Project Topics
Get Complete Project Material File(s) Now! »
Detection methods for ROS/RNS
Due to the apparent ubiquity and importance of ROS and RNS, it is of great interest to monitor their production during oxidative stress. Indeed, rather than the long-term observation of patients’ metabolites, direct measurements are much preferred in deciphering the intricate mechanisms at different oxidative stress stages. Despite the difficulties of subtle generation and transient existence of those active species, highly-sensitive and selective analytical methods have undergone great progress and some of which are introduced in the following context.
Some general analytical methods
Two types of fluorescent probes are commonly adapted in this domain, namely “oxidant-sensitive” or “non-redox” indicators [30]. The former dyes comprise aromatic compounds which can be oxidized by free radicals and then generate fluorescent end products (Figure A-6A). The other probes release masked fluorophores after oxidants’ attack on the blocking groups (Figure A-6B). The “oxidant-sensitive” dyes can detect samples at very low concentration (several to tens of nanomolar [31-33]) but react simultaneously with various oxidants, providing the global cellular redox activity rather than specifically for one analyte. In addition, misinterpreted signals usually result from unstable intermediates, side production, and autofluorescence. Famous fluorescent probes of this type include 2’,7’-dichlorodihydrofluorescein (DCFH, Figure A-7), dihydrorhodamine 123 (DHR 123, targets to H2O2, HOCl and ONOO–), dihydroethidium (DHE, targets to O●2–) and diaminofluorescein (DAF, targets to NO• [33-35]).
Chemiluminescence method
Chemiluminescence detection consists in a probe or an enhancer that provokes light emitting during oxidant reaction (Figure A-9). It is highly sensitive but has the same limitations as fluorescence method issued from the specificity and multi-step radical mechanisms.
Luminol (LM) is the most famous chemiluminescent probe which has been reported to selectively detect ROS/RNS in activated phagocytes (detection limit down to the range of femtomolar) [31]. But it cannot identify the oxidants or provide mechanistic information since a host of active species (e.g., OH•, O●2–, ONOO– or H2O2 plus peroxidase) can similarly initiate oxidation step and offer luminescence signals (Figure A-10). At the same time, luminol radicals can reduce O2 into O●2–, acting both the source and detector of O●2– and thus inevitably leading to artifactual results. Lucigenin (LC) is often thought more specific for O●2– [39], but neither react directly with O●2–. It must be first reduced to cation radical (LC•+) which then initiate reaction to generate luminescence. Some more promise and specific probes such as coelenterazine and analogues of LM and LC [40] still remain the same issues of specificity and interference, since no matter in which case, an initial oxidation step and subsequent radical reactions are involved.
Electron spin resonance (ESR) spectroscopy
ESR spectroscopy is used for measuring paramagnetic species through their transition of spin states under magnetic field (Figure A-11). For the detection of oxidative species, specific complexes “spin traps” [42] are needed to convert radicals to long-lived spin-adducts. Characteristic and quantitative information is then acquired from spectra of those stable adducts.
Figure A-11: Illustration of electron spin resonance method [43]. (A) Principle of energy levels splitting directly proportional to the magnetic field’s strength. An unpaired electron can only move between magnetic moment ms = +1/2 and ms = -1/2. (B) One example of resulting ESR spectrum. The most frequently used spin traps include pyrroline-based cyclic nitrones (e.g., 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) [44]) which react with OH• and O●2– to generate –OH and –OOH adducts; and colloid iron (II) diethyldithiocarbamate (Fe2+-DETC) [45] which is specific for bioactive NO•.
ESR spin trapping offers higher selectivity compared with previous optical methods. From the adduct spectrum, identification, concentration, and even the relevant reaction kinetics of free radicals could be obtained. However, it is not directly available for the oxidants without unpaired electrons (e.g., H2O2 and ONOO–). Some additional limitations also hamper its application in living cells, such as adverse alteration of conjugates (by endogenous metabolites), long recording times, expensive and cumbersome setup, as well as low-temperature usually required during measurement.
Ultraviolet-visible (UV-Vis) spectroscopy
UV-Vis spectroscopy provides a direct detection method of ONOO – . Unlike fluorescence or chemiluminescence detection, it does not need a probe compound; neither like SER, there is no requirement of the presence of unpaired electron). ONOO– can be easily quantified by measuring absorbance intensity at 302 nm (ɛ = 1705 M-1 cm-1)[46,47]. However, experiments are usually performed in basic solution since adequate species stability is guaranteed in such environment. Indeed, for most oxidants, special techniques are needed to circumvent their transient nature and to induce light absorbing property. One famous assay is the detection of NO• from its oxidation product NO–2 [48].
As demonstrated in Figure A-12, NO–2 reacts with the Griess reagents (sulfanilamide and N-1-napthylethylenediamine) to form a red-pink color azo dye which is then monitored spectroscopically at 540 nm. These series of chemical reactions and indirect nature of detection forbid real-time analysis of NO• generation, despite the low price and simple execution. In addition, precaution should be taken in the quantitative analysis since NO–2 from other resources can lead to the overestimation of NO•. Sensitivity of this method is highly dependent on solution composition. In ultrapure deionized distilled water, detection limit for commercially available Griess reagent kits is roughly 2.5M.
Electrochemical method
Electrochemistry has been identified since centuries and is widely accepted as a scientific branch to investigate the processes of electron-transfer-involved chemical reactions (the fundamental electrochemical principles and techniques are introduced in Appendix I.1 and I.2). Electrochemical methods solve many constraints described with previous approaches owing to the intrinsic electroactive properties of several important oxidative species. Therefore, a direct and label free investigation is allowed to study oxidative stress at its very origin.
Detection of ROS/RNS is carried out via either reduction or oxidation at the working electrode. Electro-reduction is promising but inevitably suffers from interference of oxygen in aerobic environments. Electro-oxidation is preferred in general; however, high overpotential usually precludes the accurate detection since a global signal from all feasible species is measured rather than from one particular analyte. Moreover, the traditional bare electrodes (e.g., carbon, platinum and gold) are quite easily passivated in biological medium, resulting in unstable results.
To improve the selectivity and sensitivity, functional materials were developed to modify electrode surface (Figure A-14) [5,52-54]. Immobilization of specific biomolecules on electrode surface is commonly used; specificity of this sensor is linked to reaction between analyte and those biological substances. The permselective membranes prevent diffusion of undesired molecules, by size or charge exclusion (e.g., Nafion®), to electrode surface. Thin layer of metal-containing catalytic complexes are tunably electrodeposited to increase electron-transfer kinetics and negatively shift the detection potential. More recently, metal nanoparticles attract great interest because of their favored chemical, physical and electronic properties than usual bulk materials. Improved electrons transfer as well as better surface stability is often observed at metal-particles deposited electrodes. Multiple modification layers are sometimes incorporated to minimize diverse interference; but usually at the expense of decreasing sensitivity and prolonging respond time.
In the following context, some widely used electrochemical sensors are introduced, with special attention paid to the two primary radicals (O●2– and NO•) and the three main electroactive derivatives (H2O2, ONOO– and NO–2 ).
Superoxide anion (O●2–) sensors
O ●2– is an “easily-detected” target due to the low oxidation potential at ordinary electrode surface; whereas better performances can be achieved by coating biomolecules such as cytochrome c (cyt c) or, more elegantly, its natural scavenger superoxide dismutase (SOD) [52,55,56].
As shown in Figure A-ൈ15A, Fe(III)-cyt c can be immobilized on gold or platinum surface, reduced by O●2– (k = 2 106 M-1 s-1) and then immediately re-oxidized at electrode surface at a quite low potential (15 – 25 mV vs. Ag/AgCl (silver-silver chloride reference electrode)). The oxidation current of cyt c constitutes analytical response for continuous O●2– in bulk solution. On the other side, SOD-based biosensor works through converting O●2– to H2O2 and then oxidizing this stable product (Figure A-15B). Nevertheless, this sensor may encounter the difficulty in distinguishing the previously existed H2O2 (by natural disproportionation process).
Table of contents :
ABBREVIATIONS
RÉSUMÉ DE LA THÈSE (FRENCH ABSTRACT)
GENERAL INTRODUCTION
PART A: STATE-OF-THE-ART
1. OXIDATIVE STRESS AND REACTIVE OXYGEN/NITROGEN SPECIES (ROS/RNS)
1.1. Natural process of oxidative stress
1.2. Roles of ROS/RNS in life
1.2.1. Beneficial effects
1.2.2. Deleterious effects
1.3. Conclusion of section 1
2. DETECTION METHODS FOR ROS/RNS
2.1. Some general analytical methods
2.1.1. Fluorescence method
2.1.2. Chemiluminescence method
2.1.3. Electron spin resonance (ESR) spectroscopy
2.1.4. Ultraviolet-visible (UV-Vis) spectroscopy
2.1.5. Biomarkers method
2.2. Electrochemical method
2.2.1. Superoxide anion (O●– 2) sensors
2.2.2. Hydrogen peroxide (H2O2) sensors
2.2.3. Nitric oxide (NO•) sensors
2.2.4. Peroxynitrite (ONOO– ) sensors
2.2.5. Nitrite ion (NO– 2) sensors
2.2.6. Platinum black (Pt-black) coated electrodes
2.3. Conclusion of section 2
3. ANALYTICAL PROGRESS IN EX VIVO DETECTION OF CELLULAR ELECTROACTIVE MESSENGERS
3.1. Single-cell analysis by using microelectrodes
3.1.1. Microelectrodes and their advantages
3.1.2. Detection of electroactive messengers from a single living cell
3.2. Microfluidic devices as functional tools for cell-based analysis
3.2.1. Cell culture in microfluidic devices
3.2.2. Cell manipulation in microfluidic devices
3.2.3. Cell-based analysis on microfluidic platforms
3.3. Conclusion of section 3
4. CONCLUSION OF STATE-OF-THE-ART AND PERSPECTIVES
PART B: IN VITRO ELECTROCHEMICAL DETECTION OF ROS/RNS AT HIGHLY SENSITIVE PT/PT-BLACK MICROBAND ELECTRODE INSIDE MICROFLUIDIC SYSTEM
1. CONVECTIVE-DIFFUSIVE MASS TRANSPORT ABOVE PT/PT-BLACK MICROCHANNEL ELECTRODE
1.1. Mass transport regimes above a single microband electrode
1.2. Choice of mass transport regime in our microfluidic device
1.3. Optimization and evaluation of Pt-black deposit
1.4. Conclusion of section 1
2. ROS/RNS IN VITRO DETECTION AT PT/PT-BLACK MICROBAND ELECTRODE
2.1. In vitro detection of two stable candidates: H2O2 and NO– 2
2.1.1. Current responses at Pt/Pt-black electrode
2.1.2. Oxidation mechanisms of H2O2 and NO– 2
2.1.3. Calibration curve and electrode sensitivity
2.2. In vitro detection of two unstable candidates: ONOO– and NO•
2.2.1. Detection of ONOO–
2.2.2. Detection of NO•
2.3. In vitro detection of samples mixture
2.4. Conclusion of section 2
3. CONCLUSION OF PART B
PART C: MONITORING OF CELLULAR OXIDATIVE STRESS BY PT/PT-BLACK ELECTRODES-INTEGRATED MICROSYSTEMS
1. INVESTIGATION OF OXIDATIVE STRESS IN LABORATORY
1.1. Cell models
1.2. Cell stimulations
1.2.1. Mechanical penetration
1.2.2. Biochemical stimulation
1.3. Conclusion of section 1
2. DETECTION OF OXIDATIVE BURSTS FROM CELLS POPULATIONS ON BASAL MICROBAND ELECTRODE
2.1. Experimental conditions
2.1.1. Microdevice configuration
2.1.2. Cells viability and density
2.2. Qualitative detection of ROS and RNS production
2.2.1. Characteristics of amperometric responses
2.2.2. Cells variations and successive releases
2.2.3. Cells detachment and chip reuse
2.3. Quantification of ROS and RNS production
2.3.1. Evaluation of fluxes and quantities of ROS and RNS production
2.3.2. Evaluation of primary production of O●– 2 and NO•
2.4. Conclusion of section 2
3. DETECTION OF OXIDATIVE BURSTS FROM CELLS POPULATION ON DOWNSTREAM MICROBAND ELECTRODES
3.1. Experimental conditions
3.1.1. Microdevice configuration
3.1.2. Cells manipulation inside microdevice
3.2. Detection of ROS and RNS production
3.2.1. Detection reproducibility between parallel electrodes
3.2.2. Detection reliability after biocompatible coating
3.2.3. Detection during continuous flow
3.2.4. Detection after 10-min stop flow
3.3. Conclusion of section 3
4. CONCLUSION OF PART C
GENERAL CONCLUSION AND PERSPECTIVES
APPENDIX
APPENDIX I. ELECTROCHEMICAL PRINCIPLES AND MICROELECTRODES
I.1. Electrochemical principles
I.2. General electrochemical techniques
I.3. Microelectrodes and their electrochemical performances
APPENDIX II. MICROFLUIDIC MATERIALS AND MICROFABRICATION
II.1. Materials
II.2. Microfabrication techniques
APPENDIX III. EXPERIMENTAL SECTION
III.1. Solutions preparation
III.2. Sensors preparation
III.3. Raw 264.7 macrophages preparation
III.4. Analytical measurements
REFERENCES
