6.106.10.... Profile of immobilized OR 17-40 responses to helional. Heptanal was used as a control. Data were obtained from the same A2 biofilm during 2 days of continuous work.

4. DDiscussioniscussion As it can be seen from Table 6.1 and Fig. 6.2, a total shift of the SPR angle corresponding to the formation of SAM-based architecture A1 is rather significant (about 0.8 arc degrees) mainly due to the adsorption of neutravidin and the OR 17-40-containing membrane fragments. The estimated thickness of biofilm A2 based on the randomly oriented Ab is ~30 nm being at least 27% thinner than the A1 biofilm (~41 nm; thickness of SAM was estimated from that of the Au-attached MHDA layer calculated in [60]).

The different response to the adsorption of nanosomes on A1 and A2 supporting biofilms could resulted in the high porosity of A2 biofilm, clearly observed on the AFM images (Fig. 6.6). Presence of pores in A2 initially originates from the random orientation of Abs and relates to their inability to bind a large amount of nanosomes. High porosity of nanosome layer results in significant (about two fold) increase in the biofilm surface area being exposed to the probe solution (see schema in Figs. 6.2 and 6.4), which can be considered as an advantage in comparison with relatively non-developed smooth layer A1. From this point of view, the thickness of both A1 and A2 nanosome layer can be almost similar, whereas SPR smaller response to

123 nanosome adsorption for A2 structure reflects only strong porosity of nanosome layer, resulting in the decrease of its effective index of refraction .

Low thickness of the biorecognition layer A2 and low density of nanosomes immobilized on the top of this layer resulted in better accessibility of captured OR could explain a better sensitivity of A2 to helional as it was detected by the SPR platform. Additionally, manipulating the amount of Ab attached to the surface, one can control the density of specifically immobilized receptors.

The possible reasons of signal decrease after 12 h storage of chip at room temperature are:

i) loss in receptor and/or Gαolf activity;

ii) partial depletion of Gαolf protein pool;

iii) limitation of G αolf mobilization.

The :-subunit of G protein has a molecular mass about 40-50 kDa [201, 202] comparable to that of BSA and neutravidin. However, quite low amplitude of signals measured can be ascribed to the low amount of receptors oriented in the direction allowing full access of G: olf to GTP-g-S (see also Table 6.2).

Indeed, GTP cannot penetrate the lipid bilayer to access G: olf located inside the nanosome, whereas the hydrophobic odorants can penetrate the bilayer to reach the receptor ligand binding pocket and activate the receptor.

Very recently, J. Vidic et al. have shown that anchoring of OR via the N- end leads to some attenuation of functional responses to odorant detected by means of Biacore technique [199]. This evidence should be taken into account and immobilization of OR via its C-terminus probably should be considered as preferable in the future work. Nevertheless, the amplitude of the Biacore- measured functional responses of OR 17-40 specifically immobilized via either its N- or C-terminus remained much smaller than the signals corresponding to proteins adsorption on the SPR chip [56, 199]. A similar tendency was observed in the present study.

124 Another molecular event that could contribute to the optical signal was the conformational change of activated OR. The latter is composed of a bundle of 7 transmembrane :-helices connected by loops [203]. Odorant stimulation of OR induces a rearrangement of helices 6 and 3 [203] leading to separation of transmembrane domains in the helix bundle [196]. Recently it has been suggested that the signal changes observed by SPR can be also ascribed to the protein secondary structure changes. Thus, May and Russell [16] have correlated a decrease in SPR signal to the formation of Y-sheets, turn or unordered protein secondary structures; compact helical structure was thought to possess higher refractive index and thus to increase SPR signal. In this way, the impact of OR conformational changes on SPR signals may also be taken into consideration.

The response profile for helional was endowed with double maximum (Fig. 6.10). This response pattern of immobilized OR 17-40 corroborates already reported experimental data on olfactory receptor pharmacology [56], but it differs from the single maximum dose-dependence curves presented in Part 5. The source of such a discrepancy, whether phenomenological or methodological, will be clarified by further research work.

5. Conclusion and perspectives The main findings presented in Part 6 of this work have demonstrated that implementation of complex “well-oriented” architecture of biorecognition elements does not necessarily lead to better biosensing properties. The most crucial points in design of odorant-sensitive biofilm seem to be related to the density of OR-bearing nanosomes adsorption and/or to the total thickness of the biofilm. The characteristics of OR-containing biofilm responses to helional observed by the SPR platform could be ascribed to two main molecular events actually undistinguishable from SPR signals: desorption of G: protein from membrane bilayer and intrinsic conformational changes in activated olfactory receptor itself. Further more precise studies are needed to clarify the mechanisms involved in odorant recognition by immobilized OR.

125 Double maximum response pattern of immobilized OR 17-40 established in the present study is in good agreement with already reported experimental data on OR 17-40 pharmacology and is consistent with the hypothesis about the functional states of OR mobilized by various concentrations of agonist [Dr. E. Pajot; personal communication] proposed by the research team of Prof. R. Salesse (INRA). The model on the biological nature of such response profile is under testing.

126

ConclusConclusionion générale et perspectives

Les résultats de recherches présentés dans ce manuscript ont été ciblés sur l’application de biocapteurs électrochimiques et optiques sans marquage pour suivi direct des interactions bioaffines et biocatalytiqes de certains xénobiotiques (odorants et glycoalcaloïdes) avec des biomacromolécules immobilisées telles que le récepteur olfactif humain RO 17-40 couplé à la protéine G et les cholinestérases de sérum humain et équin. Nous avons démontré que les butyryl cholinestérases humaine et equine réticulées à l’albumine bovine sur la surface de transistors à éffet de champ sensibles au pH (pH-FETs) ont suivi une cinétique de Michaelis pour l’hydrolyse de leurs substrats. Les glycoalcaloïdes de pomme de terre, l’:- solanine et l’:-chaconine, et celui des tomates, la tomatine, ont inhibé l’enzyme équine de manière réversible et compétitive tandis que l’inhibition réversible mixte de la cholinestérase humaine a été révélée. Considérant Kiapp en tant que caractéristique d’affinité/force d’inhibition, l’:-chaconine a été démontrée être l’inhibiteur le plus fort des deux cholinestérases étudiées ; l’:-solanine est le plus faible inhibiteur d’enzyme équine tandis que dans le cas de l’enzyme humaine c’est la tomatine. Les données obtenues en utilisant le biocapteur sont concordantes avec les données de la littérature sur la cinétique de la butyryl cholinestérase native et la toxicité des glycoalcaloïdes. Puisque les pH- FETs sont compatibles avec les technologies de la microélectronique et qu’ils peuvent être biofonctionnalisés facilement, ils peuvent être la base de plateformes « multibiocapteurs » à haute productivité visant au sélection de médicaments pour la supression de l’activité cholinésterasique ainsi que de xénobiotiques toxiques.

127 Les interactions affines de l’:-chaconine et l’:-solanine avec la cholinestérase équine ont été étudiées en utilisant la spectroscopie d’impédance électrochimique. Le couplage des glycoalcaloïdes et de l’enzyme a induit une diminution spécifique de l’impédance électrochimiqie tandis qu’aucun effet n’a été observé sur l’albumine de sérum bovin ou la carboxylestérase bactérienne qui, en fait, ne sont pas des molécules cibles aux glycoalcaloïdes. Ces résultats ont été interprétés en termes de modification de répartition de charges à travers un film enzymatique démontrant une bonne perspective pour une détection directe de petits xénobiotiques. Le récepteur olfactif humain RO 17-40 couplé à la protéine G a été employé avec succès dans deux biocapteurs sans marquage: électrochimique et optique en tant qu’unité complexe capable de la bioreconnaissance de molécules odorantes. La réponse de RO 17-40 en fonction de son agoniste spécifique l’hélional sondée par deux techniques a montré des courbes en forme de cloche. Ces données ont été discutées en termes de libération de protéine G de la bicouche lipidique et changement de conformation de RO 17- 40 lui-même. La possibilité du suivi direct des événements moléculaires déclenchés par un récepteur stimulé par son agoniste sera un premier pas vers un dispositif multibiocapteur de type « nez bioélectronique » pour sélectionner des ligands pour des récepteurs couplés à la protéine G (GPCRs) et pour supprimer le caractère orphelin de ces derniers.

De nos jours, la bioélectronique se basant sur l’enzyme et les GPCRs devient une discipline à haut potential biomédical et pharmacologique. Des avancées réalisées dans les techniques d’éxpression de GPCRs et de production d’enzymes recombinantes, dans la diminution de taille des transducteurs et le dévelopment de modéles de traitement de données permettront l’élaboration de nouveaux dispositifs biocapteurs pour le sélection de médicaments.

128

General conclusion an andd perspectives

Investigations reported in this manuscript were focused on the use of label-free electrochemical and optical biosensors for direct probing bioaffinity and biocatalytic interactions of some xenobiotics (odorants and glycoalkaloids) with immobilized macromolecules, namely, G protein coupled human olfactory receptor OR 17-40 and cholinesterases from human and equine serum. It has been shown that human and equine butyryl cholinesterases cross- linked with bovine serum albumin on pH-sensitive field-effect transistors (pH- FETs) follow Michaelis kinetics of hydrolysis of their substrates. Potato glycoalkaloids :-solanine and :-chaconine, as well as tomato glycoalkaloid tomatine, inhibit equine enzyme reversibly and competitively while for the human cholinesterase the mixed mode of reversible inhibition has been suggested. Considering Kiapp as a characteristic of affinity/inhibition potency, :-chaconine is shown to be the strongest inhibitor for the both enzymes studied; :-solanine was the feeblest inhibitor for equine enzyme while tomatine – for the human one. Data obtained by means of biosensor are consistent with literature data on native butyryl cholinesterase kinetics and glycoalkaloids toxicity. As pH-FETs are compatible with technologies of microelectronics and can be easily biofunctionalized, they can serve as a basis for the design of high throughput biosensing platforms capable of screening anti cholinesterase drugs and toxic xenobiotics. The affinity interactions of :-chaconine and :-solanine with equine cholinesterase have been probed by the electrochemical impedance spectroscopy. Binding of glycoalkaloids to enzyme induces a specific dose- dependent decrease of the electrochemical impedance; and any effect has been observed neither on bovine serum albumin nor on bacterial carboxylesterase,

129 which are not target molecules of glycoalkaloids. These results have been interpreted in terms of modification of charge repartition in the enzymatic film revealing a promising approach for the direct detection of small xenobiotics. The human G protein coupled olfactory receptor OR 17-40 has been successfully employed as a bio recognition part of label-free impedimetric and surface plasmon resonance biosensor platforms; thus electrochemical and optical detection of some odorants has been carried out. Dose-dependent bell- shaped pattern of OR 17-40 response to its specific agonist helional is established by means of both techniques and discussed in terms of G protein liberation from the lipid bilayer and conformational changes of OR 17-40 itself. Possibility of monitoring molecular events triggered by agonist-stimulated receptor is the first step towards label-free biosensor platforms opening new possibilities in the field of “bioelectronic noses” capable of ligands profiling and G protein coupled receptors (GPCRs) deorphanization.

Nowadays, enzyme and GPCR based bioelectronics becomes a discipline with exceptionally prominent biomedical and pharmacological potential. New achievements in expression of GPCRs and production of recombinant enzymes, reductions in transducer size and development of robust models of data treatment will result in elaboration of new biosensing platforms for drug screening.

130 Annex A

PotentiomPotentiometryetry based on ISISFETsFETs (pH(pH----FETsFETsFETs)))) The pH-sensitive field effect transistors, or pH-FETs (Fig. A-1, A, B, C, D) were fabricated at the Research Institute of Microdevices (Kyiv, Ukraine). The potentiometric sensor chip (p-type Si substrate, 3 mm × 10 mm) contained two identical Si 3N4-pH-FETs; the bare substrate of the sensor chip was used as a quasi-reference electrode (Fig. A-1). This makes unnecessary the use of any conventional reference electrode or of noble metal quasy-reference electrode to render the biosensor operation [204]. The schema of differential operation mode of such potentiometric transducer [205, 206] is depicted in Fig. A-1, A. The pH-FETs operated at a constant drain current and drain-source voltage mode (I d=100 fA, V ds =1 V). The photo of experimental device is given in Fig. A-2.

AAA

BBB CCC DDD

Fig. AAA-A---1111 (A) Schema of differential pair of ISFETs (pH-FETs); (B) General view of transducer; (C) Transducer connected to the support over a stationary glass cell; (D) Schematic view of ISFET [19].

131

Fig. AAA-A---2222... Experimental potentiometric device: General view: transducer is connected to differential ISFET amplifier and recorder;

A differential pair of ISFETs (one being functionnalized with enzyme and the second one – with inert protein, BSA) was employed in this work to compensate for common interferences, bulk pH, light and temperature fluctuations [204]. The sensitivity of pH-FETs was 30-40 mV/pH being sufficient for registration of pH changes inside the biorecognition film occurring upon catalytic hydrolysis of butyryl- and acetyl choline by butyryl cholinesterase (see Chapter 1, Part 2).

132 Annex B

Electrochemical impedance spectroscopy (EIS) In EIS, small amplitude perturbing sinusoidal voltage signal is applied to the electrochemical cell, and resulting current is measured [157]. The complex impedance is calculated as the ratio between the system voltage phasor, U (B) and the current phasor, I (B) which are generated by a frequency response analyzer. Otherwise, impedance is the sum of the real, Zre (q) and imaginary,

Zim (q) components: U (ω) Z(ω) = = Z + jZ (ω) ; (1) I(ω) re im where ω = 2πf ; j = −1 ; q and f (excitation frequency) have dimensions of

rad ⋅ s −1 and Hz, respectively. To understand electrochemical processus occurring in cell, impedance data are usually fitted with an equivalent electrical circuit. In bioelectrochemistry, the most widely used one consists of an ohmic resistance of the bulk electrolyte ( Rs) in series with a parallel combination of the capacitive element (double layer capacitance, C dl or constant phase element,

CPE) and the polarization resistance ( Rp), representing interfacial resistance in the absence of any redox species [74], Fig. B-1, A. In case of faradaic impedance (i.e. probed in the presence of any redox couple), Ret is applied instead of Rp, representing interfacial electron transfer resistance.

Rs CPE Rs CPE

Rp Rp Zw

AAA BBB

Fig. BBB-B---1111... Equivalent circuits applied for impedance data fitting, see in text.

Double-layer capacitance of electrode interface is defined as follows: ε ⋅ε ⋅ A C = 0 ; (2) dl d ε ε where is dielectric constant of biofilm, 0 is dielectric constant of the vacuum, A is the electrode area and d – thickness of biofilm (considered as dielectric).

133 It is well known that at real biofunctionalized electrodes the double layer capacitance shows a phase angle r<90º and this effect is usually attributed to the surface heterogeneity (roughness, porosity and so on). To improve the quality of data fitting, it was proposed to introduce the CPE element (instead of pure capacitance Cdl ) in the model circuit [20, 157]. CPE is an empirical impedance function:

− CPE = Y −1 ⋅ ( j ⋅ω) n ; (3) where Y is the numerical value of admittance at ω =1 rad ⋅ s −1 . When a coefficient n is close to 0, CPE is essentially a resistance, and if n=1, the CPE is equal to Cdl and electrode can be considered as ideal. An introduction of the CPE into the equivalent electric circuit is important for describing the electrical properties protein layers on solid electrodes [157]. Values of the n between 0.9 and 1 bring to the capacitive behavior of CPE and reflect good insulating properties of modified interface. It should be mentioned that bare electrode capacitance also contributes to the CPE value extracted from a simple equivalent circuit, i.e. CPE is not a “pure” capacitance of biofilm [192].

Interface resistance can also be presented as a serie of Relectrode and

Rbiofilm , generally it is defined as follows: R ⋅T R = ; (4) et ⋅ ⋅ n F i0 where R is gas constant, T – temperature in K, F – Faraday constant, i0 – exchange current under equilibrium, n – number of electrons transferred per molecule of redox probe. When faradaic reaction occurs at the electrode, diffusion of redox species from bulk electrolyte to electrode will contribute to the total impedance, therefore so-called Warburg impedance, Zw, should be introduced into the equivalent circuit:

R ⋅T − Z = ⋅ D ⋅ω 1 2 ; (5) w n 2 ⋅ F 2 () where D is diffusion coefficient. Thus, Fig. B-1, B illustrates widely used

Randles-Ershler electrical circuit containing Zw [207, 208].

134 Rs and Zw are usually considered as “bulk” parameters because indeed they are not affected by biorecognition events occurring at the interface (see

Fig. B-2). Alterations of CPE or/and Rp (Ret ) are usually ascribed to the biochemical reactions at the electrode surface.

Fig BBB-B---2222... Nyquist impedance diagrams, adapted from [157]. (1) Total impedance Z is controlled by interfacial and bulk processes, presented by Ret and Zw; (2) Z is mainly controlled by bulk diffusion processes, Z w; (3) Z is entirely controlled by interfacial processes, Ret .

In this work EIS was carried out in a classical three-electrode cell (Fig. B- 3, A), V=5 mL, placed into a Faraday cage in order to improve the signal-to- noise ratio.

SCE

AE WE

V=5 ml

AAA B

Fig. BBB-B---3333... (A) Three-electrode glass cell of the VoltaLab 40 impedance analyzer, WE – working electrode, AE – auxiliary electrode, SCE – reference electrode. (B) Schema of saturated calomel electrode (SCE).

135

136 Annex C

CCC-C---1.1. Single channel SPR spectrometer and goldgold----coatedcoated chips Surface plasmon resonance (SPR) technique is responsive to changes that occur in the vicinity of sensor surface and thereby is one of the most potent tools in biomolecular events probing. The main element of substrate for SPR platforms is a thin metal (Au, Ag, Al etc.) film with negative dielectric permittivity deposed onto a transparent dielectric support. SPR spectrometer “Biosuplar 3” possessing single optical channel used in the Part 5 of this thesis was developed at the Institute of Semiconductor Physics of National Academy of Sciences of Ukraine (Kyiv). This optoelectronic device based on the phenomenon of the surface plasmon resonance in the Kretchmann’s optical configuration (Fig. C-1.1 ) was controlled by a computer via self-developed software.

θθθ000 εεε3

SP εεε2

εεε1

Fig. C-CCC-1.1--1.1.1.11.1 ... Kretchmann’s optical configuration. SP – surface plasmon, s1 is dielectric constant of biofilm/bulk medium, s 2 – that of metal, s3 – that of prism; r 0 is the angle of incidence.

Gold film (45 nm) deposited through Cr adhesion layer (1-5 nm) onto a glass chip represents a surface of a sensor chip (Fig. C-1.2 ).

Fig. CCC-C---1.2.1.2. Glass substrates coated with gold were applied in SPR spectroscopy

137 An incident beam of p-polarized light from a semiconductor laser diode (t=650 nm) excited an oscillation of electronic plasma (i.e. surface plasmon, SP) in the metallic film of chip. A special prism (Fig. C-1.3 , B) capable of rotation on a computer-defined angle provided optimal conditions for the plasmon excitation at the metal-dielectric interface.

A plasmon resonance by itself was registered as a drastic decrease in the intensity of the light reflected (see Fig. C-1.4 , A). In Kretchmann’s geometry, SP resonance angle is defined as follows: ω ω ε ε ε θ = 1 2 3sin 0 ; (1) c c ε2 + ε 1 where c = λ ⋅ν , ν is a frequency of light wave with length λ , and ϖ = 2π ⋅ν . In the present work, SPR angle shift (ur) was taken as a sensor output.

Resonance angle r (i.e. angle of p-polarized light incidence at which intensity of reflection is minimal) is a specific characteristic of surface state, sensitive to variations of mass, thickness, dielectric properties within the biorecognition layer but also to the bulk optical properties of external medium (refractive index n of solution).

Location of the SPR angle also depends on the refractive indexes of: glass prism n p, glass support of a chip n s and immersion liquid n il used for better adhesion of glass side of chip to the top of the prism. Mismatch between n p, ns and n il should be absolutely avoided.

In order to minimize non-specific shift of SPR angle caused by the bulk variations of n (especially in case of a single channel SPR spectrometer), dilution of all biorecognition partners to be studied should be performed in the same buffer if possible.

138 In the investigations reported in Part 5, the SPR spectrometer flow cell

(V ∼20 µl) was connected to a Gilson Minipuls 3 pump (Fig. C-1.3 , A, B).

AAA BBB

Fig. C-CCC-1.3.--1.3. (A) Single channel SPR spectrometer connected to a pump. (B) Flow cell of a single channel SPR spectrometer coupled to a prism.

A dependence of the SPR angle on analysis time registered by the device is called sensogram (Fig. C-1.4 , B).

AAA BBB

Fig. CCC-C---1.4.1.4. Two types of SPR data presentation used in this work: (A) Reflectance vs angle of incidence; (B) Sensogram: SPR angle (estimated from (A)) vs time.

139 CCC-C---2.2. DoDoDoubleDo uble channel SPR spectrometer and gold coated chipschips

The SPR Kretschmann-type double channel spectrometer NanoSPR-6 (model 421, NanoSPR, USA) with light-emitting diode light source (t = 650 nm) was used in the Part 6 of this work (Fig. C-2.1 ). A high refraction index of the prism (n = 1.61) and a broad dynamic range (up to 19º in air) of the SPR instrument enabled high quality of the computer fitting of the experimental data with a theoretical curve. The SPR data were processed by means of the NanoSPR-6 software (version 6.0). SPR sensogram similar to that shown in Fig. C-1.4 , B represented real time changes in the SPR angle and were recorded both in custom and differential mode (delta between working and reference channels) using double channel teflon flow cell. Glass supports (TF-1 glass, 20×20 mm) coated with a polycrystalline gold layer (50 nm) via thin chromium sublayer (5 nm) were provided by NanoSPR and used for the SPR measurements. The intersensor reproducibility was found to be 5% for n=5.

Fig. CCC-C---2.12.12.12.1 ... Double channel SPR sensor platform “NanoSPR”. The photo was kindly provided by Dr. Vladimir Chegel (ISP NASU, Ukraine).

140 Annex D

Plasmids designed for the heterologous expexpressionression of human olfactorolfactoryy receptor 1740 and protein GI olfolfolf in yeast Saccharomyces cerevisiae (strain MC18)

Plasmid cmyccmyc----1717171740.40. Schema was kindly provided by Dr. M.-A. Persuy (INRA)

Plasmid GolfGolf.... [209] .

141

142 Annex E

EEE-E---1.1. Preparation of helional, heptanal and blank probesprobes for screening in the double channel SPSPRR spectrometer

Mol/L Helional Blank Heptanal Blank for helional for heptanal 10 -1 10 fl stock 10 fl PBS 10 fl stock 10 fl PBS +510 fl DMSO +510 fl DMSO +700 fl DMSO +700 fl DMSO 10 -4 1 fl 10 -1 1 fl “10 -1“ 1 fl 10 -1 1 fl “10 -1“ +999 fl PBS +999 fl PBS +999 fl PBS +999 fl PBS 10 -5 70 fl 10 -4 111 fl “10 -4“ 70 fl 10 -4 111 fl “10 -4“ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS 10 -6 70 fl 10 -5 111 fl “10 -5“ 70 fl 10 -5 111 fl “10 -5“ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS 10 -7 70 fl 10 -6 111 fl “10 -6“ 70 fl 10 -6 111 fl “10 -6“ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS 10 -8 70 fl 10 -7 111 fl “10 -7“ 70 fl 10 -7 111 fl “10 -7“ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS 10 -9 70 fl 10 -8 111 fl “10 -8“ 70 fl 10 -8 111 fl “10 -8“ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS 10 -10 70 fl 10 -9 111 fl “10 -9“ 70 fl 10 -9 111 fl “10 -9“ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS 10 -11 70 fl 10 -10 111 fl “10 -10 “ 70 fl 10 -10 111 fl “10 -10 “ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS 10 -12 70 fl 10 -11 111 fl “10 -11 “ 70 fl 10 -11 111 fl “10 -11 “ +630 fl PBS +1000 fl PBS +630 fl PBS +1000 fl PBS

Adding of 1 mM GTP* to the final concentration 10 GM:GM:

Mol/L Helional Blank Heptanal Blank for helional for heptanal 10 -5 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP 10 -6 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP 10 -7 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP 10 -8 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP 10 -9 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP 10 -10 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP 10 -11 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP 10 -12 0.6 ml+6 fl GTP 1 ml+10 fl GTP 0.6 ml+6 fl GTP 1 ml+10 fl GTP

*This procedure should be carried out on ice 1 mM GTP (or GTPGTP----LLLL----S)S)S)S) is prepared from the aqueous 100 mM stock solutionsolution

143 EEE-E---2.2. ProtocoProtocoll of odorant screening in the double channel SPR spectrometer in differential mode

RRR – Area of functionalized surface corresponding to the reference channel; WWW – Area of functionalized surface corresponding to the working channel; FRFRFR – flow rate. In the configuration of the probe injection system in our SPR device, injected solution reaches the chip surface in less than 2 min at FR 40 fl/min.

1. R + W : washing with PBS, 15-20 min at FR 40 fl/min, until differential baseline is stabilized.

2. R + W : blank probe #1, v = 1 ml, FR 40 fl/min until differential baseline is stabilized.

3. WWW – odorant solution #1, v = 0.6 ml, FR 40 fl/min ; R – blank probe #1, v ≈ 0.6 ml, FR 40 fl/min.

4. R + W : washing with PBS, at least 20 min at FR 40 fl/min.

EEE-E---3.3. Preparation of octanal, nonanal and vanillin

Mol/L Octanal Nonanal Vanillin 10 -1 10 fl stock 10 fl stock 0.0152 g vanillin +630 fl DMSO +565 fl DMSO +1000 fl DMSO 10 -4 1 fl 10 -1 1 fl 10 -1 1 fl 10 -1 +999 fl PBS +999 fl PBS +999 fl PBS 10 -5 70 fl 10 -4 70 fl 10 -4 70 fl 10 -4 +630 fl PBS +630 fl PBS +630 fl PBS 10 -6 70 fl 10 -5 70 fl 10 -5 70 fl 10 -5 +630 fl PBS +630 fl PBS +630 fl PBS 10 -7 70 fl 10 -6 70 fl 10 -6 70 fl 10 -6 +630 fl PBS +630 fl PBS +630 fl PBS 10 -8 70 fl 10 -7 70 fl 10 -7 70 fl 10 -7 +630 fl PBS +630 fl PBS +630 fl PBS 10 -9 70 fl 10 -8 70 fl 10 -8 70 fl 10 -8 +630 fl PBS +630 fl PBS +630 fl PBS 10 -10 70 fl 10 -9 70 fl 10 -9 70 fl 10 -9 +630 fl PBS +630 fl PBS +630 fl PBS 10 -11 70 fl 10 -10 70 fl 10 -10 70 fl 10 -10 +630 fl PBS +630 fl PBS +630 fl PBS 10 -12 70 fl 10 -11 70 fl 10 -11 70 fl 10 -11 +630 fl PBS +630 fl PBS +630 fl PBS

144

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162 188. Hou, Y., Jaffrezic-Renault, N., Martelet, C., Zhang, A., Minic-Vidic, J., Gorojankina, T., Persuy, M.A., Pajot-Augy, E., Salesse, R., Akimov, V., Reggiani, L., Pennetta, C., Alfinito, E., Ruiz, O., Gomila, G., Samitier, J., and Errachid, A. (2006). A novel detection strategy for odorant molecules based on controlled bioengineering of rat olfactory receptor I7. Biosens. Bioelectron. 189. Nobs, L., Buchegger, F., Gurny, R., and Allemann, E. (2004). Poly(lactic acid) nanoparticles labeled with biologically active Neutravidine for active targeting. Eur. J. Pharmaceut. Biopharmaceut. 58 , 483-490. 190. Minic, J., Grosclaude, J., Aioun, J., Persuy, M.A., Gorojankina, T., Salesse, R., Pajot-Augy, E., Hou, Y., Helali, S., Jaffrezic-Renault, N., Bessueille, F., Errachid, A., Gomila, G., Ruiz, O., and Samitier, J. (2005). Immobilization of native membrane-bound rhodopsin on biosensor surfaces. Biochim. Biophys. Acta 1724 , 324-332. 191. Hays, H.C., Millner, P.A., and Prodromidis, M.I. (2006). Development of capacitance based immunosensors on mixed self-assembled monolayers. Sens Actuat B 114 , 1064-1070. 192. K'Owino, I.O., and Sadik, O.A. (2005). Impedance spectroscopy: a powerful tool for rapid biomolecular screening and cell culture monitoring. Electroanalysis 17 , 2101-2113. 193. Jones, D.T., and Reed, R.R. (1989). Golf: an olfactory neuron specific G- protein involved in odorant signal transduction. Science 244 , 790-795. 194. Nakamura, T., and Gold, G.H. (1987). A cyclic nucleotide gated conductance in olfactory receptor cilia. Nature 325 , 442-444. 195. Bakalyar, H.A., and Reed, R.R. (1990). Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science 250 , 1403- 1406. 196. Bourne, H.R. (1997). How receptors talk to trimeric G proteins. Curr. Opin. Cell Biol. 9, 134-142. 197. Gomila, G., Casuso, I., Errachid, A., Ruiz, O., Pajot, E., Minic, J., Gorojankina, T., Persuy, M.A., Aioun, J., Salesse, R., Bausells, J., Villanueva, G., Rius, G., Hou, Y., Jaffrezic, N., Pennetta, C., Alfinito, E., Akimov, V., Reggiani, L., Ferrari, G., Fumagalli, L., Sampietro, M., and Samitier, J. (2006). Advances in the production, immobilization, and

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166

PPPublicationsPublications et communications scientiscientifiquesfiques

Revues

1. I. Benilova, V. I. Chegel, Yu. V. Ushenin, J. Vidic, E. Pajot, A. P. Soldatkin, C. Martelet, N. Jaffrezic-Renault, Stimulation of human olfactory receptor OR 17-40 with odorants probed by surface plasmon resonance , submitted to the Journal of Biotechnology.

2. I. Benilova , J. Minic Vidic, E. Pajot-Augy, A.P. Soldatkin, C. Martelet, N. Jaffrezic-Renault, Electrochemical study of human olfactory receptor OR 17-40 stimulation by odorants in solution , Materials Science and Engineering C, accepted.

3. I. V. Benilova , A. P. Soldatkin, C. Martelet, N. Jaffrezic-Renault. Non- faradaic impedance probing of potato glycoalkaloids interaction with butyrylcholinesterase immobilized onto gold electrode , Electroanalysis, 2006 , v. 18, #19-20, p.1950-1956.

4. I. V. Benilova , V. N. Arkhypova, S. V. Dzyadevych, N. Jaffrezic-Renault, C. Martelet, A. P. Soldatkin. Kinetics of human and horse sera cholinesterases inhibition with solanaceous glycoalkaloids: study by potentiometric biosensor , Pesticide Biochemistry and Physiology, 2006 , v. 86, #3, p. 203-210.

167 5. I. V. Benilova, V. M. Arkhypova, S. V. Dzyadevych, N. Jaffrezic-Renault, C. Martelet, O. P. Soldatkin. Kinetic properties of butyrylcholinesterases immobilized on pH-sensitive field-effect transistor surface and inhibitory action of steroidal glycoalkaloids on these enzymes , Ukrainian Biochemical Journal , 2006 , v.78, #2, p.131-139 (in Ukrainian).

6. Ya. I. Korpan, E. A. Nazarenko , I. V. Skryshevskaya, C. Martelet, N. Jaffrezic-Renault, A. V. El’skaya. Potato glycoalkaloids: true safety or false sense of security? Trends in Biotechnology, 2004 , v.22, #3, p. 148- 151.

ConfConféééérencesrences

Communications orales 1. I. Benilova, C. Martelet, J. Minic, E. Pajot, V. Chegel, Yu. Ushenin, A. Soldatkin, , N. Jaffrezic-Renault, Mise au point d’un capteur olfactif bioélectrochimique , acceptée à les Journées d'électrochimie 2007 – 2-6 juillet 2007, Lyon, France.

2. I. Benilova , J. Minic Vidic, E. Pajot-Augy, A.P. Soldatkin, C. Martelet, N. Jaffrezic-Renault. Elaboration d’un biocapteur impédimétrique basé sur les récepteurs olfactifs humains OR 1740 pour la détection sensible des odorants en milieu liquide , 19 èmes entretiens Jacques Cartier “Nanobiotechnologies pour l’analyse et la conversion d’énergie”, 4-5 décembre 2006 , La Tronche, France .

3. I. Benilova, S. Gavrylenko, O. Yakovets, O. Rudenko, A. Rachkov, V. Chegel, Yu. Ushenin, A. Soldatkin. Elaboration of affinity biosensor based on the SPR technique for monitoring interactions between calmodulin and eEF1 , 2 nd International Scientific and Technical Conference “Sensor Electronics and Microsystems Technology”, June 26-30, 2006 , Odessa, Ukraine.

168 CommunicationCommunicationssss par afficaffichehe (posters) 4. I. Benilova , J. Minic Vidic, E. Pajot-Augy, A.P. Soldatkin, C. Martelet, N. Jaffrezic-Renault. SPR and impedance spectrometry study of nanobiofilms based on the human olfactory receptor OR 1740 , Ukrainian-German Symposium on Nanobiotechnology, December 14-16, 2006 , Kyiv, Ukraine.

5. I. Benilova , J. Minic Vidic, E. Pajot-Augy, A. P. Soldatkin, C. Martelet, N. Jaffrezic-Renault. Elaboration d’un biocapteur impédimétrique basé sur les récepteurs olfactifs humains OR 1740 pour la détection sensible des odorants en milieu liquide , 5 èmes Journées Maghreb-Europe MADICA- 2006, 30 octobre - 1 novembre 2006 , Mahdia, Tunisie.

6. I. V. Benilova, E. A. Nazarenko, T. O. Kishko, Y. I. Korpan, N. P. Dmitrenko. Cytotoxic and membrane-acting effects of glycoalkaloids on erythrocytes and thymocytes of rats , Abstr. 10 th International Congress of Toxicology, July 11-15, 2004 , Tampere, Finland, printed in Toxicology and Applied Pharmacology, 2004 , v.197, #3, p.312.

PrixPrix/A/A/A/Awardwardwardssss

Le Prix de la meilleure publication de jeune chercheur dans le domaine des biotechnologies, attribué en 2006 par le jury scientifique de l’Institut de biologie moléculaire et génétique d’Ukraine (copie de diplôme ci-jointe).

169

Résumé De nos jours, la bioélectronique devient une discipline à haut potential biomédical et pharmacologique. Les résultats de recherches présentés dans ce manuscript sont ciblés sur l’application de biocapteurs électrochimiques et optiques sans marquage pour suivi des interactions bioaffines et biocatalytiqes de certains petits xénobiotiques (odorants et glycoalcaloïdes stéroïdiques) avec des biomacromolécules immobilisées telles que le récepteur olfactif humain RO 17-40 couplé à la protéine G et les cholinestérases de sérum humain et équin. Les butyryl cholinestérases immobilisées sur la surface de transistors à l’effet de champ sensibles au pH suivent une cinétique de Michaelis pour l’hydrolyse de leurs substrats. Les glycoalcaloïdes l’--solanine, l’-- chaconine et la tomatine inhibent l’enzyme équine de manière réversible et compétitive tandis que pour cholinestérase humaine, l’inhibition réversible mixte a été démontrée. L’--chaconine est l’inhibiteur le plus fort de deux enzymes. Les interactions affines des glycoalcaloïdes avec la butyryl cholinestérase équine ont été étudiées en utilisant la spectroscopie d’impédance électrochimique. La détection directe des inhibiteurs faibles et compétitifs (l’--solanine) peut être beaucoup plus sensible en absence des substrats enzymatiques. Le récepteur olfactif RO 17-40 a été employé avec succès dans deux plateformes de biocapteurs: impédimétrique et à base de résonance plasmonique de surface en tant qu’unité complexe capable de la bioreconnaissance des odorants. La possibilité du suivi direct des événements moléculaires déclenchés par un récepteur stimulé par son agoniste est un premier pas vers le « nez bioélectronique » .

BIOSENSOR APPROACH TO PROBE BIOAFFINITY AND BIOCATALYTIC INTERACTIONS OF SSMALLMALL XENOBIOXENOBIOTICSTICS

Abstract Nowadays, bioelectronics becomes a discipline with prominent biomedical and pharmacological potential. Investigations reported in this manuscript are focused on the use of label-free electrochemical and optical biosensors for the study of bioaffinity and biocatalytic interactions of some small xenobiotics (odorants and steroid glycoalkaloids) with immobilized macromolecules, namely, G protein-coupled human olfactory receptor OR 17-40 and cholinesterases from human and equine serum. The butyryl cholinesterases immobilized on pH-sensitive field-effect transistors follow Michaelis kinetics of hydrolysis of their substrates. Glycoalkaloids --solanine, --chaconine and tomatine inhibit the equine enzyme reversibly and competitively while for the human cholinesterase a mixed mode of reversible inhibition is suggested. --Chaconine is the most potent inhibitor of both enzymes. The affinity interactions of glycoalkaloids and equine butyryl cholinesterase have been probed with electrochemical impedance spectroscopy. The absence of enzymatic substrate can significantly improve label-free detection of weak and competitive inhibitors (--solanine). Olfactory receptor OR 17-40 has been successfully employed as odorant-recognition part of impedimetric and surface plasmon resonance-based platforms. Possibility of direct monitoring molecular events triggered by agonist-stimulated receptor is the first step towards the “bioelectronic nose”.

DISCIPLINES : biotechnologie, biochimie, chimie de surface

Mots clés: biocapteur ; petite molécule ; transistors à l’effet de champ ; impédance ; résonance plasmonique de surface ; odorant ; cholinestérase ; récepteur olfactif .

Keywords: biosensor; small molecule; field-effect transistor; impedance; surface plasmon resonance; odorant; cholinesterase; olfactory receptor.

INTITULES ET ADDRESSES DES LABORATOIRELABORATOIRESSSS :::

Laboratoire AMPERE, Ecole Centrale de Lyon, bât. H9, 36 avenue Guy de Collongue, 69134-Ecully Cedex, FRANCE

Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotnogo str., 03143-Kyiv, UKRAINE