Adsorption of charged macromolecules on a gold electrode

In this thesis we have examined the role of electrostatic interactions in the adsorption of charged macromolecules from aqueous solution to a solid surface and the possibilitiesfor manipulatingthis process through the electric potential of the interface. Gold has been used as the adsorbent surface, and before studying theadsorption ofseveral types of macromolecules onto this substrate we have determined the dependence of the double layer potential of the gold on the pH and on the externally applied potential across the gold/electrolyte solution interface.

Chapter 2 describes a colloidal probe atomic force microscopy study on the electric double layer of a gold electrode in aqueous solutions. The double layer potentials of the gold surface were obtained by fitting force-distance curves for the interaction with a spherical silica particle to the DLVO theory. It was found that the gold electrodeombinesthe features of reversible andpolarizableinterfaces, i.e., its charge and potential are determined by both the solution pH and the external potential. The pHlependenceis attributed to proton adsorption anddesorptionfromoxidicgroups at the gold surface. In the potential range studied, the double layer potentialyfvaries linearly with the applied potential; at a background electrolyte concentration of 1mM, the variation inyi* is roughly 10% of that in the applied potential. The potential of zero force (the external potential at which ^ - 0) varies with pH. The various features of the gold/electrolyte interface are described well by anamphifunctionaldouble layer model, which takes into account the simultaneous effects of the external potential across the interface and the association/dissociation of functional surface groups. The results of this study form the basis of the interpretation of adsorption studies on gold as a function of pH and externally applied potential, described in chapters 4, 5 and 6.

Chapter 3 addresses the sensitivity of thereflectometerset-up for our adsorption studies as .well as the electro-optic effect. The electro-optic effect involves a change in the optical properties of the gold electrode as a result of applying a potential. This leads to a change in thereflectometersignal. Furthermore, calculations and measurements concerning the sensitivity ofreflectometermeasurements in studying the adsorption of charged polymers from aqueous solution onto gold are presented. This provides a measure for the reproducibility of the adsorption data obtained withreflectometry. The gold substrate is in fact a thin gold film (15 nm) on a silicon wafer. Between the gold film and the waferaS am layer of titanium has been deposited for better attachment. The sensitivity-factor/4,was calculated by modelling the system as a stack of layers of uniform refractive index. The influence of several parameters, such as the layer thickness of the gold and the angle of incidence of the laser beam, on the sensitivity of thereflectometerset-up was examined In the range of potentials applied (-0.2 to +0.6 V vs. Ag/AgClreference electrode) it was round thattrieelectro-optic effect is significant compared to the change in thereflectometeroutput signal •esultingfrom adsorption. The sensitivity factorAs, however, is much less affected by the electro-optic effect than by the effect of inaccuracies in the system parameters. Therefore, by recording a baseline at the same applied potential as at which adsorption is monitored, adsorption data can be obtained without interference of the electro-optic effect. The experimental error in the adsorption measurements is generally between 2-7% and is dominated by the uncertainty in the thickness of the gold film.

In chapters 4, 5 and 6 the adsorption of charged macromolecules onto gold as measured usingreflectometry, is described. Generally, the adsorption kinetics anddsorbedamounts are determined as a function of the concentrationotthe macromolecule, the electrolyte concentration and the double layer potential of the gold. The double layer potential of the gold is varied through the pH as well as by applying an external potential. By comparing the results of adsorption measurements performed under pH control and under external potential control, effects of conformational changes and changes in the charge of the molecule in solution can beeparatedfrom effects of the variation electrostatic interactions between the molecule and the adsorbing surface.

Chapter 5 describes the adsorption of a fifth generationdendrimer, 1,4-diaminobutanepoly(propyleneimine). The charge of this molecule is determined by the solution pH. It is a spherical molecule and conformational changes are limited. The electrolyte concentration and the pH of the solution do not have any significant effect on the total adsorbed amount. Neitherdothe initial adsorption rates as a function of pH and as a function of applied potential follow the electrostatic interactions, on the contrary. From this, it was concluded that electrostatics is not the dominant factor in the adsorption process. However, the total adsorbed amount shows linear decrease with increasing applied potential, similar to want was found for the adsorption of PVP* onto gold. We assume that the effect of the applied potential on the adsorbed amounts is an indirect one, e.g., stemming from an increase in binding strength betweendendrimerand metallic surface sites with decreasing applied potential.

Protein adsorption onto gold was examined in chapter 6.Lysozymewas chosen as theadsorbatebecause the structure of this protein is relatively stable. For such a "hard" protein electrostatic interactions in the adsorption process are believed to be more important than for proteins with low internal stability, especially when thesorbentsurface is hydrophilic. The charge and structural stability of the protein varies with the solution pH. As for thedendrimersit was found thatlysozymeadsorption from aqueous solution onto gold is not controlled by electrostatic interactions. The initial adsorption rate is neither dependent on the electrolyte concentration and pH, nor on the externally applied potential. Quite unexpectedly, we found that the adsorbed amounts at constant pH exhibit a minimum around the potential of zero charge of the gold surface and increase more than linearly for decreasing as well as increasing potential. The curves of adsorbed amounts versus applied potential show a remarkable similarity with so-calledelectrocapillarycurves for metallic electrodes. This is a strong indication that the interfacial tension of the gold/solution interface is an important factor in the adsorption process. At the positive and negative sides of the double layer potential window that could be studied, the adsorbed amount approaches the value corresponding to a full monolayer of undisturbedlysozymemoleculesidsorbed'side-on'. Therefore, it is concluded that at the high-energy gold surface thelysozymemolecules undergo conformational changes, but as the interface becomes more polarized and the interfacial tension decreases, the extent of the surface-induced conformational changes decreases. This conclusion is corroborated by the dependence of the adsorbed amount on the pH, which reflects the combined effects of the change in interfacial tension with double layer potential and the decrease in structural stability with decreasing pH.

Chapter 7 contains a number of general conclusions and suggestions for furtheresearch. It was concluded that our approach has been successful for determining the relative importance of electrostatic interactions in the adsorption process of charged macromolecules onto the gold substrate. The choice fix gold as thesorbentsurface has proven to be very suitable for our adsorption studies because of itsamphifunctionalcharacter. A disadvantage is die high surface energy of gold, winch makes that many kinds of molecules readily adsorb and that the role of electrostatics in adsorption processes is somewhat limited rather than dominantThismay be avoided by surface modification, for example attaching athiollayer with proton binding functional groups to the gold surface. By doing so, the surface is stillaraphifimctionaland the surface energy is lowered at the same time. Another possible advantage of surface modification is that the range in which the double layer potential can be varied is larger, because the density of proton binding sites can be made high andredoxreactions are somewhat suppressed. In this thesis only one type of protein was investigatedSincethe properties of proteins vary quite substantially from one protein to another, it is recommended to perform similar experiments with different types of proteins, possibly using a less high-energetic surface. Thepossibilities to control adsorption of macromolecules on metal electrodes seemsto be limited.Desorptionby changing the applied potential never occurred. Neither was it possible to affect the kinetics by raising an electrostatic barrier. For PVP" anddendrimerthe latter is presumably resulting from charge compensation by the presence ofcounterionswithin the molecules. Self-consistent field model calculations may help to get more insight in these aspects.