Interactions between proteins and the solid support are highly dependent on surface chemistry (reactive groups, wetting properties) and on physicochemical characteristics of proteins at defined pH. The global charge of a protein at a given pH is defined by its isoelectric point (pI), the pH value at which the global charge of the protein is neutral. For pH below the pI, the protein is positively charged, and negatively charged for pH above the pI. Surface chemistries developed in this thesis includes two groups according to the immobilization process: (1) physical adsorption via amine groups (APDMES, Jeffamine, chitosan) or carboxylic groups (COOH) on the surface; (2) covalent immobilization through amine-reactive surfaces (NHS, NHS-activated CMD, MAMVE and GPDMES).
In order to optimize protein-surface interactions, we evaluated pH effects on protein immobilization capacity on the various surface chemistries. Three fluorescent labeled proteins, streptavidin-Cy3, myoglobin-Cy3 and IgG-Cy3, were spotted at 0.6 µM in various spotting buffer containing 0.05% PVA according to previous results: sodium acetate (Ac) pH 4.5, PBS pH 7.4 and sodium carbonate (Car) pH 9.6. The relative immobilization capacity of proteins was evaluated by calculating the signal-to-noise ratio (SNR) of immobilized fluorescent labelled proteins versus surface chemistry (Figure 3-6).
SNR of myoglobin was overall higher than that of streptavidin and IgG. First, the molecular weight of myoglobin is 3 times and 9 times lower than that of streptavidin and IgG, respectively. Thus, more myoglobin than streptavidin and IgG could bind to surfaces leading to higher fluorescent signal. Secondly, the degree of labelling of myoglobin is about 2 times higher than that of streptavidin (see Table 3-1) giving a higher fluorescent signal whereas it is in the same range with IgG. Then regarding myoglobin and streptavidin, surfaces functionalized with carboxylic groups (COOH surface) or with amine reactive groups (NHS, CMD and MAMVE surfaces) displayed higher protein immobilization levels with acetate buffer (pH 4.5) than that with the other two buffers. By contrast, on chitosan surface, both proteins were more efficiently immobilized with carbonate buffer (pH 9.6). APDMES surface showed a middle behaviour, with best protein immobilization using PBS (pH 7.4). Regarding IgG, the highest immobilization level was always obtained with carbonate buffer (pH 9.6) whatever the surface chemistry except on MAMVE surface where acetate buffer (pH 4.5) gave the best results. These results pointed up the importance of electrostatic interactions between proteins and the surface chemistry.
A B
C
Figure 3-6 Signal-to-noise ratio (SNR) of fluorescent labeled protein myoglobin-Cy3 (A), streptavidin-Cy3 (B) and IgG-Cy3 (C) spotted at 0.6 µM in three different pH buffers on various surface chemistries; Standard deviation (SD) is in the range of 6%-18% of the mean value SNR.
Typically, in PBS (pH 7.4), myoglobin bears neutral global charge because pH is very close to its isoelectric point (pI=7.29) whereas in acetate buffer (pH 4.5) and in carbonate buffer (pH 9.6), myoglobin is positively and negatively charged, respectively. Concerning streptavidin (pI=6.1), in acetate buffer, the protein is positively charged whereas in PBS and carbonate buffer, it is negatively charged. For IgG, no precise pI was given by the company. At the same time, the net charge of the chemically functionalized surfaces was also affected by the pH of spotting buffers. COOH, NHS and CMD surfaces displayed similar pKA values,
determination by acidimetric titration), making them uncharged at pH 4.5 and negatively charged at pH 7.4 and 9.6. MAMVE surface bears two acidic functions with pKA values 3.5
and 7.5 [11] . Thus it is highly negatively charged at pH 9.6. Whereas chitosan is highly aminated polysaccharide, its pKA is 6.4, therefore it is protonated only at pH 4.5 and not
charged at pH 7.4 and 9.6. Jeffamine was described with pKA = 9.4 shifting to 7.1 when
grafted on graphite surface [12]. We can suppose that Jeffamine surface is positively charged only in acetate buffer (pH 4.5). Finally, APDMES surface, with pKA = 9.5 [13] , is
deprotonated only in carbonate buffer (pH 9.6). Thus, when both surface and protein show the same charge, protein immobilization level is low (low SNR) due to repulsive forces. As it can be shown in Figure 3-6, this is the case with myoglobin (charge -) and streptavidin (charge -) on MAMVE surface (2 negative charges) in carbonate buffer (pH 9.6), and with myoglobin (charge +) and streptavidin (charge +) on APDMES (charge +) and chitosan (charge +) surfaces in acetate buffer (pH 4.5). On the contrary, the best conditions for high level of protein immobilization (high SNR) were obtained when the surface was not charged and the protein was positively or negatively charged. Thus, the highest level of immobilization was obtained for:
- myoglobin on CMD or NHS surface, via covalent binding, in acetate buffer;
- streptavidin on NHS surface, via covalent binding, in acetate buffer or on chitosan surface, via adsorption, in carbonate buffer;
- IgG on MAMVE surface, via covalent binding, in acetate buffer or on chitosan surface, via adsorption, in carbonate buffer.
According to these results, not only electrostatic interactions but also hydrogen bonds (related to polar energy higher on polymer surfaces), Van der Waals interactions (related to dispersive energy) and binding surface area (related to molecular weight, higher for polymer surfaces) are involved at the solid-liquid interface. Indeed, MAMVE and chitosan are high molecular weight polymers (216 000 g/mol and 470 000 g/mol, respectively) which were grafted on silanized surface displaying the highest polar energies (19.5 mJ/m2 and 12.4 mJ/m2, respectively). Furthermore, MAMVE is a highly amine reactive copolymer due to the maleic anhydride moieties. CMD and NHS surfaces displayed similar polar energy (about 7 mJ/m2) and bear the same amine reactive group (NHS group) giving them the same reactivity towards protein immobilization. At least, for GPDMES surface, immobilization level of IgG was very low for all buffers and is the same range as NHS surface. We suggested that low amount of IgG was immobilized on these two surfaces due to its high molecular weight (150 000 g/mol) leading to steric hindrance of surface reactive groups. Hence, for each type of protein, it is essential to screen various immobilization conditions (surface chemistry, spotting buffer, concentration) in order to define the best one.