Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Université de Bourgogne Franche-Comté

FEMTO-ST Institute

Since it was demonstrated that nanostructured surfaces are more efficient for the detection based on the specific capture of analytes, there is a real need to develop strategies for grafting nanoparticles onto flat surfaces. Among the different routes for the functionalization of a surface, the reduction of diazonium salts appears very attractive for the covalent immobilization of nanoparticles because this method does not require a pre-treatment of the surface. For achieving this goal, gold nanoparticles coated by precursor of diazonium salts were synthesized by reduction of gold salt in presence of mercaptoaniline. These mercaptoaniline-coated gold nanoparticles (Au@MA) were successfully immobilized onto various conducting substrates (indium tin oxide (ITO), glassy carbon (GC) and gold electrodes with flat terraces) after addition of sodium nitrite at fixed potential. When applied onto the gold electrodes, such a grafting strategy led to an obvious enhancement of the luminescence of luminol used for the biodetection.

Enhanced chemiluminescence-based detection on gold substrate after electrografting of diazonium precursor-coated gold nanoparticles.

Estrothiazine (ESTZ) is a weak estrogen sharing structural similarities with coumestrol. ESTZ failed to compete with [H]17β-estradiol ([H]17β-E) for binding to the estrogen receptor α (ERα), questioning its ability to interact with the receptor. However, detection by atomic force spectroscopy (AFS) of an ESTZ-induced ERα dimerization has eliminated any remaining doubts. The effect of the compound on the proliferation of ERα-positive and negative breast cancer cells confirmed the requirement of the receptor. The efficiency of ESTZ in MCF-7 cells was weak without any potency to modify the proliferation profile of estradiol and coumestrol. Growth enhancement was associated with a proteasomal degradation of ERα without substantial recruitment of LxxLL coactivators. This may be related to an unusual delay between the acquisition by the receptor of an ERE-binding capacity and the subsequent estrogen-dependent transcription. A complementary ability to enhance TPA-induced AP-1 transcription was observed, even at concentrations insufficient to activate the ERα, suggesting a partly independent mechanism. ESTZ also rapidly and transiently activated ERK1/2 likely through membrane estrogenic pathways provoking a reorganization of the actin network. Finally, the systematic absence of biological responses with an ESTZ derivative unable to induce ERα dimerization stresses the importance of this step in the action of the compound, as reported for conventional estrogens. In view of the existence of many other ERα modulators (endocrine disruptors such as, for example, pesticides, environmental contaminants or phytoestrogens) with extremely weak or similar apparent lack of binding ability, our work may appear as pilot investigation for assessing their mechanism of action.

ERα dimerization: a key factor for the weak estrogenic activity of an ERα modulator unable to compete with estradiol in binding assays.

Widely used in microelectronics and optoelectronics; Gallium Arsenide (GaAs) is a III-V crystal with several interesting properties for microsystem and biosensor applications. Among these; its piezoelectric properties and the ability to directly biofunctionalize the bare surface, offer an opportunity to combine a highly sensitive transducer with a specific bio-interface; which are the two essential parts of a biosensor. To optimize the biorecognition part; it is necessary to control protein coverage and the binding affinity of the protein layer on the GaAs surface. In this paper; we investigate the potential of a specific chemical interface composed of thiolate molecules with different chain lengths; possessing hydroxyl (MUDO; for 11-mercapto-1-undecanol (HS(CH₂)OH)) or carboxyl (MHDA; for mercaptohexadecanoic acid (HS(CH₂)CO₂H)) end groups; to reconstitute a dense and homogeneous albumin (Rat Serum Albumin; RSA) protein layer on the GaAs (100) surface. The protein monolayer formation and the covalent binding existing between RSA proteins and carboxyl end groups were characterized by atomic force microscopy (AFM) analysis. Characterization in terms of topography; protein layer thickness and stability lead us to propose the 10% MHDA/MUDO interface as the optimal chemical layer to efficiently graft proteins. This analysis was coupled with  MALDI-TOF mass spectrometry measurements; which proved the presence of a dense and uniform grafted protein layer on the 10% MHDA/MUDO interface. We show in this study that a critical number of carboxylic docking sites (10%) is required to obtain homogeneous and dense protein coverage on GaAs. Such a protein bio-interface is of fundamental importance to ensure a highly specific and sensitive biosensor.

Influence of a Thiolate Chemical Layer on GaAs (100) Biofunctionalization: An Original Approach Coupling Atomic Force Microscopy and Mass Spectrometry Methods.

We report on the formation kinetics of mixed self-assembled monolayers (SAMs) comprising 16-mercaptohexadecanoic acid (MHDA) and 11-mercapto-1-undecanol (MUDO) thiols on GaAs(100) substrates. These compounds were selected for their potential in constructing highly selective and efficient architectures for biosensing applications. The molecular composition and quality of one-compound and mixed SAMs were determined by the Fourier transform infrared absorption spectroscopy measurements. The formation of enhanced-quality mixed SAMs was investigated as a function of the molecular composition of the thiol mixture and the proportion of ethanol/water solvent used during their arrangement. Furthermore, the formation of mixed SAMs has been carried out by successive immersion of MHDA SAMs in MUDO thiol solutions and MUDO SAMs in MHDA thiol solution through the process involving thiol-thiol substitution. Our results, in addition to confirming that water-ethanol-based solvents improve the packing density of single thiol monolayers, demonstrate the attractive role of water-ethanol solvents in forming superior quality mixed SAMs.

Formation Kinetics of Mixed Self-Assembled Monolayers of Alkanethiols on GaAs(100).

N-cadherin is a transmembrane glycoprotein expressed by mesenchymal origin cells and is located at the adherens junctions. It regulates also cell motility and contributes to cell signaling. In previous studies, we identified that its anomalous expression in bladder carcinoma was a tumor progression marker. A pharmacological approach to inhibit N-cadherin expression or to block its function could be relevant to prevent disease progression and metastasis development. The morphological exploration of T24 invasive bladder cancer cells by atomic force microscopy (AFM) revealed a spindle-like shape with fibrous structures. By engaging force spectroscopy with AFM tip functionalized with anti-E or anti-N-cadherin antibodies, results showed that T24 cells expressed only N-cadherin as also demonstrated by Western blotting and confocal microscopy. For the first time, we demonstrated by RTqPCR and Western blotting analyses that the peroxisome proliferator-activated receptor β/δ (PPARβ/δ) agonist GW501516 significantly decreased N-cadherin expression in T24 cells. Moreover, high non-cytotoxic doses of GW501516 inhibited confluent T24 cell wound healing closure. By using AFM, a more sensitive nanoanalytical method, we showed that the treatment modified the cellular morphology and diminished N-cadherin cell surface coverage through the decreasing of these adhesion molecule-mediated interaction forces. We observed a greater decrease of N-cadherin upon GW501516 exposure with AFM than that detected with molecular biology techniques. AFM was a complementary tool to biochemical techniques to perform measurements on living cells at the nanometer resolution level. Taken together, our data suggest that GW501516 could be an interesting therapeutic strategy to avoid bladder cancer cell spreading through N-cadherin decrease.

Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure.

A regenerable bulk acoustic wave (BAW) biosensor is developed for the rapid, label-free and selective detection of  in liquid media. The geometry of the biosensor consists of a GaAs membrane coated with a thin film of piezoelectric ZnO on its top surface. A pair of electrodes deposited on the ZnO film allows the generation of BAWs by lateral field excitation. The back surface of the membrane is functionalized with alkanethiol self-assembled monolayers and antibodies against . The antibody immobilization was investigated as a function of the concentration of antibody suspensions, their pH and incubation time, designed to optimize the immunocapture of bacteria. The performance of the biosensor was evaluated by detection tests in different environments for bacterial suspensions ranging between 10 and 10 CFU/mL. A linear dependence between the frequency response and the logarithm of  concentration was observed for suspensions ranging between 10 and 10 CFU/mL, with the limit of detection of the biosensor estimated at 10 CFU/mL. The 5-fold regeneration and excellent selectivity towards  detected at 10 CFU/mL in a suspension tinted with  at 10 CFU/mL illustrate the biosensor potential for the attractive operation in complex biological media.

Regenerable ZnO/GaAs Bulk Acoustic Wave Biosensor for Detection of  in "Complex" Biological Medium.

Extracellular vesicles (EV) are emergent therapeutic effectors that have reached clinical trial investigation. To translate EV-based therapeutic to clinic, the challenge is to demonstrate quality, safety, and efficacy, as required for any medicinal product. EV research translation into medicinal products is an exciting and challenging perspective. Recent papers, provide important guidance on regulatory aspects of pharmaceutical development, defining EVs for therapeutic applications and critical considerations for the development of potency tests. In addition, the ISEV Task Force on Regulatory Affairs and Clinical Use of EV-based Therapeutics as well as the Exosomes Committee from the ISCT are expected to contribute in an active way to the development of EV-based medicinal products by providing update on the scientific progress in EVs field, information to patients and expert resource network for regulatory bodies. The contribution of our work group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France", created in 2020, can be positioned in complement to all these important initiatives. Based on complementary scientific, technical, and medical expertise, we provide EV-specific recommendations for manufacturing, quality control, analytics, non-clinical development, and clinical trials, according to current European legislation. We especially focus on early phase clinical trials concerning immediate needs in the field. The main contents of the investigational medicinal product dossier, marketing authorization applications, and critical guideline information are outlined for the transition from research to clinical development and ultimate market authorization.

Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France".

Extracellular vesicles (EVs) originate from eukaryotic and prokaryotic cells and play a crucial role in intercellular communications. They are found in the environment of cells and tissues, and contribute to the complexity of different biological media, in particular biofluids. Due to their high diversity of cell origin, size range, concentration and composition, EVs offer some of the most important challenges in (pre-)analytical fields. To tackle these challenges, many works deal with the development and implementation of a wide variety of approaches, technologies and methodologies to enrich, isolate, quantify and characterize EVs and their subsets. Nevertheless, other components such as lipoproteins or viruses in complex samples, can interfere with EVs qualification, and make difficult, even today, to standardize biochemical and physical approaches for this purpose. The present chapter presents EVs and the mostly used technics for their isolation and characterization. Performances of methods in terms of resolution, discrimination, throughput and also ability to be or not applied in clinics, are also discussed.

[Extracellular vesicles: Definition, isolation and characterization].

Microfluidics integration of acoustic biosensors is an actively developing field. Despite significant progress in "passive" microfluidic technology, integration with microacoustic devices is still in its research state. The major challenge is bonding polymers with monocrystalline piezoelectrics to seal microfluidic biosensors. In this contribution, we specifically address the challenge of microfluidics integration on gallium arsenide (GaAs) acoustic biosensors. We have developed a robust plasma-assisted bonding technology, allowing strong connections between PDMS microfluidic chip and GaAs/SiO at low temperatures (70 °C). Mechanical and fluidic performances of fabricated device were studied. The bonding surfaces were characterized by water contact angle measurement and ATR-FTIR, AFM, and SEM analysis. The bonding strength was characterized using a tensile machine and pressure/leakage tests. The study showed that the sealed chips were able to achieve a limit of high bonding strength of 2.01 MPa. The adhesion of PDMS to GaAs was significantly improved by use of SiO intermediate layer, permitting the bonded chip to withstand at least 8.5 bar of burst pressure. The developed bonding approach can be a valuable solution for microfluidics integration in several types of MEMS devices.

Celine Elie-Caille

FEMTO-ST Institute,
CNRS UMR-6174

Université de Bourgogne Franche-Comté

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Celine Elie-Caille

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}FEMTO-ST Institute,CNRS UMR-6174

Université de Bourgogne Franche-Comté

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

FEMTO-ST Institute,
CNRS UMR-6174