Souhir Boujday

Silane layers on silicon surfaces: mechanism of interaction, stability, and influence on protein adsorption.

In this work the mechanism of (3-aminopropyl)triethoxysilane (APTES) interaction with silicon surfaces is investigated at the molecular level. We studied the influence of experimental parameters such as time, temperature, and concentration on the quality of the APTES layer in terms of chemical properties, morphology, and stability in aqueous medium. This was achieved using a highly sensitive IR mode recently developed, grazing angle attenuated total reflection (GA-ATR). This technique provides structural information on the formed APTES layer. The topography of this layer was investigated by atomic force microscopy in aqueous medium. The hydrophilicity was also studied using contact angle measurement. Combining these techniques enables discussion of the mechanism of silane grafting. Considerable differences were observed depending on the reaction temperature, room temperature or 90 °C. The data suggest the presence of two adsorption sites with different affinities on the oxidized silicon layer. This also allows the optimal parameters to be established to obtain an ordered and stable silane layer. The adsorption of proteins on the APTES layer was achieved and monitored using in situ quartz crystal microbalance with dissipation monitoring and ex situ GA-ATR analyses.

Recent advances in aptasensors based on graphene and graphene-like nanomaterials

Graphene and graphene-like two-dimensional nanomaterials have aroused tremendous research interest in recent years due to their unique electronic, optical, and mechanical properties associated with their planar structure. Aptamers have exhibited many advantages as molecular recognition elements for sensing devices compared to traditional antibodies. The marriage of two-dimensional nanomaterials and aptamers has emerged many ingenious aptasensing strategies for applications in the fields of clinical diagnosis and food safety. This review highlights current advances in the development and application of two-dimensional nanomaterials-based aptasensors with the focus on two main signal-transducing mechanisms, i.e. electrochemical and optical. A special attention is paid to graphene, a one-atom thick layer of graphite with exceptional properties, representing a fastgrowing field of research. In view of the unique properties of two-dimensional nanostructures and their inherent advantages of synthetic aptamers, we expect that high-performance two-dimensional nanomaterials-based aptasensing devices will find extensive applications in environmental monitoring, biomedical diagnostics, and food safety.

In-depth investigation of protein adsorption on gold surfaces: Correlating the structure and density to the efficiency of the sensing layer

Protein A (PrA), mouse monoclonal anti-IgG antibody (SAb) and deglycosylated avidin (NAV) were adsorbed on gold surfaces to capture the model rabbit IgG and build three immunosensing platforms. The assembling of immunosensors, their specificity, and the receptor accessibility were monitored by polarization modulation reflection-absorption infrared spectroscopy (PM-RAIRS) and quartz crystal microbalance with dissipation measurement (QCM-D) at each step. Combining these two techniques allows us to compare both chemical and structural properties of the sensing layers with the former bringing chemical and semiquantitative information on the grafted protein layers, whereas the latter, in addition to the mass uptake, enables us to take the layer rigidity into account. Grafting of the three capture proteins to the transducer surfaces, covered with appropriate self-assembled monolayers, yielded protein layers with variable properties. NAV formed a dense and rigid molecular layer, likely containing protein aggregates, whereas the amount of PrA was below one monolayer resulting in a flexible layer. The amount of immobilized rabbit IgG was different for the three systems with the densest capture protein layer exhibiting the lowest binding capacity. The accessibility of antibodies on the resulting immunosensors measured by interaction with a secondary antirabbit IgG antibody was found to be closely dependent on their coverage as well as on the rigidity of the protein layer. The overall study provides in-depth information on three of the most common immunosensor recognition interfaces and demonstrates the crucial influence of both structure and density of the protein layer on the efficiency of the molecular recognition phenomena.

Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A (SEA) at the ng level

A direct, label-free immunosensor was designed for the rapid detection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions using quartz crystal microbalance with dissipation (QCM-D) as transduction method. The sensing layer including the anti-SEA antibody was constructed by chemisorption of a self-assembled monolayer of cysteamine on the gold electrodes placed over the quartz crystal sensor followed by activation of the surface amino groups with the rigid homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDITC) and covalent linking of binding protein (protein A or protein G). Four anti-SEA antibodies (two of which from commercial source) have been selected to set up the most sensitive detection device. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1000 or 2000 ngml(-1) with a detection limit of 20 ngml(-1). The total time for analysis was 15 min. Using a sandwich type assay, the response was ca. twice higher and consequently the lowest measurable concentration dropped down to 7 ngml(-1) for a total assay time of 25 min.

Effect of SAM chain length and binding functions on protein adsorption: β-lactoglobulin and apo-transferrin on gold.

Controlled immobilization of proteins is crucial in many applications, including biosensors. Allergen biosensing, for example, requires molecular recognition of suitably immobilized proteins by specific antibodies and sensitive measurement of this interaction. Self-assembled monolayers (SAMs), terminated by active functions, and are of great interest for the immobilization of biomolecules. The efficiency of further biorecognition involving molecules immobilized on these surfaces demonstrates an interesting dependence on the chain length and terminal function of the SAM. This motivated us to investigate adsorption of two proteins both known as milk allergens-β-lactoglobulin and apo-transferrin-on amine-terminated SAMs. We varied the chain length by using either short or long chain amine-terminated thiols (cysteamine, CEA, and 11-mercaptoundecylamine, MUAM). We also investigated the influence of the addition of a rigid cross-linker, p-phenylene diisothiocyanate (PDITC), to these amine layers prior to protein adsorption. Protein binding was studied using polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS) and atomic force microscopy (AFM) to characterize their amount and dispersion. We found that protein immobilization varies with SAM chain length and is also influenced by the presence of a cross-linker. The presence of a rigid cross-linker favours the binding of proteins on long chain SAMs, while the effect is almost nonexistent on shorter chains. In addition, the presence of the cross-linker induces a better dispersion of the proteins on the surfaces, regardless of the length of the thiols forming the SAMs. The effects of chain length and chemistry of protein binding are discussed.

Enzyme immobilization on silane-modified surface through short linkers: fate of interfacial phases and impact on catalytic activity.

We investigated the mechanism of enzyme immobilization on silanized surfaces through coupling agents (cross-linkers) in order to understand the role of these molecules on interfacial processes and their effect on catalytic activity. To this end, we used a model multimeric enzyme (G6PDH) and several cross-linking molecules with different chemical properties, including the nature of the end-group (-NCO, -NCS, -CHO), the connecting chain (aliphatic vs aromatic), and geometrical constraints (meta vs para-disubstituted aromatics). There did not seem to be radical differences in the mechanism of enzyme adsorption according to the linker used as judged from QCM-D, except that in the case of DIC (1,4-phenylene diisocyanate) the adsorption occurred more rapidly. In contrast, the nature of the cross-linker exerted a strong influence on the amount of enzyme immobilized as estimated from XPS, and more unexpectedly on the stability of the underlying silane layer. DIC, PDC (1,4-phenylene diisothiocyanate), or GA (glutaraldehyde) allowed successful enzyme immobilization. When the geometry of the linker was changed from 1,4-phenylene diisothiocyanate to 1,3-phenylene diisothiocyanate (MDC), the silane layer was subjected to degradation, upon enzyme adsorption, and the amount of immobilized molecules was significantly lowered. TE (terephtalaldehyde) and direct enzyme deposition without cross-linker were similar to MDC. The organization of immobilized enzymes also depended on the immobilization procedure, as different degrees of aggregation were observed by AFM. A correlation between the size of the aggregates and the catalytic properties of the enzyme was established, suggesting that aggregation may enhance the thermostability of the multimeric enzyme, probably through a compaction of the 3D structure.

nPEG-TiO2 Nanoparticles: A Facile Route to Elaborate Nanostructured Surfaces for Biological Applications

We report the synthesis of diacid-terminated PEG-functionalized cubic TiO(2) nanocrystals by a simple one-step solvothermal method, and their further use to form nanostructured surfaces for protein immobilization. The relevance and major interest of the so-obtained nanocrystals are the presence of terminal carboxylic acid groups at their surface, as confirmed by infrared analyses, in addition to the surrounding PEG chains, essential to avoid non specific interactions. These functional chemical groups were used to (i) immobilize the synthesized nanocubes on a cysteamine-modified Au surface, and to (ii) attach proteins via a presumable covalent link. AFM images show that the shapes and the narrow size distribution of the nanocubes, observed by TEM, were preserved after their immobilization on the modified Au surface. Moreover, the efficiency and specificity of antigen recognition were demonstrated using spectroscopic analyses. Our successful approach provides a versatile and facile way to elaborate specific and sensitive nanostructured surfaces for biosensors.

An experimental and theoretical approach to investigate the effect of chain length on aminothiol adsorption and assembly on gold.

Despite the numerous studies on the self-assembled monolayers (SAMs) of alkylthiols on gold, the mechanisms involved, especially the nature and influence of the thiol-gold interface are still under debate. In this work the adsorption of aminothiols on Au(111) surfaces has been studied by using surface IR and X-ray photoelectron spectroscopy (XPS) as well as by density functional theory (DFT) modeling. Two aminothiols were used, cysteamine (CEA) and mercaptoundecylamine (MUAM), which contain two and eleven carbon atoms, respectively. By combining experimental and theoretical methods, it was possible to draw a molecular picture of the thiol-gold interface. The long-chain aminothiol produced better ordered SAMs, but, interestingly, the XPS data showed different sulfur binding environments depending on the alkyl chain length; an additional peak at low binding energy was observed upon CEA adsorption, which indicates the presence of sulfur in a different environment. DFT modeling showed that the positions of the sulfur atoms in the SAMs on gold with similar unit cells [(2√3×2√3)R30°] depended on the length of the alkyl chain. Short-chain alkylthiol SAMs were adsorbed more strongly than long-chain thiol SAMs and were shown to induce surface reconstruction by extracting atoms from the surface, possibly forming adatom/vacancy combinations that lead to the additional XPS peak. In the case of short alkylthiols, the thiol-gold interface governs the layer, CEA adsorbs strongly, and the mechanism is closer to single-molecule adsorption than self-assembly, whereas for long chains, interactions between alkyl chains drive the system to self-assembly, leading to a higher level of SAM organization and restricting the influence of the sulfur-gold interface.

Enhanced Vibrational Spectroscopies as Tools for Small Molecule Biosensing.

In this short summary we summarize some of the latest developments in vibrational spectroscopic tools applied for the sensing of (small) molecules and biomolecules in a label-free mode of operation. We first introduce various concepts for the enhancement of InfraRed spectroscopic techniques, including the principles of Attenuated Total Reflection InfraRed (ATR-IR), (phase-modulated) InfraRed Reflection Absorption Spectroscopy (IRRAS/PM-IRRAS), and Surface Enhanced Infrared Reflection Absorption Spectroscopy (SEIRAS). Particular attention is put on the use of novel nanostructured substrates that allow for the excitation of propagating and localized surface plasmon modes aimed at operating additional enhancement mechanisms. This is then be complemented by the description of the latest development in Surface- and Tip-Enhanced Raman Spectroscopies, again with an emphasis on the detection of small molecules or bioanalytes.

Nanostructured and spiky gold in biomolecule detection: improving binding efficiencies and enhancing optical signals

Nanostructured gold can improve the ability to detect biomolecules. Whether planar nanostructured surfaces or nanostructured particles are used, similar principles governing the enhancement apply. The two main benefits of nanostructured gold are improved geometry and enhancement of optical detection methods. Nanostructuring improves the geometry by making surface-bound receptors more accessible and by increasing the surface area. Optical detection methods are enhanced due to the plasmonic properties of nanoscale gold, leading to localized surface plasmon resonance sensing (LSPR), surface-enhanced Raman scattering (SERS), enhancement of conventional surface plasmon resonance sensing (SPR), surface enhanced infrared absorption spectroscopy (SEIRAS) and metal-enhanced fluorescence (MEF). Anisotropic, particularly spiky, surfaces often feature a high density of nanostructures that show an especially large enhancement due to the presence of electromagnetic hot-spots and thus are of particular interest. In this review, we discuss these benefits and describe examples of nanostructured and spiky gold on planar surfaces and particles for applications in biomolecule detection.