Conclusion et perspectives

Cette thèse a eu pour objectif d’utiliser la microfluidique pour détecter le NO dans les milieux biologiques, stocké sous forme des RSNOs. Plusieurs méthodologies nécessaires pour cette détection ont été développées. Une méthode EC-SM pour identifier la composition et les produits de décomposition de GSNO a été développée. Ces résultats nous ont permis de proposer des voies de décomposition. La séparation de GSNO et de son produit de décomposition, de la Cysteine et de CySNO a été optimisée avec une méthode EC-C4_D

conventionnelle. Différentes techniques de décomposition (Cu2+ _{/ GSH, nanoparticules d’or,}

lumière, température…) des RSNOs ont été étudiées afin de choisir la plus rapide et la plus adaptée à une mise en œuvre en système microfluidique. Lors de ce travail d’optimisation, différentes méthodes de détection ont été utilisées comme la spectrophotométrie, l’électrochimie, et la chimiluminescence. La miniaturisation de la détection des RSNOs a ensuite été menée en microsystème papier avec une détection colorimétrique dans le but de produire un« point of care device ». Enfin, la détection de GSNO a été faite en un système microfluidique intégrant une réaction de décomposition par Hg2+_.

Les perspectives de ce travail concernent entre autres (1) l’optimisation du protocole d’injection et de l’étape de séparation des RSNOs, (2) la mise au point de l’intégration de la méthode Cu2+

/ GSH avec des électrodes à membrane sélective dans le microsystème, (3) de l’étude d’un protocole de concentration d’AuNPs pour décomposer les RSNOs dans des volumes faibles comme ceux employés en microfluidique, (4) une étape en ligne de préconcentration des

BGE

S

SW

BW

36 RSNOs en microchip en utilisant par exemple l’isotachophorèse ou des effets de différence de champs électriques pour améliorer la sensibilité du diagnostic. Ce travail sur la sensibilité pourrait aussi éventuellement venir d’améliorations des potentiostats afin de détecter des concentrations plus faibles sans être affecté par le champ électrique dû à la séparation.

37

General introduction

Nitric oxide (NO) is considered as the first gaseous signal transduction molecule [11,57]. It is a diatomic free radical that has extremely a small free trajectory (100-200 µm) and short half- life (<1 s) in biological fluids. The addition of NO to functional proteins (or S-nitrosation reaction) is as important as phosphorylation in its consequences on cellular activities [58] and this necessitates the transport of NO to the targets in biological fluids. In order to be transported and stored in biological fluids, NO binds to the sulfhydryl groups of peptides and proteins forming S-nitrosothiols (RSNOs; thionitrites). RSNOs have been shown to occur endogenously in various biological systems (respiratory, cardiovascular, neurological, digestive…). RSNOs play important roles in several physiological functions (vasodilatation and relaxation [59-61], antiplatelet aggregation [20,59,62-66], antimicrobial [67], regulation and signaling of protein function etc.) and physiopathological events (neurodegenerative deseases such as Parkinson and Alzheimer) [68,69], apoptosis [70], cancer, asthma, chronic obstructive pulmonary disease [71], preeclampsia [72] and diabetes[17]).

RSNOs can be of biological or artificial origin. They can be formed in many pathways for example through (i) radical recombination between NO and a thiyil radical ( ), (ii) transition metal catalyzed pathway, (iii) transnitrosation reaction from low molecular weight RSNOs (LMW-RSNOs, such as S-nitrosoglutathione and S-nitrosocysteine) to high molecular weight RSNOs (HMW-RSNOs, such as nitrosoalbumin and nitrosohemoglobin) [47]. HMW-RSNOs, perform their biological activity by transferring NO to LMW-SNO that can penetrate cells and act on functional proteins [73].

RSNOs can be classified based on many criteria such as natural abundance and molecular weight. They are either present in vivo (S-nitrosohemoglobin, S-nitrosoalbumin, S- nitrosoglutathione, S-nitrosocysteine) or can be synthesized (S-nitroso-N-acetylpenicillamine,

S-nitrosocaptopril, S-nitroso-N-acetyl-L-cysteine) as candidates of pharmacological molecules, acting as NO donors. In all cases, they are considered as stocks of NO and then act on functional proteins either by releasing NO [74] or mostly by transnitrosation reactions [75].

RSNOs exist in biological media at concentrations that vary between tenth of nanomolar to less than ten micromolar ([2,3] and references therein). The variation of RSNO concentration has been shown to occur in many diseases [1]. There is no gold standard method to determine the biological concentrations of RSNOs. Even presently the same sample can give different results



38 using different detection methods. This raises the challenge to develop a robust, sensitive, selective and rapid method to detect RSNOs. Several analytical methods have been developed over the years to detect RSNOs. They can be direct if the RS-NO remains intact or otherwise indirect. Indirect methods are more sensitive but can suffer from several drawbacks such as: i) length and complexity of the procedure increasing the likelihood of artefacts, ii) selectivity problems (especially from nitrite usually present in biological fluids) and iii) employment of toxic Hg2+ _{in millimolar concentration in many of these assays, which impose safety issues for}

the operator and the environment [76].

Indirect methods are based on a two-step protocol: decomposition of the RS-NO bond followed by detection of the decomposition products (NO, nitrite or thionyl moiety) using electrochemical, spectrophotometric, or fluorescent methods, gas chromatography coupled to mass spectrometry, biotin switch methods or chemiluminiscence assays [2,50,77-80]. According to the application purposes, one of these methods can be selected. For example, chemiluminescence is a very selective and sensitive real time detection method but is only applicable for in vitro studies [81]. The colorimetric method (Saville method) is widely employed as it is very reproducible, but it cannot be used for real time analysis and presents low sensitivity and selectivity [2]. Mass spectrometry (MS) requires preliminary sample treatment and can underestimate RSNO amounts due to possible signal suppression or sample decomposition [80]. Electrochemical methods represent direct, real time, and label-free detection techniques that can be used for in vivo applications ([3] and references cited herein). All the indirect detection methods benefit from the fact that RSNOs can be decomposed through different pathways such as metal cation catalysis [24], ascorbic acid assisted reduction [47], heat [24,44], infrared, ultraviolet [82,83] or visible light assisted decompositions [43,60,82,84]. However, they mainly lead to partial and non-reproducible decomposition that can be detrimental for accurate detection. Furthermore the decomposition process can be multiple: homolytic cleavage giving rise to the formation of unstable NO• _{and RS}• _{that can lead to nitrite}

and other end-products, or heterolytic cleavage leading to RS- _{and NO}+ _{which rapidly forms}

nitrite.

Direct methods include MS [80,85] and detection of phosphines after their selective reaction with RSNO by MS, NMR, or fluorescence. The former can induce loss in NO during sample handling and during MS detection. The latter, even though highly selective, can induce a reaction with nitroxyl (HNO). Also it has the limitation of the stability of the formed aza-ylide compound, which varies depending on the RSNO being determined. Other direct characterization methods of RSNOs utilize a separation method coupled to one of these

General introduction

39 detection methods, such as high performance liquid chromatography (HPLC) with UV detection[26,45,86,87], HPLC with electrochemical detection [88] capillary zone electrophoresis (CE) with UV detection [89-92], capillary gel electrophoresis with laser induced florescence [93].

The improvement of the existing detection techniques by coupling with a separation method such as high performance liquid chromatography (HPLC), gas chromatography (GC) or capillary electrophoresis (CE) is helpful in selectivity enhancement, therefore in determining different RSNOs compositions. Conventional techniques necessitate most of the time several separation steps (sample pretreatment, separation, decomposition and detection). One way to improve these methods for RSNO quantitation would be to integrate them into miniaturized devices to form a lab on a chip device. This miniaturization should integrate the separation, decomposition and detection within the same microsystem and would offer many advantages over conventional analytical devices including: i) low sample and reagent consumption, ii) faster analysis time, iii) low power consumption, iv) low cost, v) reduced risk of contamination and vi) global affordability and portability[94,95]. The optimization of the decomposition step, in the case of indirect methods, represent an additional challenge to the ones of coupling the separation with the detection.

The long time project of the team is to integrate on a micro device all analysis steps including injection, decomposition, separation and detection for RSNOs of different molecular weights in a miniaturized device (Figure 25).

Figure 25: Schematic representation of the objectives of the PhD project

HMW LMW

LMW

Electrokinetic

separation

Decomposition

NO

Detection

Micro-chip

Injection

NO

40 The objective of this PhD work was develop and optimize the methodologies for each step and then to assemble and integrate them in a microfluidic device. As we aim at developing a simple and efficient miniaturized diagnostic device, the separation will be performed electrokinetically and the detection electrochemically. As for electrokinetic separation, CE was chosen over liquid chromatography because of its higher efficacity, ease of application of electrokinetic injection in comparison to pressure injection and ease of fabrication of microchip because no filling with microparticles is required. Detection was set to be electrochemical because of its simplicity (no need to derivatization) and portability in comparison to other detection methods, which need bulky instrumental detection (fluorescence and MS). Work was done principally to determine the best decomposition pathway among the possible ones (light, metallic ions, heat and gold nanoparticles).

In the first chapter, the current knowledge about biosynthesis and biological activities of NO and RSNOs is presented. Then, the synthesis, reactivity, decomposition and analysis methods for RSNO are developed. Finally we provide a short bibliography on miniaturization: the materials and techniques used and what is achieved for RSNO detection in microfluidic devices.