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Table of contents
I. Introduction
II. Etat de l’art
A. Biologie de NO et des RSNOs
B. Décomposition et quantification des RSNOs
C. Miniaturisation et microfluidique
III. Résultats
A. Analyse de la décomposition dans le temps de GSNO par électrophorèse capillaire : cinétique et identification des produits de décomposition
B. Analyse de la décomposition des RSNOs par le Cu+ en milieu réducteur ou en présence de nanoparticules d’or
1) Décomposition des RSNOs par Cu+ en milieu réducteur
2) Décomposition des RSNOs en présence de nanoparticules d’or
C. Miniaturisation
1) Détection colorimétrique dans un dispositif microfluidique d’analyse à base de papier
2) Détection électrochimie des RSNOs en microsystème après séparation par électrophorèse de zone.
IV. Conclusion et perspectives
Chapter I: State of Art on Nitric oxide and S-nitrosothiols
I. Chemo and Bio-Properties of Nitric Oxide and Nitrosothiols
A. Nitric Oxide (NO)
1) History of NO discovery
2) NO and NO derivatives characteristics
3) Biological synthesis of NO
i. Enzymatic Synthesis of NO by Nitric Oxide Synthase
ii. Enzymatic synthesis of NO by reduction of nitrite and nitrate [105]
iii. Non-enzymatic NO synthesis
4) Biological effects of NO
i. Role in cardiovascular system
ii. Role in digestive system
iii. Role in inflammation
iv. Role in cancer
v. Role in central nervous system and neurodegenerative disorders
vi. Role in diabetes
vii. Role in immunity
5) Targets of NO in biological system
i. Reactions of NO with metals / metalloproteins
ii. Reactions of NO with low molecular weight chemicals
iii. Reaction with thiols:
B. NO-donors drugs
1) Organic nitrates (RONO2s)
2) NONOates (Diazeniumdiolates):
3) C-nitroso compounds:
4) Iron nitrosyl complexes:
5) Furoxans
6) S-nitrosothiols
7) Other NO-hybrid donors
C. S-nitrosothiols
1) Formation of RSNOs in-vivo
i. Auto-oxidation of NO followed by addition to thiolate
ii. Oxidative nitrosylation
iii. Direct nitrosylation
iv. Transition metal ion / protein nitrosation
v. Transnitrosation
vi. Decomposition of low molecular weight DNICs with thiolate ligand
vii. Nitrite mediated S-nitrosation
2) RSNOs trans-membrane trafficking
3) Decomposition of RSNOs
i. Enzymatic denitrosylation
ii. Decomposition by metal ions
iii. Decomposition by ascorbate
iv. Decomposition by light
v. Decomposition by heat
4) RSNOs in health and disease
i. RSNOs therapeutic effects
ii. RSNOs as diagnosis indicator
II. Methods of quantification of RSNOs
A. Sample pretreatment
B. Direct vs indirect methods
1) Direct Methods
i. Phosphines-based detection method
2) Indirect methods
i. Colorimetric (Saville reaction)
ii. Fluorescence detection
iii. Chemiluminescence
iv. Biotin Switch Assay (BSA) and derived methods
v. Electrochemistry
3) Separation techniques (HPLC, GC, CE) coupled to direct or indirect methods
III. Miniaturization and microfluidics
A. Introduction
B. RSNOs detection using microsystems
C. Materials for microfluidic devices
1) Silicon and glass
2) Polymers
3) Paper
4) Comparison
D. Separation on microfluidic devices
1) Microchip liquid chromatography
2) Microchip capillary electrophoresis (MCE)
i. Injection techniques in MCE
a) Floating injection
b) Pinched injection
c) Gated injection
ii. Detection techniques
a) Optical detection methods
b) Mass spectrometry
c) Electrochemical detection methods
Chapter II: Analysis of GSNO decomposition and reactivity by capillary electrophoresis: kinetics and decomposition products identification
I. EC and MS techniques for the analysis of decomposition products of GSNO at solid state 122
A. Experimental
1) Chemicals
2) Sample synthesis
3) CE apparatus and measurements
4) MS detection
B. Results and discussion
C. Conclusion
II. EC and C4D for the analysis of the decomposition of GSNO solution under light and heat 132
A. Experimental
1) Samples, reagents and solutions
2) Capillary Electrophoresis Instrumentation
3) Decomposition and transnitrosation protocols
B. Results and discussion
1) Characterization of GSNO sample
2) Decomposition of GSNO using light.
3) Decomposition by heat
4) Transnitrosation reaction between GSNO and Cysteine
C. Concluding remarks
Chapter III: Decomposition of S-nitrosoglutathione by Cu2+ / GSH and by gold nanoparticles
I. Quantitation of S-nitrosoglutathione using Saville and electrochemical detection upon its Cu+-catalyzed decomposition
A. Experimental
1) Chemicals
2) Microsensor fabrication and NO detection
3) Colometric assays
B. Results and discussion
C. Conclusion
II. Quantification of GSNO using gold nanoparticles
A. Experimental section
1) Materials.
2) Preparation of gold nanoparticles.
3) S-nitrosoglutathione synthesis.
4) Reconstituted human and mice plasma manipulation.
5) Preparation of the NO selective Pt ultramicroelectrode (UME).
6) Amperometric detection of NO.
B. Results and discussion
1) Effect of AuNPs on the GSNO quantification.
2) Effect of plasma thiols on RSNOs quantification.
3) Detection of total RSNOs in plasma.
C. Conclusion of part II
III. Conclusion of chapter III
Chapter IV: Miniaturization
I. Colorimetric analysis of S-nitrosothiols decomposition on paper-based microfluidic devices
A. Experimental
1) Chemicals and materials
2) S-nitrosothiols synthesis
3) Fabrication of μPADs
4) Fabrication of a 3D printed holder
5) Lateral flow procedure and colorimetric analysis
6) Plasma RSNOs detection
B. Results and discussion
C. Conclusions
II. Electrochemical detection of RSNOs in electrophoretic micro device: preliminary studies
A. Experimental
1) Microchip configuration
i. PMMA microchip fabrication
ii. Commercial microchips
2) Operating conditions for the microchip electrophoresis of GSNO
i. PMMA and COC microchip
ii. Commercial glass microchip
3) Chemicals and GSNO synthesis
B. Results and discussions
1) Preliminary study
i. Employement of wireless potentiostat
ii. Optimization of the injection mode
iii. microchip with integrated electrodes
2) Application to the separation and quantitation of RSNOs
C. Conclusion
