Andrea Valsesia

pH-Dependent Immobilization of Proteins on Surfaces Functionalized by Plasma-Enhanced Chemical Vapor Deposition of Poly(acrylic acid)-and Poly(ethylene oxide)-like Films

The interaction of the proteins bovine serum albumin (BSA), lysozyme (Lys), lactoferrin (Lf), and fibronectin (Fn) with surfaces of protein-resistant poly(ethylene oxide) (PEO) and protein-adsorbing poly(acrylic acid) (PAA) fabricated by plasma-enhanced chemical vapor deposition has been studied with quartz crystal microbalance with dissipation monitoring (QCM-D). We focus on several parameters which are crucial for protein adsorption, i.e., the isoelectric point (pI) of the proteins, the pH of the solution, and the charge density of the sorbent surfaces, with the zeta-potential as a measure for the latter. The measurements reveal adsorption stages characterized by different segments in the plots of the dissipation vs frequency change. PEO remains protein-repellent for BSA, Lys, and Lf at pH 4-8.5, while weak adsorption of Fn was observed. On PAA, different stages of protein adsorption processes could be distinguished under most experimental conditions. BSA, Lys, Lf, and Fn generally exhibit a rapid initial adsorption phase on PAA, often followed by slower processes. The evaluation of the adsorption kinetics also reveals different adsorption stages, whereas the number of these stages does not always correspond to the structurally different phases as revealed by the D- f plots. The results presented here, together with information obtained in previous studies by other groups on the properties of these proteins and their interaction with surfaces, allow us to develop an adsorption scenario for each of these proteins, which takes into account electrostatic protein-surface and protein-protein interaction, but also the pH-dependent properties of the proteins, such as shape and exposure of specific domains.

Protein nanopatterns for improved immunodetection sensitivity.

In this work, we clearly demonstrate the capability of protein nanopatterns of improving the quality factors of immunosensing devices, such as lowering of the limit of detection and increase of sensitivity. This beneficial effect is obtained by the formation on the sensors surface of bioadhesive domains of nanometric dimensions in a nonadhesive matrix by means of colloidal lithography.

Multiplexed label-free optical biosensor for medical diagnostics

Abstract. This paper describes a new multiplexed label-free biosensor. The detection technology is based on nanostructured gold-polymer surfaces. These surfaces support surface plasmon resonance modes that can be probed by a miniaturized optical setup. The optical characterization of the sensing chip shows the sensitivity and the limit-of-detection to refractive index changes. Moreover, by studying the progressive adhesion of molecular monolayers of polyelectrolytes, the decay of the plasmonic mode electric field above the surface has been reconstructed. A multiplexed label-free biosensing device is then described and characterized in terms of sensitivity, lateral resolution, and sensitivity to a model biological assay. The sensitivity in imaging mode of the device is of the order of 10−6 refractive index units, while the measured lateral resolution is 6.25 μm within a field of view of several tenths of mm2, making the instrument unique in terms of multiplexing capability. Finally, the proof-of-concept application of the technology as a point-of-care diagnostic tool for an inflammatory marker is demonstrated.

Use of Nanopatterned Surfaces To Enhance Immunoreaction Efficiency

In this work, we compare the immunoreaction efficiency between uniformly functionalized surface and chemically nanopatterned surfaces when applied as platforms for antigen/antibody interactions with and without the use of protein A as orienting protein. On the nanopatterned platform, the immunoreaction efficiency is higher than all the other cases with no protein A pretreatment of the surface, providing evidence of the capability of the adhesive/antiadhesive nanopatterned surface to immobilize the molecules in a reactive state, increasing their possibility to form complexes.

Plasma assisted production of chemical nano-patterns by nano-sphere lithography: application to bio-interfaces

In this work a low-cost parallel technique for creating chemical nano-patterned surfaces using nano-spheres as masks is presented. This technique, called nano-sphere lithography, makes use of different steps of plasma etching and deposition processes, for the creation of polymeric nano-structures of different chemical functionalities with relevant applications to bio-interfaces. In this study, the attention is focused on the plasma processing aspects for the etching of the polymeric masks (colloidal masks) in order to control the shape and the size of the etched nano-structures. The bio-functionality of the nano-patterned surfaces has been proved with a selective immobilization of proteins on the bioactive spots.

Large-Scale Fabrication of Bi-Functional Nanostructured Polymer Surfaces for Selective Biomolecular Adhesion†

The development of technologies allowing the fabrication of nanoscale chemical contrasts is of great interest, as it may allow study of fundamental aspects of cell adhesion at the level of single protein/receptor molecules, and for engineering a new generation of biological and chemical sensors. Recent works on bi-functional nanostructured surfaces containing chemical functions towards proteins (adhesive and repellent) have shown an increasingly immunoreaction-recognition sensitivity between antigen and antibody couples, indicating their increased availability for specific bioreactions. Patterning techniques allowing low-cost fabrication of regular arrays with chemically distinct nanoscale features, from sub-100 nm to several hundreds of nanometers, have to be developed to study this phenomenon in more detail and to favor the creation of a new generation of protein platforms. Recently, several research groups have adopted different technological approaches to create nanopatterned surfaces for biological applications, and details can be found in the recent review on the topic by Blatter et al. Among the serial techniques mentioned in the review, direct writing methods such as dip-pen lithography and electron-beam lithography have been successfully used to fabricate protein nanoarrays with well-ordered features and controlled size, shape, and pitch, hence offering the potential to develop protein biochips and biosensors. However, all these techniques suffer from limitations that are principally related to the sophistication of the required equipment and the need for clean-room facilities. Also, they are not really suitable for fabrication of patterned platforms over large areas (>few cm) and are strongly dependent on the substrate material (gold, TiO2, SiO2). A recent alternative to produce sub-100-nm nanopatterns with self-assembled monolayers (SAMs), without using lithography, has been described by H. Gao et al. They succeeded in transferring nanoscale patterns from porous alumina templates to gold and silicon surfaces. The pores in the templates spatially confine the self assembly of molecules on the exposed surfaces allowing the fabrication of SAM patterns over several cm with features down to 30 nm. The combination of plasma polymerization processes and colloidal lithography appears to be a promising technique for producing nanostructured, functionalized, polymer surfaces. This unique combination offers an inexpensive way of fabrication that may be easily transferred to industrial applications, since it is a parallel technique. The interest in plasma polymerization relies on the formation, on different substrates, of a large variety of homogeneous polymer layers with unique physico-chemical properties, which can be accurately controlled by the plasma parameters. In parallel, deposition of self-assembled layers of monodisperse nanospheres has been widely used because this technique offers the possibility of easy production of two-dimensional (2D) nanomasks over a large area at low cost. In a previous study, by combining these two methods, we succeeded in fabricating nanocraters of adhesive acrylic acid moieties inside a protein-repellent matrix. However, even if this method has shown promising results in the production of a chemical contrast on the surface with a resolution down to 100 nm, the fabrication process suffers from some limitations. Indeed, the poly(styrene) (PS) beads used for fabrication of the nanomask and oxygen-plasma etching used in this method both limit the fabrication process because the etching step is not trivial to monitor and it confers to the surface a particular shape (the crater form). Moreover, sub-100-nm structures can be difficult to obtain because the small PS particles tend to melt and merge due to the local warming of the system during the etching step. Furthermore, the reduction of the size of the nanoparticles by etching is limited because of the glass transition of the polymer beads, which occurs below a given diameter threshold. The use of inorganic nanoparticles such as silica can overcome these limitations: monodisperse silica nanoparticles, modified by a hydrophobic silane coupling agent, can be easily self-assembled into a (2D) hexagonal array at the water–air interface. This system, combined with the plasma polymerization technique, is potentially interesting for fabrication of nanostructured surfaces for several reasons: i) large-scale closely packed colloidal particle arrays can be easily obtained; ii) the size of the silica particles can be accurately tuned from a few nanometers to a few micrometers, thus allowing the control of the structural surface parameters with standard chemical laboratory facilities; iii) by controlling the growth rate of the plasma polymer deposition, the polymerization of the layer can proceed through the interstices of the colloidal particle layer; iv) the chemistry of the plasma polymer can be easily tuned. In this study, we report on a simple and inexpensive method for the fabrication of bi-functional nanostructured polymer surfaces with contrasted adhesive property over large surface areas. The method combines colloidal lithography (CL), using hydrophobic silica nanoparticles, and plasmaenhanced chemical vapor deposition (PE-CVD). In particular, we focus on the fabrication of bio-adhesive poly(acrylic acid) [!] Dr. F. Rossi, Dr. S. Mornet, Dr. F. Bretagnol, Dr. I. Mannelli, Dr. A. Valsesia, Dr. L. Sirghi, Dr. P. Colpo Nanotechnology and Molecular Imaging Unit European Commission Joint Research Centre Institute for Health and Consumer Protection Via E. Fermi, TP203, 21020 Ispra, VA (Italy) E-mail: francois.rossi@jrc.it [!!] The authors are very grateful to Mr. Takao Sasaki of the Nanotechnology and Molecular Imaging unit of the Institute for Health and Consumer Protection, Ispra, for the SEM images. This work has been performed in the framework of JRC Action 15008: ‘‘Nanobiotechnology for Health’’. : Supporting Information is available on the WWW under http:// www.small-journal.com or from the author.

Nanopatterned Surfaces for Bio-Detection

The development of advanced biosensor devices for very sensitive detection is highly required for many applications. The careful design of the bio-interface on the transducer surface is known to be one of the major bottlenecks for the development of high performance sensing devices. This paper reviews the emerging role of nanopatterned surfaces as alternative bio interface in the field of bio-detection. The different material used to functionalize this type of surfaces and the fabrication methodologies are described. Finally, the application of these surfaces in bio-detection using different biological systems and detection techniques is presented. In particular, a recent and very promising approach based on the optical interaction of nanoarrays with Surface Plasmon Resonance detection is described.

Interaction among plasmonic resonances in a gold film embedding a two-dimensional array of polymeric nanopillars

Nanostructured surfaces have proven to be effective in controlling the electric field distribution and triggering a series of interesting physical effects. In particular, ordered metallic lattices with a typical size of the same order of magnitude of the wavelength of the incident radiation exhibit extraordinary transmission and reflection properties and represent a sensitive tool to exploit surface plasmon resonance for sensing applications. We investigated, either by experimental structural and optical measurements or by modeling and calculations, samples consisting of a two-dimensional array of polymeric pillars embedded in a gold film. In particular, we analyzed the dependence of the plasmonic resonance on the pillar size. We showed that a peculiar interplay among localized modes and propagating surface plasmon polaritons exists for some selected conditions and affects the spectral distribution, lifetime, and field configuration of the plasmonic excitations.

Quantification of the cellular dose and characterization of nanoparticle transport during in vitro testing

The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods. This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane. The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.BackgroundThe constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods.ResultsThis work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane.ConclusionThe fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.

Fabrication of Bio-Functionalised Polypyrrole Nanoarrays for Bio-Molecular Recognition

The present study demonstrates that nanosphere lithography and electro-polymerization can be successfully combined to produce bioactive protein nanoarrays. In particular, we describe a method to produce well-defined nanoarrays of polypyrrole functionalized with biomolecules. The nanoarrayed surfaces were fabricated on gold coated surface plasmon resonance prisms by first creating silicon oxide or polyethylene oxide nanotemplate using nanosphere lithography. The nanotemplate was subsequently used to grow bio-functionalized polypyrrole nanoarrays by electrocopolymerization. Atomic force microscopy analysis showed that the fabricated surfaces have a well-organized 2D hexagonal geometry with nanoscale dimensions. The biological activity of the bio-functionalized polypyrrole was assessed by surface plasmon resonance detection. The results showed that the immobilized biomolecules within the nanoarrayed polypyrrole films had the necessary bioactivity for successful molecular recognition. Moreover the detection signals normalized to the bioactive area were increased by a factor 5 as compared to non-structured bio-functionalized polypyrrole in the nanoarrayed surfaces using polyethylene oxide.