Marie Henry

Surface spectroscopy of adsorbed proteins: input of data treatment by principal component analysis.

X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (ToF–SIMS), two surface-sensitive spectroscopic methods, are commonly used to characterize adsorbed protein layers. Principal component analysis (PCA) is a statistical method which aims at reducing the number of variables in complex sets of data while retaining most of the original information. The aim of this paper is to review work carried out in our group regarding the use of PCA with a view to facilitate and deepen the interpretation of ToF–SIMS or XPS spectra acquired on adsorbed protein layers. ToF–SIMS data acquired on polycarbonate membranes after albumin and, or insulin adsorption were treated with PCA. The results reveal the preferential exposure of particular amino acids at the outermost surface depending on the adsorption conditions (nature of the substrate and of the proteins involved, concentration in solution), giving insight into the adsorption mechanisms. PCA was applied on XPS data collected on three different substrates after albumin or fibrinogen adsorption, followed in some cases by a cleaning procedure with oxidizing agents. The results allow samples to be classified according to the nature of the substrate and to the adsorbed amount and, or the level of surface coverage by the protein. Chemical shifts of particular interest are also identified, which may facilitate further peak decomposition. It is useful to recall that the outcome of PCA strongly depends on data selection and normalisation.

Surface analysis of an encapsulation membrane after its implantation in mini-pigs.

The biocompatibility of membranes aiming at being a part of a bioartificial pancreas has been tested. For that purpose, we have studied a polycarbonate membrane surface after its implantation in mini-pigs. The membranes were made hydrophilic by an argon plasma surface treatment followed by a dipping in a hydrophilic polymer solution. Two polymers were tested: polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC). To test their biocompatibility, an encapsulation device for pig Langerhans islets, with external membranes treated as described above, was implanted in different mini-pigs. The pigs received no further treatment. The devices were explanted after in vivo exposure and the membranes were analysed by XPS (x-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). After this time, the substrate with the PVP or HPMC treatment was still detected on the different samples. The surface treatment signal, however, was attenuated. This is explained by the detection of other components partly covering the surface. XPS and ToF-SIMS analyses revealed the presence of biological molecules on the two faces of the membrane: the outside face in contact with the biological environment and the inside face in contact with the device. ToF-SIMS images show the inhomogeneity of the biological molecules on the membrane surface. In conclusion, biological molecules adhered to the encapsulation membrane surface after implantation but the surface treatments remained unaltered.

Characterization of Polyethylene Glycol Self-Assembled Monolayers by Means of Sum-Frequency Generation Spectroscopy for Biosensor Applications

Protein biochips are miniaturized biological sensors intended to analyze and characterize biomolecule interactions with high throughput. An important issue when developing such biochips is substrate passivation. The support is rendered inert in order to avoid non-specific adsorption. Strategic control of the non-specific protein adsorption can be achieved by creating a resistant self-assembled monolayer (SAM) based on polyethylene glycol (PEG). The degree of resistance depends on the PEG surface density, i.e. the number of PEG units the molecule contains. Infrared-visible sum-frequency generation (SFG) spectroscopy (Lambert et al., Appl Spectrosc Rev 40:103–145, 2005) is used to in-vestigate the vibrational fingerprint of a PEG self-assembled monolayer adsorbed on a flat platinum surface, in the 2,750–3,050 cm−1 frequency range. The objec-tive is to characterize the SFG baseline of the biosensor that will be further developed by mixing the PEG antifouling layer with bioactive host molecules. Nanostructures will then be implemented on the substrate in order to enhance the SFG signal through localized surface plasmon resonances (Lis et al., Adv Opt Mater 1:244–255, 2013). The ultimate goal will be to detect the SFG signature of the antigen/antibody recognition process at the interface of the above biosensing layer.