Inferring the Interfacial Reactivity of Gold Nanoparticles by Surface Plasmon Resonance Measurements.

Gold nanoparticles (GNPs) require a functionalization step in most cases to be suitable for applications. Optimizing this step in order to maintain both the stability and the plasmonic properties of the GNPs is a demanding process. Indeed, multiple analyses are required to get sufficient information on the grafting rate and the stability of the obtained suspension, leading to material and time waste. In this study, we propose to investigate ligand reactivity on a gold surface with surface plasmon resonance (SPR) measurements as a way to simulate the reactivity in GNP suspensions. We consider two thiolated ligands in this work: thioglycolic acid (TA) and 6-mercaptohexanoic acid (MHA). These thiols are grafted using different conditions on GNPs (monitored by optical absorption) and on a gold surface (monitored by SPR) and the grafting efficiency and stability are compared. The same conclusions are reached in both cases regarding the best protocol to implement, namely, the thiol molecules should be introduced in a water solution at a low concentration. This demonstrates the suitability of SPR to predict the reactivity on a GNP surface.

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 and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential.

AB186 Inhibits Migration of Triple-Negative Breast Cancer Cells and Interacts with α-Tubulin.

Breast cancer is one of the leading causes of cancer-related death among females worldwide. A major challenge is to develop innovative therapy in order to treat breast cancer subtypes resistant to current treatment. In the present study, we examined the effects of two Troglitazone derivatives Δ2-TGZ and AB186. Previous studies showed that both compounds induce apoptosis, nevertheless AB186 was a more potent agent. The kinetic of cellular events was investigated by real-time cell analysis system (RTCA) in MCF-7 (hormone dependent) and MDA-MB-231 (triple negative) breast cancer (TNBC) cells, followed by cell morphology analysis by immuno-localization. Both compounds induced a rapid modification of both impedance-based signals and cellular morphology. This process was associated with an inhibition of cell migration measured by wound healing and transwell assays in TNBC MDA-MB-231 and Hs578T cells. In order to identify cytoplasmic targets of AB186, we performed surface plasmon resonance (SPR) and pull-down analyses. Subsequently, 6 cytoskeleton components were identified as potential targets. We further validated α-tubulin as one of the direct targets of AB186. In conclusion, our results suggested that AB186 could be promising to develop novel therapeutic strategies to treat aggressive forms of breast cancer such as TNBC.

Epidemiological Impact of GII.17 Human Noroviruses Associated With Attachment to Enterocytes.

For the last 30 years, molecular surveys have shown that human norovirus (HuNoV), predominantly the GII.4 genotype, is one of the main causative agents of gastroenteritis. However, epidemiological surveys have revealed the worldwide emergence of GII.17 HuNoVs. Genetic analysis confirmed that GII.17 strains are distributed into three variants (i.e., Kawasaki 308, Kawasaki 323, and CS-E1). Here, virus-like particles (VLPs) were baculovirus-expressed from these variants to study putative interactions with HBGA. Qualitative analysis of the HBGA binding profile of each variant showed that the most recent and predominant GII.17 variant, Kawasaki 308, possesses a larger binding spectrum. The retrospective study of GII.17 strains documented before the emergence of the dominant Kawasaki 308 variant showed that the emergence of a new GII.17 variant could be related to an increased binding capacity toward HBGA. The use of duodenal histological sections confirmed that recognition of enterocytes involved HBGA for the three GII.17 variants. Finally, we observed that the relative affinity of recent GII.17 VLPs for HBGA remains lower than that of the GII.4-2012 variant. These observations suggest a model whereby a combination of virological factors, such as polymerase fidelity and increased affinity for HBGA, and immunological factors was responsible for the incomplete and non-persistent replacement of GII.4 by new GII.17 variants.

[Extracellular vesicles: Definition, isolation and characterization]. / Les vésicules extracellulaires - Définition, séparation, caractérisation.

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.

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

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.

Assessment of Shear-Dependent Kinetics of Primary Haemostasis With a Microfluidic Acoustic Biosensor.

Primary haemostasis is a complex dynamic process, which involves in-flow interactions between platelets and sub-endothelial matrix at the area of the damaged vessel wall. It results in a first haemostatic plug, which stops bleeding, before coagulation ensues and consolidates it. The diagnosis of primary haemostasis defect would benefit from evaluation of the whole sequence of mechanisms involved in platelet plug formation in flow. This work proposes a new approach that is based on characterization of the shear-dependent kinetics that enables the evaluation of the early stages of primary haemostasis. We used a label-free method with a quartz crystal microbalance (QCM) biosensor to measure the platelet deposits over time onto covalently immobilized type I fibrillar collagen. We defined three metrics: total frequency shift, lag time, and growth rate. The measurement was completed at four predefined shear rates prevailing in small vessels (500, 770, 1000 and 1500 s-1) during five minutes of perfusion with anticoagulated normal whole blood. The rate of the frequency shift over the first five minutes was strongly influenced by shear rate conditions, presenting a maximum around 770 s-1, and varying by a factor larger than three in the studied shear rate range. To validate the biosensor signal, the total frequency shift was compared to results obtained by atomic force microscopy (AFM) on final platelet deposits. The results show that shear-dependent kinetic assays are promising as an advanced method for screening of primary haemostasis.

Topology Challenge for the Assessment of Living Cell Deposits with Shear Bulk Acoustic Biosensor.

Shear bulk acoustic type of resonant biosensors, such as the quartz crystal microbalance (QCM), give access to label-free in-liquid analysis of surface interactions. The general understanding of the sensing principles was inherited from past developments in biofilms measurements and applied to cells while keeping the same basic assumptions. Thus, the biosensor readouts are still quite often described using 'mass' related terminology. This contribution aims to show that assessment of cell deposits with acoustic biosensors requires a deep understanding of the sensor transduction mechanism. More specifically, the cell deposits should be considered as a structured viscoelastic load and the sensor response depends on both material and topological parameters of the deposits. This shifts the paradigm of acoustic biosensor away from the classical mass loading perspective. As a proof of the concept, we recorded QCM frequency shifts caused by blood platelet deposits on a collagen surface under different rheological conditions and observed the final deposit shape with atomic force microscopy (AFM). The results vividly demonstrate that the frequency shift is highly impacted by the platelet topology on the bio-interface. We support our findings with numerical simulations of viscoelastic unstructured and structured loads in liquid. Both experimental and theoretical studies underline the complexity behind the frequency shift interpretation when acoustic biosensing is used with cell deposits.

NanoBioAnalytical characterization of extracellular vesicles in 75-nm nanofiltered human plasma for transfusion: A tool to improve transfusion safety.

Plasma transfusion induces some transfusion related acute lung injury (TRALI) mediated through neutrophil extracellular traps (NETs). We investigated whether extracellular vesicles (EVs) present in plasma or obtained from resting (N-PEVs) or thrombin activated platelets (T-PEVs) can trigger NETs, and whether 75 nm-nanofiltration, to partially remove EVs, prohibits NETs formation. EVs size and concentration were determined by conventional biophysical approaches and by an original NanoBioAnalytical (NBA) platform based on EV immunocapture biochip, combining Surface Plasmon Resonance Imaging (SPRi) and Atomic Force Microscopy (AFM) exploration. EVs effective diameter was in the 25-1000 nm range, with a majority (≈ 90%) ≤ 100 nm. Both T-PEVs in buffer (but not N-PEVs) and non-nanofiltered plasma containing T-PEVs triggered NETs formation. Nanofiltration depleted large EVs (> 70 nm) and decreased NETs formation. The NBA platform was found to be a suitable tool to investigate the safety of plasma for transfusion.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Development of a NanoBioAnalytical platform for "on-chip" qualification and quantification of platelet-derived microparticles.

Blood microparticles (MPs) are small membrane vesicles (50-1000nm), derived from different cell types. They are known to play important roles in various biological processes and also recognized as potential biomarkers of various health disorders. Different methods are currently used for the detection and characterization of MPs, but none of these methods is capable to quantify and qualify total MPs at the same time, hence, there is a need to develop a new approach for simultaneous detection, characterization and quantification of microparticles. Here we show the potential of surface plasmon resonance (SPR) method coupled to atomic force microscopy (AFM) to quantify and qualify platelet-derived microparticles (PMPs), on the whole nano-to micro-meter scale. The different subpopulations of microparticles could be determined via their capture onto the surface using specific ligands. In order to verify the correlation between the capture level and the microparticles concentration in solution, two calibration standards were used: Virus-Like Particles (VLPs) and synthetic beads with a mean diameter of 53nm and 920nm respectively. The AFM analysis of the biochip surface allowed metrological analysis of captured PMPs and revealed that more than 95% of PMPs were smaller than 300nm. Our results suggest that our NanoBioAnalytical platform, combining SPR and AFM, is a suitable method for a sensitive, reproducible, label-free characterization and quantification of MPs over a wide concentration range (≈107 to 1012 particles/mL; with a limit of detection (LOD) in the lowest ng/µL range) which matches with their typical concentrations in blood.

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

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.

Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation.

UNLABELLED: Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF(4-23) mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression. IMPORTANCE: The bacterium Pseudomonas aeruginosa is a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites of P. aeruginosa infection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this study, we showed that the CNP hormone counteracts P. aeruginosa biofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein, which is an ortholog of the human receptor NPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factor production while reducing biofilm formation.

Chemotherapy-triggered cathepsin B release in myeloid-derived suppressor cells activates the Nlrp3 inflammasome and promotes tumor growth.

Chemotherapeutic agents are widely used for cancer treatment. In addition to their direct cytotoxic effects, these agents harness the host's immune system, which contributes to their antitumor activity. Here we show that two clinically used chemotherapeutic agents, gemcitabine (Gem) and 5-fluorouracil (5FU), activate the NOD-like receptor family, pyrin domain containing-3 protein (Nlrp3)-dependent caspase-1 activation complex (termed the inflammasome) in myeloid-derived suppressor cells (MDSCs), leading to production of interleukin-1ß (IL-1ß), which curtails anticancer immunity. Chemotherapy-triggered IL-1ß secretion relied on lysosomal permeabilization and the release of cathepsin B, which bound to Nlrp3 and drove caspase-1 activation. MDSC-derived IL-1ß induced secretion of IL-17 by CD4(+) T cells, which blunted the anticancer efficacy of the chemotherapy. Accordingly, Gem and 5FU exerted higher antitumor effects when tumors were established in Nlrp3(-/-) or Casp1(-/-) mice or wild-type mice treated with interleukin-1 receptor antagonist (IL-1Ra). Altogether, these results identify how activation of the Nlrp3 inflammasome in MDSCs by 5FU and Gem limits the antitumor efficacy of these chemotherapeutic agents.

Immuno-MALDI-MS in human plasma and on-chip biomarker characterizations at the femtomole level.

Immuno-SPR-MS is the combination of immuno-sensors in biochip format with mass spectrometry. This association of instrumentation allows the detection and the quantification of proteins of interest by SPR and their molecular characterization by additional MS analysis. However, two major bottlenecks must be overcome for a wide diffusion of the SPR-MS analytical platform: (i) To warrant all the potentialities of MS, an enzymatic digestion step must be developed taking into account the spot formats on the biochip and (ii) the biological relevancy of such an analytical solution requires that biosensing must be performed in complex media. In this study, we developed a procedure for the detection and the characterization at ~1 µg/mL of the LAG3 protein spiked in human plasma. The analytical performances of this new method was established, particularly its specificity (S/N > 9) and sensitivity (100% of LAG3 identification with high significant mascot score >68 at the femtomole level). The collective and automated on-chip MALDI-MS imaging and analysis based on peptidic fragments opens numerous applications in the fields of proteomics and diagnosis.

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein.

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.

Neuropilin-2 expression promotes TGF-ß1-mediated epithelial to mesenchymal transition in colorectal cancer cells.

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-ß1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-ß1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFß-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFß-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-ß1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.

Adhesion forces controlled by chemical self-assembly and pH: application to robotic microhandling.

Robotic microhandling is a promising way to assemble microcomponents in order to manufacture a new generation of hybrid microelectromechanical systems. However, at the scale of several micrometers, the adhesion phenomenon highly perturbs the micro-object release and positioning. This phenomenon is directly linked to both the object and the gripper surface chemical composition. We propose to control the adhesion by using a chemical self-assembled monolayer on both surfaces. Different types of chemical functionalization have been tested, and this paper focuses on the presentation of aminosilane-grafted 3-(ethoxydimethylsilyl)propylamine and (3-aminopropyl)triethoxysilane. We show that the liquid pH can be used to modify the adhesion and to switch from an attractive behavior to a repulsive behavior. The pH control can thus be used to increase the adhesion during handling and cancel the adhesion during release. Experiments have shown that the pH control is able to control the release of a micro-object. This paper shows the relevance of a new type of reliable submerged robotic microhandling principle, which is based on adjustment of the chemical properties of the liquid.

Nanobioengineering and Characterization of a Novel Estrogen Receptor Biosensor.

We constructed an original supramolecular assembly on a surface of sensor composed of an innovative combination of an engineered cytochrome b5 and a modified nucleic acid bound to a synthetic lipid hemimembrane. The protein/DNA block, called (PDNA) 2, was synthesized and purified before its immobilization onto a hybrid bilayer reconstituted on a gold surface. Surface plasmon resonance (SPR) and atomic force microscopy (AFM) were engaged in parallel on the same substrates in order to better understand dynamic events that occur at the surface of the biosensor. Good correlations were obtained in terms of specificity and reversibility. These findings allow us to present a first application of such biosensor in the study of the interaction processes between nuclear receptor and DNA.