Fanny d'Orlyé

Charge-based characterization of nanometric cationic bifunctional maghemite/silica core/shell particles by capillary zone electrophoresis.

In view of employing functionalized nanoparticles (NPs) in the context of an immunodiagnostic, aminated maghemite/silica core/shell particles were synthesized so as to be further coated with an antibody or an antigen via the amino groups at their surface. Different functionalization rates were obtained by coating these maghemite/silica core/shell particles with 3‐(aminopropyl)triethoxysilane and 2‐[methoxy(polyethyleneoxy)propyl]‐trimethoxysilane at different molar ratios. Adequate analytical performances with CE coupled with UV‐visible detection were obtained through semi‐permanent capillary coating with didodecyldimethyl‐ammonium bromide, thus preventing particle adsorption. First, the influence of experimental conditions such as electric field strength, injected particle amount as well as electrolyte ionic strength and pH, was evaluated. A charge‐dependent electrophoretic mobility was evidenced and the separation selectivity was tuned according to electrolyte ionic strength and pH. The best resolutions were obtained at pH 8.0, high ionic strength (ca. 100 mM), and low total particle volume fraction (ca. 0.055%), thus eliminating interference effects between different particle populations in mixtures. A protocol derived from Kaisers original description was performed for quantitation of the primary amino groups attached onto the NP surface. Thereafter a correlation between particle electrophoretic mobility and the density of amino groups at their surface was established. Eventually, CE proved to be an easy, fast, and reliable method for the determination of NP effective surface charge density.

Functionalization and characterization of persistent luminescence nanoparticles by dynamic light scattering, laser Doppler and capillary electrophoresis.

Zinc gallate nanoparticles doped with chromium (III) (ZnGa1.995O4:Cr0.005) are innovative persistent luminescence materials with particular optical properties allowing their use for in vivo imaging. They can be excited in the tissue transparency window by visible photons and emit light for hours after the end of the excitation. This allows to observe the probe without any time constraints and without autofluorescence signals produced by biological tissues. Modification of the surface of these nanoparticles is essential to be colloidally stable not only for cell targeting applications but also for proper distribution in living organisms. The use of different methods for controlling and characterizing the functionalization process is imperative to better understand the subsequent interactions with biological elements. This work explores for the first time the characterization and optimization of a classic functionalization sequence, starting with hydroxyl groups (ZGO-OH) at the nanoparticle surface, followed by an aminosilane-functionalization intermediate stage (ZGO-NH2) before PEGylation (ZGO-PEG). Dynamic light scattering and laser doppler electrophoresis were used in combination with capillary electrophoresis to characterize the nanoparticle functionalization processes and control their colloidal and chemical stability. The hydrodynamic diameter, zeta potential, electrophoretic mobility, stability over time and aggregation state of persistent luminescence nanoparticles under physiological-based solution conditions have been studied for each functional state. Additionally, a new protocol to improve ZGO-NH2 stability based on a thermal treatment to complete covalent binding of (3-aminopropyl) triethoxysilane onto the particle surface has been optimized. This thorough control increases our knowledge on these nanoparticles for subsequent toxicological studies and ultimately medical application.

Capillary electrophoresis coupled to contactless conductivity detection for the analysis of S-nitrosothiols decomposition and reactivity

S‐Nitrosothiols (RSNO) are composed of a NO group bound to the sulfhydryl group of a peptide or protein. RSNO are very important biological molecules, since they have many effects on human health. RSNO are easily naturally decomposed by metal ions, light, and heat, with different kinetics. They can furthermore undergo transnitrosation (NO moieties exchange), which is a crucial point in physiological conditions since the concentration ratios between the different nitrosothiols is a key factor in many physiopathological processes. There is therefore a great need for their quantitation. Many S‐nitrosothiol detection and quantitation methods need their previous decomposition, leading thus to some limitations. We propose a direct quantitation method employing the coupling of capillary electrophoresis with a homemade capacitively coupled contactless conductivity (C4D) detector in order to separate and quantify S‐nitrosoglutathione and its decomposition products. After optimization of the method, we have studied the kinetics of decomposition using light and heat. Our results show that the decomposition by light is first order (kobs = (3.40 ± 0.15) × 10−3 s−1) while that using heat (at 80°C) is zeroth order (kobs,80°C = (4.34 ± 0.14) × 10−6 mol L−1 s−1). Transnitrosation reaction between S‐nitrosoglutathione and cysteine was also studied, showing the possibility of separation and detection of all the products of this reaction in less than 2.5 min.

Colorimetric analysis of the decomposition of S-nitrosothiols on paper-based microfluidic devices

A disposable microfluidic paper-based analytical device (μPAD) was developed to easily analyse different S-nitrosothiols (RSNOs) through colorimetric measurements. RSNOs are carriers of nitric oxide (NO) that play several physiological and physiopathological roles. The quantification of RSNOs relies on their decomposition using several protocols and the colorimetric detection of the final product, NO or nitrite. μPADs were fabricated by wax printing technology in a geometry containing one central zone for the sample inlet and eight circular detection zones interconnected by microfluidic channels for decomposition and posterior detection of decayed products. Different decomposition protocols including mercuric ions and light (UV, visible, and infrared) were tested on μPADs. For this purpose, a 3D printed holder was coupled with μPADs to easily design a simultaneous decomposition procedure using different light sources. The Griess reagent was added to detect NO and nitrite produced by the different decomposition methods. μPADs were then scanned using a flat board scanner and calibration curves based on color intensity were plotted. The limit of detection (LOD) values achieved for nitrite (used as a reference compound) and S-nitrosoglutathione (GSNO) using mercuric decomposition were 3 and 4 μM, respectively. The LOD reported herein for nitrite is considered among the lowest LODs already reported for this compound using μPADs. The results also show that low-molecular-weight RSNO, namely S-nitrosocysteine, decomposes more easily than high-molecular-weight RSNOs with light. As a proof of concept, RSNOs in human plasma were successfully detected on μPADs. For this purpose, a preliminary treatment step was optimized and the presence of high-molecular-weight (HMW) RSNOs was evidenced in the available plasma samples. The concentrations of HMW-RSNOs and nitrite in the various samples ranged from 5 to 16 μM and from 37 to 58 μM, respectively.

Aptamer entrapment in microfluidic channel using one-step sol-gel process, in view of the integration of a new selective extraction phase for lab-on-a-chip

There is a great demand for integrating sample treatment into μTASs. In this context, we developed a new sol‐gel phase for extraction of trace compounds in complex matrices. For this purpose, the incorporation of aptamers in silica‐based gel within PDMS/glass microfluidic channels was performed for the first time by a one‐step sol‐gel process. The effective gel attachment onto microchannel walls and aptamer incorporation in the polymerized gel were evaluated using fluorescence microscopy. A good gel stability and aptamer incorporation inside the microchannel was demonstrated upon rinsing and over storage time. The ability of gel‐encapsulated aptamers to interact with its specific target (either sulforhodamine B as model fluorescent target, or diclofenac, a pain killer drug) was assessed too. The binding capacity of entrapped aptamers was quantified (in the micromolar range) and the selectivity of the interaction was evidenced. Preservation of aptamers binding affinity to target molecules was therefore demonstrated. Dissociation constant of the aptamer‐target complex and interaction selectivity were evaluated similar to those in bulk solution. This opens the way to new selective on‐chip SPE techniques for sample pretreatment.