Characterization and Whole Genome Sequencing of AR23, a Highly Toxic Bacillus thuringiensis Strain Isolated from Lebanese Soil.

The demand for sustainable and eco-friendly control methods of pests and insects is increasing worldwide. From this came the interest in Bacillus thuringiensis, an entomopathogenic bacterium capable of replacing chemical pesticides. However, the possibility of pests developing resistance to a particular strain may impair its use, and there is a need to identify novel strains of this species as potential commercial biopesticides. B. thuringiensis sv. israelensis is one of the most successful serovars, widely commercialized for its activity against black fly and mosquito larvae. In this study, we isolated, characterized, and sequenced a new Lebanese B. thuringiensis sv. israelensis isolate, strain AR23. Compared to the commercialized reference strain AM65-52 (Vectobac®, Sumitomo), AR23 showed an increased activity against several mosquito species. The genomic analysis revealed that this strain, compared to AM65-52, possesses a simplified plasmid content and an additional functional cry4Ba coding gene that most likely accounts for the increased effectiveness of this strain in mosquito larvae killing.

Vegetable oil hybrid films cross-linked at the air-water interface: formation kinetics and physical characterization.

Vegetable oil based hybrid films were developed thanks to a novel solvent- and heating-free method at the air-water interface using silylated castor oil cross-linked via a sol-gel reaction. To understand the mechanism of the hybrid film formation, the reaction kinetics was studied in detail by using complementary techniques: rheology, thermogravimetric analysis, and infrared spectroscopy. The mechanical properties of the final films were investigated using nano-indentation, whereas their structure was studied using a combination of wide-angle X-ray scattering, electron diffraction, and atomic force microscopy. We found that solid and transparent films form in 24 hours and, by changing the silica precursor to castor oil ratio, their mechanical properties are tunable in the MPa-range by about a factor of twenty. In addition to that, a possible optimization of the cross-linking reaction with different catalysts was explored, and finally cytotoxicity tests were performed on fibroblasts proving the absence of film toxicity. The results of this work pave the way to a straightforward synthesis of castor-oil films with tunable mechanical properties: hybrid films cross-linked at the air-water interface combine an easy and cheap spreading protocol with the features of their thermal history optimized for possible future micro/nano drug loading, thus representing excellent candidates for the replacement of non-environmentally friendly petroleum-based materials.

Estrogen receptor preparation effects on the receptor-DNA interaction by surface plasmon resonance.

Up to now, several studies have investigated estrogen receptor (ER)-estrogen response element (ERE) interaction using biosensors such as surface plasmon resonance. These strategies have aimed to understand the molecular mechanism of such interaction as well as the effect of the ligand on this interaction. These approaches start to be used to determine the mechanisms of protein/DNA interaction, in particular in the context of drug discovery or environmental applications. However, some physical and biochemical parameters (incubation time, temperature, protease inhibitor cocktail, and bovine serum albumin (BSA)) are not completely described in the literature and could deeply modify the obtained results. This paper aims to focus not only on the preliminary steps of sample preparation such as protein thawing and incubation conditions (time and temperature) but also on the evaluation of protease inhibitor cocktail and BSA effect on the measurement of ER-ERE interactions.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer.

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.

Characterization of a membrane-active peptide from the Bordetella pertussis CyaA toxin.

Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1-384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1-489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375-485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454-485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454-485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane.

Kinetics of interaction between ADP-ribosylation factor-1 (Arf1) and the Sec7 domain of Arno guanine nucleotide exchange factor, modulation by allosteric factors, and the uncompetitive inhibitor brefeldin A.

The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg(2+), and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg(2+) potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg(2+)-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex.

Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation.

UNLABELLED: Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. CONCLUSION: VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis.

Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology.

The estrogen receptor (ER) is a transcription factor that binds under 17-ß-estradiol (E2) stimulation as homodimer to a short DNA consensus sequence named estrogen response element (ERE). The ER/ERE interaction has been assessed by several research groups through different methodologies notably by surface plasmon resonance (SPR) techniques. The biochemical parameters and conditions (solvent, ER concentration, salt, time and temperature) used to prepare samples before analysis were very different from one study to another. But no studies have aimed to compare the effect of these modifications on ER/ERE interaction. Therefore the main objective of the present paper was to assess the influence of biochemical parameters onto the ER/ERE interaction with the final aim to improve the comprehension of this interaction. Our results highlighted that parameters like solvent, ER concentration, salt and surfactant concentration, temperature and time deeply modify ER/ERE interaction. Nevertheless, the dimer formation under E2 stimulation occurred with all tested conditions. Altogether, incubation parameters of ER with E2, deeply modify its binding level onto ERE. These data constitute an important key point to consider for the improvement of ER/ERE detection method depending upon the aim of the study (interaction measurement, environmental detection, development of new technologies or devices).

Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae.

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC50 of Lip and HD1 were 33.27 and 128.61 µg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.

Rapid communication: insights into the role of extracellular vesicles during Auger radioimmunotherapy.

PURPOSE: Non-targeted effects, including bystander and systemic effects, play a crucial role during Auger targeted radionuclide therapy. Here, we investigated whether small extracellular vesicles (sEVs) produced by irradiated cells could contribute to the bystander cytotoxic effects in vitro and also to therapeutic efficacy in vivo, after their injection in tumor xenografts. MATERIALS AND METHODS: B16F10 melanoma donor cells were exposed to radiolabeled antibodies (Auger radioimmunotherapy, RIT) for 48 h or to X-rays (donor cells). Then, donor cells were incubated with fresh medium for 2 h to prepare conditioned medium (CM) that was transferred onto recipient cells for bystander effect assessment, or used for sEVs enrichment. Resulting sEVs were incubated in vitro with recipient cells for determining bystander cytotoxicity, or injected in B16F10 melanoma tumors harbored by athymic and C57BL/6 mice. RESULTS: In vitro analysis of bystander cytotoxic effects showed that CM killed about 30-40% of melanoma cells. SEVs isolated from CM contributed to this effect. Moreover, the double-stranded DNA (dsDNA) content was increased in sEVs isolated from CM of exposed cells compared to control (not exposed), but the difference was significant only for the X-ray condition. These results were supported by immunodetection of cytosolic dsDNA in donor cells, a phenomenon that should precede dsDNA enrichment in sEVs. However, sEVs cytotoxicity could not be detected in vivo. Indeed, in athymic and in immunocompetent mice that received four intratumoral injections of sEVs (1/day), tumor growth was not delayed compared with untreated controls. Tumor growth was slightly (not significantly) delayed in immunocompetent mice treated with sEVs from X-ray-exposed cells, and significantly with sEVs purified from CM collected after 48 h of incubation. These results highlight the need to determine the optimal conditions, including radiation absorbed dose and sEVs collection time, to obtain the strongest cytotoxic effects. CONCLUSIONS: This study demonstrates that sEVs could play a role during Auger RIT through bystander effects in vitro. No systemic effects were observed in vivo, under our experimental conditions. However, X-rays experiments showed that sEVs collection time might be influencing the nature of sEVs, a parameter that should also be investigated during Auger RIT.

A facile approach to modify cellulose nanocrystal for the adsorption of perfluorooctanoic acid.

Cellulose-based materials are a sustainable alternative to polymers derived from petroleum. Cellulose nanocrystal (CNC) is a biopolymer belonging to this family; it is commonly known for its important physical and chemical properties and ability to form a film. Modifying CNC via electrostatic interaction provided by cationic polymers is a facile and promising technique to enlarge the application of CNC. Herein, we report the preparation of films, from blends of negatively charged CNC and positively charged poly (trimethyl aminoethyl methacrylate) (PTMAEMA). The interaction between CNC and PTMAEMA was verified by using a quartz crystal microbalance with dissipation monitoring (QCM-D), as well as by measuring the particle size and ζ-potential of the casting mixture. To favor the application of the nanocomposite film in water treatment, the film was supported on Whatman™ paper, and adsorption tests were conducted using perfluorooctanoic acid (PFOA) as a model compound for the family of persistent fluorinated pollutants known as PFAS (per- and polyfluoroalkyl substances).

Novel trehalose-based excipients for stabilizing nebulized anti-SARS-CoV-2 antibody.

COVID-19 is caused by the infection of the lungs by SARS-CoV-2. Monoclonal antibodies, such as sotrovimab, showed great efficiency in neutralizing the virus before its internalization by lung epithelial cells. However, parenteral routes are still the preferred route of administration, even for local infections, which requires injection of high doses of antibody to reach efficacious concentrations in the lungs. Lung administration of antibodies would be more relevant requiring lower doses, thus reducing the costs and the side effects. But aerosolization of therapeutic proteins is very challenging, as the different processes available are harsh and trigger protein aggregation and conformational changes. This decreases the efficiency of the treatment, and can increase its immunogenicity. To address those issues, we developed a series of new excipients composed of a trehalose core, a succinyl side chain and a hydrophobic carbon chain (from 8 to 16 carbons). Succinylation increased the solubility of the excipients, allowing their use at relevant concentrations for protein stabilization. In particular, the excipient with 16 carbons (C16TreSuc) used at 5.6 mM was able to preserve colloidal stability and antigen-binding ability of sotrovimab during the nebulization process. It could also be used as a cryoprotectant, allowing storage of sotrovimab in a lyophilized form during weeks. Finally, we demonstrated that C16TreSuc could be used as an excipient to stabilize antibodies for the treatment against COVID-19, by in vitro and in vivo assays. The presence of C16TreSuc during nebulization preserved the neutralization capacity of sotrovimab against SARS-CoV-2 in vitro; an increase of its efficacy was even observed, compared to the non-nebulized control. The in vivo study also showed the wide distribution of sotrovimab in mice lungs, after nebulization with 5.6 mM of excipient. This work brings a solution to stabilize therapeutic proteins during storage and nebulization, making pulmonary immunotherapy possible in the treatment of COVID-19 and other lung diseases.

ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro.

The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched-preparations of (H)ANT (ANT from heart, mainly ANT1) and (L)ANT (ANT from liver, mainly ANT2) isoforms interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform identity and on the conformation of ANT.

One step synthesis of gold-loaded radial mesoporous silica nanospheres and supported lipid bilayer functionalization: towards bio-multifunctional sensors.

A simple synthetic route is developed to achieve gold functionalized radial mesoporous silica nanoparticles (Au-MsNP) synthesized by a one step procedure fully compatible with basic conditions required for the preparation of monodispersed nanospheres. In a second step, Au-MsNP particles have been coated with phospholipid bilayers in order to design an advanced biofunctional platform with the gold metallic nanoparticles previously grown into the pore channels and responsible for a plasmonic activity relevant for biosensing. The size of Au-MsNP is checked by dynamic light scattering while zeta potential measurements reflect their surface charge. The particle morphology is characterized by transmission and scanning electron microscopy and the Si/Au ratios are obtained from energy dispersive X-ray analysis. The textural properties of Au-MsNP, specific surface area and pore size, are determined from N(2) adsorption. The supported bilayers are achieved from vesicles of different phospholipids incubated with Au-MsNP particles. The coating efficiency is investigated by zeta potential and cryo- transmission electron microscopy. The plasmonic activities of bare Au-MsNP particles and coated lipid bilayer Au-MsNP platform are evidenced for two model systems: direct adsorption of bovine serum albumin and molecular recognition events between avidin molecules and biotin receptors integrated in the supported lipid bilayer.

A Novel Approach to the Facile Growth and Organization of Photothermal Prussian Blue Nanocrystals on Different Surfaces.

We report here a novel "one-pot" approach for the controlled growth and organization of Prussian blue nanostructures on three different surfaces: pure Au0, cysteamine-functionalized Au0, and SiO2-supported lipid bilayers with different natures of lipids. We demonstrate that fine control over the size, morphology, and the degree and homogeneity of the surface coverage by Prussian Blue (PB) nanostructures may be achieved by manipulating different parameters, which are the precursor concentration, the nature of the functional groups or the nature of lipids on the surfaces. This allows the growth of isolated PB nanopyramids and nanocubes or the design of thin dense films over centimeter square surfaces. The formation of unusual Prussian blue nanopyramids is discussed. Finally, we demonstrate, by using experimental techniques and theoretical modeling, that PB nanoparticles deposited on the gold surface exhibit strong photothermal properties, permitting a rapid temperature increase up to 90 °C with a conversion of the laser power of almost 50% for power source heat.

Interest of extracellular vesicles in regards to lipid nanoparticle based systems for intracellular protein delivery.

Compared to chemicals that continue to dominate the overall pharmaceutical market, protein therapeutics offer the advantages of higher specificity, greater activity, and reduced toxicity. While nearly all existing therapeutic proteins were developed against soluble or extracellular targets, the ability for proteins to enter cells and target intracellular compartments can significantly broaden their utility for a myriad of exiting targets. Given their physical, chemical, biological instability that could induce adverse effects, and their limited ability to cross cell membranes, delivery systems are required to fully reveal their biological potential. In this context, as natural protein nanocarriers, extracellular vesicles (EVs) hold great promise. Nevertheless, if not present naturally, bringing an interest protein into EV is not an easy task. In this review, we will explore methods used to load extrinsic protein into EVs and compare these natural vectors to their close synthetic counterparts, liposomes/lipid nanoparticles, to induce intracellular protein delivery.

Vegetable oil-based hybrid microparticles as a green and biocompatible system for subcutaneous drug delivery.

The aim of this study was to evidence the ability of vegetable oil-based hybrid microparticles (HMP) to be an efficient and safe drug delivery system after subcutaneous administration. The HMP resulted from combination of a thermostabilized emulsification process and a sol-gel chemistry. First of all, castor oil was successfully silylated by means of (3-Isocyanatopropyl)trimethoxysilane in solvent-free and catalyst-free conditions. Estradiol, as a model drug, was dissolved in silylated castor oil (ICOm) prior to emulsification, and then an optimal sol-gel crosslinking was achieved inside the ICOm microdroplets. The resulting estradiol-loaded microparticles were around 80 µm in size and allowed to entrap 4 wt% estradiol. Their release kinetics in a PBS/octanol biphasic system exhibited a one-week release profile, and the released estradiol was fully active on HeLa ERE-luciferase ERα cells. The hybrid microparticles were cytocompatible during preliminary tests on NIH 3T3 fibroblasts (ISO 10993-5 standard) and they were fully biocompatible after subcutaneous injection on mice (ISO 10993-6 standard) underlining their high potential as a safe and long-acting subcutaneous drug delivery system.

A rational study of the influence of Mn2+-insertion in Prussian blue nanoparticles on their photothermal properties.

We investigated a series of Mn2+-Prussian blue (PB) nanoparticles NazMnxFe1-x[Fe(CN)6]1-y□y·nH2O of similar size, surface state and cubic morphology with various amounts of Mn2+ synthesized through a one step self-assembly reaction. We demonstrated by a combined experimental-theoretical approach that during the synthesis, Mn2+ substituted Fe3+ up to a Mn/Na-Mn-Fe ratio of 32 at% in the PB structure, while for higher amounts, the Mn2[Fe(CN)6] analogue is obtained. For comparison, the post-synthetic insertion of Mn2+ in PB nanoparticles was also investigated and completed with Monte-Carlo simulations to probe the plausible adsorption sites. The photothermal conversion efficiency (η) of selected samples was determined and showed a clear dependence on the Mn2+amount with a maximum efficiency for a Mn/Na-Mn-Fe ratio of 10 at% associated with a dependence on the nanoparticle concentration. Evaluation of the in vitro photothermal properties of these nanoparticles performed on triple negative human breast adenocarcinoma (MDA-MB-231) cells by using continuous and pulsed laser irradiation confirm their excellent PTT efficiency permitting low dose use.

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.

Critical parameters in surface plasmon resonance biosensor development: The interaction between estrogen receptor and estrogen response element as model.

Estrogenic compounds are contaminants that may be active at low concentrations and are a major concern for environmental quality. They interact with organisms via Estrogen Receptors (ER). Some detection methods which have been developed use the ability of ER to interact with short consensus DNA sequences known as Estrogen Response Elements (ERE). Surface Plasmon Resonance (SPR) based techniques allow detection of interaction without labelled molecule use. Such optical transductors are widely used to convert the biological recognition signals into electric quantifiable signals. In this study, SPR is used to assess signal variation in the presence of estrogenic compounds. The combination of physical properties and biological recognition events (e.g. ER/ERE) permits the development of biosensors. These require several steps: activation of the surface, DNA sequence binding, ERE sequence evaluation, ER preparation, characterization of binding properties and regeneration of the surface. This article focuses on the mode of surface activation, protein-DNA binding conditions and the regeneration of ERE. After giving a summary of the literature concerning the usual conditions employed in these steps, an evaluation of some key parameters is given.