Chelating Surfaces for Oriented Human Serum Albumin Molecules

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Interfaces: Adsorption, Reactions, Films, Forces, Measurement Techniques, Charge Transfer, Electrochemistry, Electrocatalysis, Energy Production and Storage

Chelating Surfaces for Oriented HSA molecules Nunzio Tuccitto, Grazia Maria Lucia Messina, Giovanni Li Destri, Aleksandra Wietecka, and Giovanni Marletta Langmuir, Just Accepted Manuscript • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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SCHEME 1- Gold-coated quartz crystal activation and functionalization with GHK peptide and GHK-Cu(II) 108x30mm (300 x 300 DPI)

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Figure 1- Frequency shifts a) and dissipation shifts b) for activation of carboxylic group by using EDC/NHS solution, frequency shift c) and dissipation shift d) for the adsorption of GHK peptide on activated carboxylic groups. Only the third overtone is shown, due to its higher sampling depth.


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Figure 2- Tapping mode 3D AFM images of a) GHK; b) HSA adsorbed on GHK and c) HSA adsorbed on GHKCu(II) complex. Roughness of samples was reported.


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Figure 3- a) Typical force curve acquired on bare GHK monolayer anchored on surface, b) experimental histogram of the related adhesion force. 199x76mm (300 x 300 DPI)


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Figure 4- Experimental histogram of adhesion force of HSA molecules adsorbed on a) GHK b) GHK-Cu(II) complex.


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Figure 5- Changes in frequency and dissipation plotted against each other, D-f plot, of HSA on GHK (blue circle) and on GHK-Cu(II) complex (red square). The lines indicate the changes in slope, i.e., different protein rearrangement processes on surface. 289x209mm (300 x 300 DPI)


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Figure 6- QCM-D measurements of HSA and anti-HSA on a) and b) GHK, c) and d) GHK-Cu(II) complex. Blue traces: normalized frequency changes; red traces: corresponding changes of dissipation for different overtones (3rd-13th). The bold solid traces belong to the 3rd overtone. The first trace (until 1 h) represents protein adsorption, while the trace at higher time represents antibody adsorption.


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Figure 7- a) HSA ng/cm2 as a function of time calculated by the standard RSA model during HSA adsorption onto GHK surfaces. b) HSA ng/cm2 as a function of time calculated by the modified RSA model during HSA adsorption onto the chelating surface.


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TOC 73x44mm (300 x 300 DPI)


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Figure S1- Drops of water dispensed on GHK peptide and GHK-Cu(II) complex


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Figure S2- Tapping mode 2D AFM images of a) GHK; b) HSA adsorbed on GHK and c) HSA adsorbed on GHK-Cu(II) complex.


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Chelating Surfaces for Oriented HSA molecules N. Tuccitto1, G.M.L. Messina1*, G. Li-Destri1, A. Wietecka1,2, G. Marletta1 1 Laboratory

for Molecular Surfaces and Nanotechnology (LAMSUN), Department of Chemical

Sciences, University of Catania and CSGI, Viale Andrea Doria 6, 95125, Catania, Italy 2 Adam Mickiewicz University in Poznan, Faculty of Chemistry, Umultowska 89b, PL-61-614, Poznan,

Poland

Abstract The protein immobilization in a specific conformation or orientation at interface is influenced by specific interactions with the outer layer of surface. A strategy to build-up a complex construct able to orient protein molecules, based on metal cation chelation processes, is reported. The proposed methodology implies the formation of a mercaptoundecanoic acid monolayer on gold surface that is activated to attach covalently the tripeptide GHK on surface, whose sites are then employed to chelate copper ions, providing a selective platform for the orientation of Human Serum Albumin molecules. The protein adsorption process on GHK and GHK-Cu(II)-complex surfaces was monitored by in situ quartz crystal microbalance with dissipation monitoring and force spectroscopy technique. The changes in frequency and dissipation factor as well as the D-f plots from QCM-D measurements helps to characterize the changes in the protein conformation and are confirmed by force curve spectroscopy results. An improved kinetic model, based on Random Sequential Adsorption with variable protein footprints, has been developed to predict and simulate the experimentally found HSA average surface coverage onto GHK and GHK-Cu(II)-complex surfaces.


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Introduction The control of proteins adsorption at solid surfaces, i.e., their immobilization in the desired conformation and/or orientation, has been extensively studied because of its widespread application in the ﬁelds of biocatalysis, biomedicine and biomaterials and, in general, in the broader area of biotechnology [1]. Thus, the success of biomedical devices, as different as prosthesis, biosensors, scaffolds for tissue engineering or bioreactors, and of industrial biotechnological processes is based on the optimization of the biological response of immobilized enzymes, yeasts and antibodies, i.e., it critically depends on the maintenance of their “originary” structure, i.e., to the native state conformation [2]. However, it is well known that the protein adsorption on surfaces, is generally associated to a severe structural reorganization, prompted by a balanced enthalpic and entropic gain [3-5]. Indeed, protein molecules approaching to solid surfaces do not behave like rigid particles, but rather they may undergo many reorganization processes simultaneously, including structural rearrangement [6], cooperative effect [7], different adsorption kinetics [8], size exclusion effects [9], change surface affinities or surface aggregation [10], following the synergic effects of many different proteinsurface interactions, including – among the others – the presence of electrically charged domains, the substrate morphology and surface free energy [11-14]. Thus, controlling the conformation of the immobilized proteins, is a prerequisite to control and drive both the cell response [15-18], as well as the efficiency of biomolecule-based reactions at biofunctional surfaces [19-20]. In this framework, in previous papers we have shown that proteins can be oriented, i.e., determining the exposure of suitable epitopes and/or keeping the proper biological functionality, by handling different factors, including the coadsorption from binary solutions [21-22], nanostructuring of surfaces [23]; the surface curvature at nanoscale [24-25] and the chelation action onto layers exposing transition metal cations [26-29]. With particular reference to the last technique mentioned above, we have shown that biomedically relevant proteins can be adsorbed in their quasi-native state onto functionalized surfaces consisting ACS Paragon Plus Environment

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of complexing transition metal cations [21, 30], including Ni (II), Co (II), Fe (II) and Cu (II), in turn anchored onto suitable SAM layers. It turns out that the chelating transition metal cations seem able to promote the protein adsorption in native-like state, through the formation of intrinsically oriented (and relatively strong) bonds [27-29]. In this paper, the “chelation strategy” has been further developed by building constructs with orienting sites for the adsorbing biomolecules. In particular, a mercaptoundecanoic acid SAM has been deposited on gold surface and the carboxylic groups have been activated to link covalently the glycyl-L-histidyl-L-Lysine (GHK), a tripeptide with an anchoring site with well-defined coordination (see below), which shows a high affinity for Cu(II) cations, forming a planar coordination GHK-Cu complex. It is also known to form binary and ternary structures which can involve histidine and/or the copper binding region of the albumin molecule [30-32]. The origins of this behaviour are not yet understood, but their unravelling would prompt the possible extension of the methodology as a general method to drive protein adsorption in a desired conformation, by carefully selecting the metal cation, and in turn the coordination structure of interest. The multistep construction of the GHK-Cu complex and the related HSA-adsorption features have been studied by means of Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) [33-34], providing unique semiquantitative information on the formation and viscoelastic properties of the chelation layer and the subsequent protein adsorption processes [21, 27], and Dynamic Force Spectroscopy (DFS), which allowed to determine the strength of interaction between the tip and the protein adsorbed on chelating layer [35-36]. Finally, we have implemented a simple kinetic model, based on Random Sequential Adsorption (RSA) [37-38], accounting for the formation of a protein film with a native-like character.

Experimental Surface preparation Gold-coated Silicon wafer (100nm coating Sigma-Aldrich, UK) and gold-coated sensors crystals ACS Paragon Plus Environment

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for QCM-D analysis (Q-Sense) were used as substrate. The surfaces were cleaned individually by 20 minutes of UV-Ozone treatment, then they were rinsed by ethanol and gentle dried with nitrogen. The substrates of gold on which the experiments were carried out have a comparable roughness (about 1.1 ± 0.1 nm). Surface functionalization Monolayers of 11-mercaptoundecanoic acid (Sigma-Aldrich, UK) were deposited from ethanolic solution, at a concentration of 1mM, onto cleaned gold substrates, which were immersed in thiol solution for about 18 hours, rinsed with ethanol, dried by nitrogen. Carboxylic groups of thiol SAM were activated in situ with a solution of 1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide (ECD) 0.4M, in phosphate buffer solution (PBS) and N-Hydroxy succinimide (NHS) 0.1M, in phosphate buffer solution (PBS) in ratio 1:1. A solution of glycyl-L-histidyl-L-lysine (GHK) 0.1mM, in phosphate buffer solution (PBS) was used to anchor covalently the tripeptide on the activated groups. To obtain the GHK-Cu complex an aqueous solution of CuSO4 (0.1 mM) has been used. The substrate with the tripeptide was immersed into the copper solution for 120 seconds to coordinate the copper (II) cation with the histidine and glycine aminoacids of GHK peptide, followed by rinsing step. Protein adsorption Measurements of adsorption kinetics were performed by using a Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) instrument (Q-Sense AB, Sweden) with AT-cut gold crystals sensors. The measurement chamber was operating in phosphate buffer saline (PBS, 0.01M, 0.137 M NaCl, pH 7.4) at 25 ± 0.1° C. The simultaneous measurements of frequency, f, and energy dissipation, D, were performed for the fundamental resonance frequency (n = 1, i.e., f ~ 5 MHz) and the six overtones (n = 3, 5, 7, 9, 11 and 13 corresponding to f ~ 15, ~ 25, ~ 35, ~ 45, ~ 55 and ~ 65 MHz, respectively). In the cases of rigid, evenly distributed and sufficiently thin adsorbed layers, the frequency to mass conversion was simply obtained by using Sauerbrey equation [39]: M= −(C/n)·f, where f is the decrease in resonant frequency, M is the mass uptake at the sensor surface, ACS Paragon Plus Environment

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C is a constant depending on the intrinsic properties of quartz slab (in our case C = 17.7 ng/cm2 Hz−1 at f = 5 MHz) and n (=1, 3,. . .) is the overtone number. The resolution in f and D is ±0.1 Hz and 1 × 10−7, respectively [40]. Each QCM-D experiment started with the sensor running in buffer solution (PBS) (outgassed with 30 min of sonication), then the addition of the human serum albumin (HSA, Sigma-Aldrich, UK) protein solution (1 mg/ml) and, after 1 hour the exchange of the protein solution being measured with PBS, to check both desorption and stability of the adsorbed layer. Then the antibody solution (Anti-HSA), specific for denaturated HSA recognition (10 µg/ml) was added and its interaction with the adsorbed proteins was followed for one hour at 25 °C and then rinsed twice with PBS. All the experiments were performed in buffer solution at 25°C and the flow rate was 150 μL min−1. Three replicas for each experiments were performed for data reproducibility Experimental techniques The surface wettability of each sample was determined by sessile drop measurements of static water contact angle (), using an OCA30 instrument (Dataphysics) at 25°C and 65% relative humidity. Probe liquid drops of 2 μl of volume were applied on different zones of each sample surface and by digital image analysis the static contact angles were measured on both sides of the two-dimensional projection of the droplet. At least three measurements were made for each sample and are presented as mean  standard deviation. The water adhesion tension () was calculated by  =  cos  where  is the measured water contact angle and  = 72.8 mN/m is the surface tension of water[13]. Topographical images were obtained using a commercial Nanoscope IIIa Multimode AFM Instrument (Digital Instruments, Santa Barbara, CA, USA). The device was equipped with a calibrated scanner using grating manufacturers. All samples were observed in tapping mode (TM) using 0.5-2 Ωcm Phosphorous n-doped silicon tips mounted on cantilevers with a nominal force constant of 40 N/m and a resonant frequency of 300 kHz. Image analysis was carried out using DI ACS Paragon Plus Environment

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software, version 4.23r6. The images were flattened to remove background slopes. Average roughness (Rq) was analysed by Nanoscope software. For adhesion force measurements, AFM microscope (NT-MDT) was operated in contact mode with silicon nitride tips, Au coated. The spring constants of cantilevers used were in the range 0.003-0.13 N/m. Individual spring constants were calibrated using the Sader method. The adhesion force is taken as the pull-off force required to separate the AFM tip from surface. The surfaces were probed in different areas and about three hundred approaches-retract cycles were acquired for each system. The tip-sample approaching velocity was set to 0.3 µs-1 for all force curves. The adhesion force between tip and surface can be calculated from the deflection distance of cantilever and the cantilever spring constant by applying the Hook’s law: F= k x ∆L Where F is the force (nN), k is the spring constant of cantilever and ∆L is the deflection distance (nm). The cantilever spring constants were equal to 0.02 N/m for GHK on functionalized gold surface and 0.127 N/m for HSA on GHK and HSA on GHK in presence of copper ions. Montecarlo simulation The RSA model was simulated by means of the Python-based script reported in supplementary information. We used the Python 2.7.

Results and Discussions Preparation of the functionalized substrates Many efforts have been done to develop approaches for coating metal surfaces before immobilization to minimize non-specific adsorption and to introduce reactive groups for specific immobilization. The most successful methods are based on the use of molecular self-assembly of thiol molecules on the metal surface. In this work 11-mercaptoundecanoic acid has been used to obtain a self-assembled monolayer on gold surface. The monolayer has been further chemically


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processed to attach to the carboxylic groups a reactive nucleophile functionality, to covalently bind in turn the GHK peptide. Scheme 1 reports a sketch of the process of GHK anchoring onto surface by means of the welldefined EDC/NHS stepwise protocol [41] onto carboxylic acid terminated monolayer selfassembled onto gold-coated quartz crystal sensor for QCM-D. Two different substrates have been prepared to investigate the protein behaviour in presence or in absence of GHK complex (hereafter GHK-Cu(II)) obtained by interaction of peptide with a solution of copper ions.

EDC/NHS + GHK

Cu(II)

Gold

Gold

Gold

Gold

QCM quartz

QCM quartz

QCM quartz

QCM quartz

SCHEME 1- Gold-coated quartz crystal activation and functionalization with GHK peptide and GHK-Cu(II)

In the GHK-Cu(II) complex, the copper ion is coordinated by three nitrogen atoms, one from the imidazole side chain of the histidine, the other one from the alpha-amino group of glycine, and the last one from the deprotonated amide nitrogen of the glycine-histidine peptide bond. The ion is also coordinated by the oxygen from the carboxyl group of the lysine from the neighbouring complex and another carboxyl group of lysine from a neighbouring complex provides the apical oxygen, resulting in the square-planar pyramid configuration [31-32]. The activation process of the carboxylic group anchored on gold sensor surface was monitored by using in-situ QCM-D measurements, as shown in Fig. 1a-b). The success of the activation process is clearly demonstrated by the frequency and dissipation change with the reaction progress. Upon addition of EDC/NHS solution (at t=0), a rapid frequency decrease (Fig. 1a)) corresponding to NHS anchoring was observed, quickly reaching saturation. The value of D increases with a similar profile (Fig. 1b)), indicating the higher energy dissipated as more molecules are immobilized. Mild ACS Paragon Plus Environment

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rinsing easily induces the wash-off of the physically adsorbed molecules, leaving only the covalently anchored thiols functionalized with NHS groups. Since the final dissipation value is very low, the experimental condition satisfies the requirement for the application of the Sauerbrey equation, enabling to simply calculate the mass uptake and the related number of anchored molecules, i.e., respectively 159.3 ± 12.3 ng/cm2, corresponding to about 6.2x1014 NHS/cm2, in fair agreement with the expected number of immobilized thiol molecules in a regular self-assembled monolayer (~1014 molecules) [42]. This agreement reflects the fact that all carboxylic groups present to the surface may have been activated in this first step. After the activation of carboxylic groups, the glycyl-L-histidyl-L-lysine (GHK) peptide anchoring has been investigated by QCM-D experiments. Fig. 1c shows a very low frequency due to the reaction between NHS and GHK molecules. The rinsing step does not affect the adsorbed mass, suggesting a strong stability of the GHK-Thiol constructs. Again, the very low dissipation, (Fig. 1d)) enabled to applying the Sauerbrey equation, obtaining an adsorbed mass of 59.7 ± 21.3 ng/cm2, corresponding to ~1.1x1014 GHK/cm2, confirming the effective formation of a peptidefunctionalized stable and rigid monolayer at the gold surface.

Figure 1- Frequency shifts a) and dissipation shifts b) for activation of carboxylic group by using EDC/NHS solution, frequency shift c) and dissipation shift d) for the adsorption of GHK peptide on activated carboxylic groups. Only the third overtone is shown, due to its higher sampling depth. ACS Paragon Plus Environment

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The thiol-GHK substrate has been finally treated with a solution of Cu(II) ions, in order to complex the specific Cu(II)-binding site of GHK, preparing a suitable substrate for the oriented Human Serum Albumin recognition [27]. Indeed, GHK-Cu(II) complexes can form binary and ternary structures which may involve amino acid histidine and/or the copper binding region of the albumin molecule. Structure and properties of the functionalized substrates Among the properties of surface, it is known that wettability affects protein adsorption, generally hydrophobic surfaces are considered to be more protein-sorbent than the hydrophilic ones because of the strong hydrophobic interactions occurring at these surfaces, in direct contrast to the adhesionhindering solvation forces arising from strongly bound water at the hydrophilic surface [14]. The investigated surfaces have different wettability, that is related to the adhesion tension of water. In particular, the surface functionalized with GHK peptide, with a water contact angle of about 30°, is characterized by an adhesion tension of 62.0 mN/m, whereas in presence of copper ions the surface wettability increases (13°), and the adhesion tension increases becoming 70.9 mN/m (Fig. S1 and Table S1).

Figure 2- Tapping mode 3D AFM images of a) GHK; b) HSA adsorbed on GHK and c) HSA adsorbed on GHK-Cu(II) complex. Roughness of samples was reported.

The morphology has been analysed by Atomic Force Microscopy. Figure 2 shows the topography images of GHK layer, covalently bound on thiol-SAM previously anchored on surface (Fig. 2a), ACS Paragon Plus Environment

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showing very small aggregates that confer to the surface a roughness of 1.2 nm. The adsorption of HSA on both surfaces, i.e., in absence and in presence of copper ions (Fig. 2b-c) replicate a very similar morphology and roughness, suggesting the homogeneous coverage of the adsorbed protein layers. Figure 3 shows a representative force-distance curve, acquired on the bare surface functionalized with GHK, and the corresponding histogram of adhesion forces. It represents a classical force curve characterized by a single-peak pull-off force. This indicates that the contact topology between the tip and the surface does not change significantly during the measurement. As the GHK peptide is covalently anchored on functionalized surface, forming a well-packed monomolecular layer on the top of the surface, we assume that the interaction force with the tip probe is somewhat limited by its blocked conformation. Accordingly, the measured adhesion values have a narrow distribution, with an average adhesion force of 1.2 ± 0.1 nN.

Figure 3- a) Typical force curve acquired on bare GHK monolayer anchored on surface, b) experimental histogram of the related adhesion force.

Protein adsorption processes on the functionalized substrates The distribution of pull-off forces measured after the adsorption of HSA on GHK and GHK-Cu(II) monolayers respectively is reported in figure 4. It can be seen that the average adhesion on GHKHSA is about 5.1 ± 0.2 nN, whereas on the GHK-Cu(II)-HSA complexes it decreases to a value of 3.4 ± 0.2 nN. This result shows that the presence of copper ions modifies both the adhesion force ACS Paragon Plus Environment

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distribution and their average value. More important, the observed change of the adhesion force with the substrate confirms that the features of the protein adsorption process, and in particular the molecular conformation and the layer density, are strongly affected by the substrate nature.

Figure 4- Experimental histogram of adhesion force of HSA molecules adsorbed on a) GHK b) GHK-Cu(II) complex.

In order to investigate in more detail, the above results, QCM-D has been employed for the proteinsubstrate interaction, owing to its capacity of discriminating whether the surface nature mainly affects the amount of adsorbed protein or its conformation, since it allows to simultaneously measure the wet mass and the viscosity of the thin layer at the surface of the crystal. Figure 5 reports the plots of ∆D against ∆f (D-f plot) which provides information on the adsorption kinetics of the HSA protein on GHK and GHK-Cu(II) complex and on the subsequent conformational rearrangement. These data eliminate time as an explicit parameter and they make possible to directly compare the ratio between dissipation and frequency shifts [43], i.e., the induced energy dissipation per coupled unit mass, and in turn, the influence of the protein adsorption on the viscoelastic damping of the crystals resonance, related to the viscoelastic properties of the overall GHK-HSA and GHK-Cu(II)-HSA constructs. A low ∂D/∂f value indicates mass addition without significant dissipation increase, characteristic of a fairly rigid layer, whereas a large ∂D/∂f value indicates the formation of a soft, dissipative layer. The data in figure 5 show that the adsorption process can be described with two subsequent linear steps, with two different values of the slope ∂D/∂f for both substrates. It is to note that the ∂D/∂f data for step 1 of HSA


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adsorption onto the two surfaces are very close, i.e., 3.8x10-8 Hz-1 and 4.7x10-8 Hz-1for GHK and GHK-Cu(II) complex respectively, suggesting that, in step 1, HSA is adsorbed on both surfaces with a similar average conformation. At variance of this, the ∂D/∂f slope of step 2 for GHK-Cu(II) substrates decrease from 4.7 x 10-8 Hz-1 to 0.5 x 10-8 Hz-1 at |∆f | = 9 Hz and t = 304 sec suggesting that the further adsorption produces a very rigid layer, while the ∂D/∂f slope of step 2 for GHK substrate steeply increases from 3.8 x 10-8 Hz-1 to 9.1 x 10-8 Hz-1 at |∆f | = 10.5 Hz and t = 300 sec, indicating that HSA adsorbs in a soft conformation.

Figure 5- Changes in frequency and dissipation plotted against each other, D-f plot, of HSA on GHK (blue circle) and on GHK-Cu(II) complex (red square). The lines indicate the changes in slope, i.e., different protein rearrangement processes on surface.

In order to gain further insight on the different conformation adopted by HSA on the different substrates, we have studied by QCM-D the adsorption of a monoclonal antibody, able to detect the unfolded state of HSA, on GHK and GHK-Cu(II).


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Figure 6- QCM-D measurements of HSA and anti-HSA on a) and b) GHK, c) and d) GHKCu(II) complex. Blue traces: normalized frequency changes; red traces: corresponding changes of dissipation for different overtones (3rd-13th). The bold solid traces belong to the 3rd overtone. The first trace (until 1 h) represents protein adsorption, while the trace at higher time represents antibody adsorption.

The QCM-D adsorption trends for HSA and its monoclonal antibody (anti-HSA) are reported in figure 6. It can be seen that the antibody is quickly adsorbed onto the pre-adsorbed protein, producing a marked decrease in frequency shift and a small increase in dissipation (D < 5 x 10-6). Since the dissipation shift value satisfies the requirement for the application of Sauerbrey equation [44], the adsorbed mass was calculated accordingly (see table 1). The adsorbed mass of HSA is quite similar both for adsorption on GHK (~323.9 ng/cm2) and GHKCu(II) complex (~385.6 ng/cm2), while anti-HSA, normalized to the corresponding adsorbed HSA, is roughly a factor 2 more adsorbed on GHK monolayers (~467.9 ng/cm2), with respect to GHKCu(II) (241.9 ± 13.5 ng/cm2).


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This fact can be explained by assigning the higher antibody adsorption, specific for denaturated albumin molecules, onto GHK to the higher probability of a higher exposure of the antibodyrecognition epitopes for mostly denatured conformations, adopted by HSA molecules when interacting with GHK monolayers. In the same way, the lower number of bound antibody molecules for GHK-Cu(II) can be assigned to the much higher percentage of unfolded molecules adsorbed on the chelating layers. In short, the results suggest that about 50% of the adsorbed HSA molecules onto GHK-Cu(II)-complexes do not adopt any conformation enabling the interaction with Anti-HSA. Furthermore, the overtones for HSA and its monoclonal antibody adsorbed onto bare GHK are largely spread out, with respect to the overlapping overtones observed for GHK-Cu(II) complex. The large spread of overtones found on bare GHK monolayers (fig.6a) suggests that, as far as the overtone sampling depth is different for each overtone number, a vertically inhomogeneous adsorbed layer is formed in this case. This inhomogeneity, which is also maintained with rinsing treatments, is in agreement with the different conformational stats of HSA onto GHK surfaces, owing to the random orientation and the weak protein binding on these monolayers, mainly characterized by van der Waals and dipolar forces. At variance of this, the much smaller spread of the ∆f and ∆D overtones for the HSA and anti-HSA adsorption onto the GHK-Cu(II) layers (Fig. 6b) suggests a strongly bound and densely packed layer, in agreement with the stronger and orientationally constrained binding due to the inherent binding symmetry of the Cu(II) chelation sites. It is to mention that these results are well cogent with the above reported differences in the adhesion forces on the two substrates. Table 1- QCM-D data of frequency shifts (f), dissipation (D), the calculated Sauerbrey masses and number of molecules (expressed as mean and standard deviation)

∆f (Hz)

∆D (10-6)

Mass (ng/cm2)

Molecules/cm2

EDC/NHS

-9.0 ± 0.7

0.4 ± 0.3

159.3 ± 12.3

6.2x1014 ± 0.5x1014

GHK

-3.4 ± 1.2

0.2 ± 0.3

60.2 ± 21.2

1.1x1014 ± 0.4x1014

HSA/GHK

-18.3 ± 1.0

0.5 ± 0.3

323.9 ± 17.7

2.9x1012 ± 0.2x1012

Anti-HSA/GHK

-26.4 ± 3.8

1.5 ± 0.9

467.4 ± 67.9

1.9x1012 ± 0.3x1012


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HSA/GHK-Cu(II)

-21.8 ± 0.2

0.6 ± 0.1

385.6 ± 4.4

3.5x1012 ± 0.4x1011

Anti-HSA/GHK-Cu(II)

-13.7 ± 0.8

1.2 ± 0.1

241.9 ± 13.5

1.0x1012 ± 0.5x1011

In order to analyse the time dependence of the adsorption processes on the different surfaces, we have performed MonteCarlo simulations introducing a kinetic model by implementing the standard random sequential adsorption (RSA) model. Indeed, the standard RSA model, the increase of the adsorbed protein amount  with respect to adsorption time is given by: ∂𝛤(𝑡) = 𝐾 𝛷(𝑡) ∂𝑡 In the model the molecules sequentially interact with randomly distributed binding sites with a rate K and a sticking probability (t). RSA model can be analytically solved in 1 dimension but the solution in 2D cannot be obtained without approximations. In order to simulate the experimental protein/surface interaction, we have used a numerical procedure. In particular, the standard model assumes that: a) only monolayer deposition is allowed, b) the protein footprint is circular with diameter d, and c) that each new arriving protein molecule must interact with a free binding site, fitting suitably empty areas. In other words, the c) assumption implies that a new landing protein can adsorb only if it does not overlap any previously adsorbed molecule. In figure 7a) we report the output obtained from simulating a protein surface collision frequency of about 3·1011 molecules·s1·cm-2

and around 30 nm2 footprint, in agreement with the diffusion-controlled spontaneous

adsorption of protein molecules reported in literature [45]. It confirms that the HSA adsorption follows the mechanism leading to the formation of the so-called "jammed-state", where there is no more room for further molecules adsorption.


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Figure 7- a) HSA ng/cm2 as a function of time calculated by the standard RSA model during HSA adsorption onto GHK surfaces. b) HSA ng/cm2 as a function of time calculated by the modified RSA model during HSA adsorption onto the chelating surface.

The adsorption kinetic onto GHK-Cu(II) complexes is markedly different from the one in which the HSA spontaneously adsorbs on GHK. Indeed, in this case an initial abrupt adsorption of about 60% of molecules is observed, followed by a slower adsorption of further molecules, reaching saturation at extremely long time corresponding to the adsorption of 3.5·1012 molecules/cm2. Such trend cannot be explained by classic RSA mechanism. Accordingly, we have modified the classic RSA model in order to account for this peculiar adsorption kinetics. We hypothesize that, given the high HSA affinity for binding to Cu(II), if the protein hits the surface with the proper orientation, it can tightly bind to the Cu(II) sites by chelation without strong conformational reorganizations. In particular, HSA contains four metal binding sites, namely, the N-terminal site (NTS, also known as ATCUN), Cys34 residue, and its environment, site A identified in NMR studies (and later shown to be identical with the multimetal binding site (MBS)), located at the interface of domains I and II, and site B. The most important Cu(II)- binding site is the N-terminal site (NTS), composed of the first three residues of albumin sequence, e.g., Asp-Ala-His in human protein. [46-49]. In the assumption that adsorbed native-like proteins have smaller footprint (d1) than denatured ones (d2), the simulation iteratively works following four steps: a) protein orientation is randomly extracted,


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b) two circular footprints are allowed, d1 and d2 (we impose d2 > d1), c) the percentage of the two footprint probabilities, P, is a priori imposed: c1) if the protein arrives at the surface with the proper orientation, then the smaller circular footprint is used, c2) if not, the bigger circular footprint is used d) new landing protein can adsorb only if it does not overlap any previously adsorbed molecule. Figure 7b) shows the results obtained by means of such modified-RSA simulation. In particular, after minimization running to adjust model parameters, the best experimental data fitting was obtained by using the values of d2/d1=2.2 and 5% of incoming molecules having the proper orientation to interact with the Cu(II) chelating site [46]. Although the model is rather simplified and approximated, the overlap between the simulated curve and the experimental ones (see QCM-D data fig.S4, SI) is quite satisfactory. The trend of adsorption of molecules having different footprints suggests that, in a first phase, adsorbed molecules are mostly randomly adsorbed with a larger footprint (blue line). Indeed, the probability of the protein sticking with the smaller footprint is only 5%. Since the denatured protein occupies larger space on the surface, with increasing surface crowding, the area available for adsorption of more denatured proteins quickly decreases and the jammed state is readily achieved (within 250 s). Conversely, proteins adsorbing in the native-like state can occupy interstitial spaces, thanks to their lower footprint, matching the free spaces among the unfolded molecules. Therefore, the trend of adsorption of native-like proteins is slower but, at the steady state, up to about of 60% of adsorbed molecules is in the native-like state. Noticeably, this amount is very close to the percentage of non-anti-HSA-binding proteins adsorbed onto GHK-Cu(II) (see Table 1) suggesting that the observed conformational differences with substrate nature may be partly due to the adsorption of higher amounts of native-like proteins.


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Conclusions In the present paper we have reported a methodology, based on metal ion chelation processes, that can be used to vary the conformation of proteins during surface adsorption and to provide information about average adsorbed protein orientation. The surface has been functionalized by covalently anchoring tripeptide GHK onto activated mercaptoundecanoic acid SAM, and further complexing the GHK moieties with Cu(II), giving rise to a chelating GHK-Cu(II)-complex, having a well-defined planar coordinating site for HSA. QCM-D and AFM force spectroscopy techniques showed that HSA protein molecules, while adsorbed in similar amount on both GHK and GHKCu(II)-complex sites, adopt different average conformation on the two substrates, as tested by using a monoclonal antibody that selectively recognize specific epitopes on the unfolded proteins. The conformational differentiation occurs with the adsorption time, as the D-f plot shows that, initially, adsorbed HSA adopts the same conformation on both substrates. A new kinetic model based on the modification of the Random Sequential Adsorption (RSA) approach, suggest that this conformational difference may be due to the crowding of the surface when denatured molecules adsorb facilitating, if the substrates bears the proper functional groups, the adsorption proteins in the native-like conformation.

Acknowledgements N. Tuccitto, G. Marletta acknowledge the Programme “Piano della ricerca di ateneo 2016-2018” (University of Catania) for partial financial support. G.M.L. Messina acknowledges FIRB 2013-Future in Research by MIUR for financial support.

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Figure S3- Frequency shifts (left hand side axis) and dissipation shifts (right hand side axis) for a) activation of carboxylic group by using carbodiimide solution, b) adsorption of GHK peptide on activated carboxylic groups.


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Figure S4- HSA molecules/cm2 as a function of time calculated by the standard RSA model (blue line) and experimentally measured by QCM during HSA adsorption onto NO-chelating surface


Chelating Surfaces for Oriented Human Serum Albumin Molecules

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