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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 ß/δ (PPARß/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.
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Antígenos CD/metabolismo , Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Microscopia de Força Atômica/métodos , PPAR delta/agonistas , PPAR beta/agonistas , Tiazóis/farmacologia , Neoplasias da Bexiga Urinária/metabolismo , Antígenos CD/ultraestrutura , Caderinas/ultraestrutura , Linhagem Celular Tumoral , Movimento Celular , Humanos , Transdução de Sinais , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/ultraestruturaRESUMO
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.
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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 75â¯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 25-1000â¯nm range, with a majority (≈ 90%)â¯≤â¯100â¯nm. Both T-PEVs in buffer (but not N-PEVs) and non-nanofiltered plasma containing T-PEVs triggered NETs formation. Nanofiltration depleted large EVs (> 70â¯nm) and decreased NETs formation. The NBA platform was found to be a suitable tool to investigate the safety of plasma for transfusion.
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Transfusão de Sangue , Vesículas Extracelulares/metabolismo , Nanotecnologia/métodos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Agregação Celular/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Filtração , Humanos , Nanopartículas/química , Nanoporos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Trombina/farmacologiaRESUMO
Estrothiazine (ESTZ) is a weak estrogen sharing structural similarities with coumestrol. ESTZ failed to compete with [3H]17ß-estradiol ([3H]17ß-E2) 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.
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Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/metabolismo , Tiazinas/administração & dosagem , Transcrição Gênica , Sítios de Ligação , Neoplasias da Mama/genética , Dimerização , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Células MCF-7 , Fitoestrógenos , Ligação Proteica/genética , Espectrofotometria Atômica , Tiazinas/química , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
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.
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Técnicas Biossensoriais , Vesículas Extracelulares , Ressonância de Plasmônio de Superfície , Vesículas Extracelulares/química , Microscopia de Força Atômica/métodos , Comunicação CelularRESUMO
The skin is the first physiological barrier, with a complex constitution, that provides defensive functions against multiple physical and chemical aggressions. Glyphosate is an extensively used herbicide that has been shown to increase the risk of cancer. Moreover there is increasing evidence suggesting that the mechanical phenotype plays an important role in malignant transformation. Atomic force microscopy (AFM) has emerged within the last decade as a powerful tool for providing a nanometer-scale resolution imaging of biological samples. Peak Force Tapping (PFT) is a newly released AFM-based investigation technique allowing extraction of chemical and mechanical properties from a wide range of samples at a relatively high speed and a high resolution. The present work uses the PFT technology to investigate HaCaT keratinocytes, a human epidermal cell line, and offers an original approach to study chemically-induced changes in the cellular mechanical properties under near-physiological conditions. These experiments indicate glyphosate induces cell membrane stiffening, and the appearance of cytoskeleton structures at a subcellular level, for low cytotoxic concentrations whereas cells exposed to IC50 (inhibitory concentration 50%) treatment exhibit control-like mechanical behavior despite obvious membrane damages. Quercetin, a well-known antioxidant, reverses the glyphosate-induced mechanical phenotype.
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Membrana Celular/efeitos dos fármacos , Glicina/análogos & derivados , Queratinócitos/efeitos dos fármacos , Queratinócitos/ultraestrutura , Microscopia de Força Atômica/métodos , Linhagem Celular , Membrana Celular/ultraestrutura , Citoesqueleto/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Epiderme/ultraestrutura , Glicina/toxicidade , Humanos , Queratinócitos/metabolismo , Quercetina/farmacologia , GlifosatoRESUMO
Resonant microelectromechanical systems are promising devices for real time and highly sensitive measurements. The sensitivity of such sensors to additional mass loadings which can be increased thanks to the miniaturisation of devices is of prime importance for biological applications. The miniaturisation of structures passes through a photolithographic process and wet chemical etching. So, this paper presents new results on the anisotropic chemical etching of the gallium arsenide (GaAs) crystal used for this application, in several solutions. This paper focuses on the micro/nanostructuration of the sensing surface to increase the sensor sensitivity. Indeed, this active surface will be biofunctionalized to operate in biological liquid media in view of biomolecules detection. Several experimental conditions of etching bath composition, concentration and temperature were examined to obtain a large variety of geometrical surfaces topographies and roughness. According to the orientation dependence of the chemical etching process, the experiments were also performed on various GaAs crystal plates. The bath 1 H3PO4:9 H2O2:1 H2O appeared to be particularly adapted to the fabrication of the GaAs microstructured membrane: indeed, the bath is highly stable, anisotropic, and, as a function of temperature, it allows the production of a large variety of GaAs surface topographies.
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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/SiO2 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 SiO2 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.
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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.
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Vesículas Extracelulares , Células Eucarióticas , Células ProcarióticasRESUMO
A regenerable bulk acoustic wave (BAW) biosensor is developed for the rapid, label-free and selective detection of Escherichia coli 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 E. coli. 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 103 and 108 CFU/mL. A linear dependence between the frequency response and the logarithm of E. coli concentration was observed for suspensions ranging between 103 and 107 CFU/mL, with the limit of detection of the biosensor estimated at 103 CFU/mL. The 5-fold regeneration and excellent selectivity towards E. coli detected at 104 CFU/mL in a suspension tinted with Bacillus subtilis at 106 CFU/mL illustrate the biosensor potential for the attractive operation in complex biological media.
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Técnicas Biossensoriais , Escherichia coli/isolamento & purificação , Som , Anticorpos , Arsenicais , Eletrodos , Gálio , Ouro , Limite de Detecção , Regeneração , Óxido de ZincoRESUMO
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.
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Técnicas Biossensoriais , Microfluídica , Acústica , Plaquetas , Hemostasia , Humanos , CinéticaRESUMO
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.
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Desenvolvimento de Medicamentos/organização & administração , Drogas em Investigação/farmacologia , Vesículas Extracelulares/fisiologia , Técnicas de Química Analítica/métodos , Ensaios Clínicos como Assunto/organização & administração , Vias de Administração de Medicamentos , Composição de Medicamentos , Estabilidade de Medicamentos , Europa (Continente) , Humanos , Controle de Qualidade , Secretoma/fisiologiaRESUMO
Microtubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in beta-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The observation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabilizers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtubule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymerization. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single protofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of protofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration.
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Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Paclitaxel/farmacologia , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animais , Guanosina Trifosfato/metabolismo , Hidrólise , Técnicas In Vitro , Cinesinas/metabolismo , Microscopia de Força Atômica , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Complexos Multiproteicos , Conformação Proteica , Subunidades Proteicas , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/ultraestruturaRESUMO
Among the molecules to which the human skin is exposed, glyphosate is used as an herbicide. Glyphosate has been shown to induce in vitro cutaneous cytotoxic effects, concomitant with oxidative disorders. In this following study, we focused on dynamic events of the loss of HaCaT cell integrity appearing after a glyphosate treatment. In these conditions, we showed that glyphosate is able to disrupt HaCaT cells and to induce intracellular oxidative cascade. In this aim, we optimized the conditions of cell treatment playing on exposure time (from 24 h to 30 min), which directly modify the cell viability profile (glyphosate 50% inhibition concentration from 28 to 53 mM) and allow to track cells along the treatment as an "induction and visualization" process. The combination of atomic force and fluorescence microscopic approaches offered opportunities to lead in parallel an investigation of the membrane surface and of the intracellular disorders, through cytoskeleton, nuclear, and oxidative stress marker targeting. The originality of our approach relies on monitoring all events derived from oxidative stress in process and performed by simultaneous cytotoxic induction and nanoscale cell visualization. We revealed a transition from spread and globular to elongated cell morphology, with a drastic cell size reduction, after a dose- and time-dependent glyphosate treatment; a redistribution of cell surface protrusions was also pointed out. All these membrane damages, added to observations of disorganized cytoskeleton, condensed chromatin, and overproduction of oxidative reactive species, lead us to conclude that glyphosate acts in induction of apoptotic process.
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Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Glicina/análogos & derivados , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Microscopia de Força Atômica/métodos , Nanoestruturas/análise , Morte Celular , Linhagem Celular , Membrana Celular/ultraestrutura , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Glicina/toxicidade , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração Inibidora 50 , GlifosatoRESUMO
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.
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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.
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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.
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Técnicas Biossensoriais , Plaquetas/ultraestrutura , Micropartículas Derivadas de Células/ultraestrutura , Plaquetas/química , Micropartículas Derivadas de Células/química , Citometria de Fluxo , Humanos , Microscopia de Força Atômica , Ressonância de Plasmônio de SuperfícieRESUMO
Wet chemical processes were investigated to remove alkanethiol self-assembled monolayers (SAMs) and regenerate GaAs (001) samples studied in the context of the development of reusable devices for biosensing applications. The authors focused on 16-mercaptohexadecanoic acid (MHDA) SAMs that are commonly used to produce an interface between antibodies or others proteins and metallic or semiconductor substrates. As determined by Fourier transform infrared absorption spectroscopy, among the investigated solutions of HCl, H2O2, and NH4OH, the highest efficiency in removing alkanethiol SAM from GaAs was shown by NH4OH:H2O2 (3:1 volume ratio) diluted in H2O. The authors observed that this result was related to chemical etching of GaAs that even in a weak solution of NH4OH:H2O2:H2O (3:1:100) proceeded at a rate of 130 nm/min. The surface revealed by a 2-min etching under these conditions allowed depositing successfully a new MHDA SAM with comparable quality and density to the initial coating. This work provides an important view on the perspective of the development of a family of cost-effective GaAs-based biosensors designed for repetitive detection of a variety of biomolecules immobilized with dedicated antibody architectures.
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Anticorpos/metabolismo , Arsenicais/química , Gálio/química , Ácidos Palmíticos/metabolismo , Propriedades de Superfície , Fenômenos Químicos , Eletrólitos , Ligação Proteica , Solventes , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
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.
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Compostos de Diazônio/química , Técnicas Eletroquímicas , Ouro/química , Medições Luminescentes/métodos , Nanopartículas Metálicas/química , Compostos Organoáuricos/análise , Compostos de Diazônio/síntese química , Eletrodos , Concentração de Íons de Hidrogênio , Luminescência , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
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)11OH)) or carboxyl (MHDA; for mercaptohexadecanoic acid (HS(CH2)15CO2H)) 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 insitu 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.