ANALISIS DE LOS RESULTADOS

Experimental characterization of bio-interfacial systems, such as determining the free energy of adsorption or adsorbate-surface geometry, presents a significant chal- lenge in itself. At present, surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) are the methods most commonly used to monitor the kinetics of, and infer thermodynamic data about, biomolecule adsorption. In the first, SPR, photons of light are passed through a glass prism, exciting surface electrons in the

conduction band of a thin sheet of metal attached. The resonance frequency for surface plasmon adsorption is dependent on the refractive index of the media in contact with the metal film. Therefore the rate of biomolecule-surface adsorption and desorption can be determined by monitoring SPR signal intensity. Fundamen- tal to SPR is the use of a metal which supports surface plasmons (e.g. Ag or Au) although techniques to extend this method to alternative inorganic substrates have been pioneered. For example, Sarikaya andco-workershave quantified peptide-silica adsorption by applying a thin layer of SixO to the gold surface of an SPR chip [Oren

et al. [2007, 2010]], while Cohavi et al. immobilized AuNPs onto a NeutrAvidin- coated surface to monitor protein-NP binding. However, the range of non-metallic interfaces to which SPR can be applied is limited to those which are topologically flat and thinner than the penetration depth of the incident light source.

A QCM device is composed of a piezoelectric quartz wafer placed between two gold plates. The resonance frequency of the quartz, in the presence of an AC electric field, is dependent on the mass of adsorbate adhered to the surface. Like SPR, with QCM, the range of substrates, alternative to quartz, is limited to those which can form a uniform coating of nanoscale dimensions.

Wei and Latour recently devised a protocol for using Atomic Force Mi- croscopy (AFM) to measure the binding affinity of peptides to substrates which cannot be studied by SPR or QCM [Wei and Latour [2010]]. Previously there were two fundamental obstacles to relating the desorption force measured in an AFM experiment to the free energy of adsorption: determining the number of peptides attached to an AFM probe tip that actually interact with a surface; and removing the contribution from the tip alone to surface adhesion. The latter can be overcome by using long flexible tethers between AFM tip and peptide of interest. To tackle the former hurdle, host-guest TGTG-X-GTCT peptides were used to calibrate AFM data against SPR measurements [Wei and Latour [2010]]. The aqueous alkanethiol self-assembled monolayers (SAMs) interface, suitable for SPR experiments as well as AFM, was used in this calibration stage. This enabled the free energy of adsorption of the same host-guest peptides to a range of other surfaces, including (100) quartz and fused silica glass, to be estimated from AFM experiment alone [Thyparambil et al. [2012]].

A full description of both the internal conformation of a biomolecule and its geometry in relation to a substrate, at the level of detail required for designing material-binding peptide sequences, remains elusive to obtain experimentally in all but a small number of cases. Many of the techniques commonly used to deter- mine peptide conformation in solution can not be extrapolated to the bound state.

Circular Dichroism (CD) spectroscopy is one such example. Using this technique, adsorption in the far UV region of the electromagnetic spectrum is monitored. The ensemble- and sequence-averaged likelihood that a peptide has a particular sec- ondary structural characteristic is found using reference databases containing the CD spectra of proteins of known conformation. However, the method relies on light passing all the way through a sample, making it intrinsically unsuitable for interfa- cial systems. However, promising recent advances have been made in two alternative fields–Sum Frequency Generation (SFG) Spectroscopy and Nuclear Magnetic Reso- nance (NMR).

Two photons of light are projected at an interface–one in the infra red (IR) and the other in the visible region–to obtain a SFG spectrum. Vibrational transi- tions arise from resonance in the second order susceptibility, χ, of the system. In- terfacial media can be selectively studied over bulk sinceχ=0 for all entities which include a point of inversion. By assigning adsorption bands to different X-H bonds (X=C, O, N), the average orientation of a biomolecule with respect to an interface can be determined. For example, Somorjai and co-workers used SFG to contrast the adsorption of amino acids [Holinga et al. [2011]] and homopeptides [York et al. [2009]] to a hydrophobic (polystyrene) and a hydrophilic (fused silica) surfaces under aqueous conditions. The absence of C-H stretching bands in a SFG spectrum can mean one of two things: no adsorbate is present; or that the adsorbate is randomly oriented. One of the major drawbacks of SFG therefore, is that it cannot be used to elucidate information on a single molecule level.

Out of all the experimental methods discussed, NMR perhaps holds the most promise for future experimental characterization of biointerfacial systems, with the possibility of obtaining both spatial and orientational data. Unlike SPR, however, it can not intrinsically discriminate between interfacial and bulk phases. This at present therefore limits its use to substrates with high surface areas (e.g. porous materials) and consequently necessitates with use of solid-state magic angle spinning (MAG) NMR, for which the resolution is much lower. Overcoming these difficulties, Ben Shiret al. studied the adsorption of alanine [Ben Shir et al. [2010]] and glycine [Ben Shir et al. [2012]] to amorphous silica in dry and microsolvated conditions using13C and15N labelled amino-acids. Adsorbate-silica geometry was determined in each case from inter- and intra-molecular distances.

Nuclear Overhauser Effect NMR spectroscopy (NOSEY), a technique com- monly used to investigate the native state of proteins in solution, was in 2011 suc- cessfully used by Mirauet al. to elucidate the adsorbed conformation of a peptide for the first time [Mirau et al. [2011]]; saturation-transfer difference (STD) NMR was

used in the same study to determine peptide-substrate orientation. The TBP-12 [Sano et al. [2005]] peptide adsorbed to either silica or titania NPs under condi- tions of rapid exchange was investigated. In this cutting-edge study, the sign and magnitude of the NOE signal–dependent on the rate of molecular reorientation in solution–was used to discriminate between bound and free states of the adsorbate. Cross peaks in a NOSEY spectrum identify residues in close spatial proximity to each other, while protons bound to a surface can be recognized from the difference between the standard NMR spectrum and one in which the signal from the hydroxyl groups and water is saturated. In a similar vein to STD, Calzolaiet al. had previ- ously employed chemical shift perturbation (CSP) NMR to identify patches on the surface of an isotopically labelled protein (ubiquitin) through which AuNP-binding was mediated [Calzolai et al. [2010]].