After the conditions for monolayer adsorption of the two variant forms of CPR had been established the effect of potential on the adsorption process has been investigated.
Two electrochemical QCM-D modules have been used to investigate the adsorption of CPR. However difficulties in producing accurate data under potential
control were associated with the design of the modules. One of the modules used has much larger volume than the modules used in the concentration and pH investigations. Pumping protein solution through this larger module frequently resulted in a frothing of the protein sample, which is associated with aggregation of the protein. This made it extremely difficult to pump the solution through the cell and produce an accurate trace for both f and D. The other electrochemical module used was a new design cell produced by Q-Sense which incorporated electrochemical control within a window cell. Again this cell was problematic in that it proved extremely difficult to pump through solution without trapping small air bubbles in the module. These trapped bubbles can cause shifts in both f and D and also introduce severe inaccuracies in controlling the applied potential, thus the potential can not be controlled properly and the experiment had to be abandoned.
Despite these difficulties early electrochemical QCM-D experiment indicated the potential did not affect the adsorption conditions for a monolayer coverage.
5.8 Summary
The adsorption of protein from a sample of similar concentration as used in the preliminary RAS experiments discussed in chapter 4 was monitored using the QCM-D instrument. The QCM-D results discussed in this chapter suggest that the concentration of protein used in the preliminary RAS experiments results in the adsorption of multiple layers of protein, therefore indicating that the RAS data discussed in chapter 4 may have been produced after the adsorption of multiple layers of CPR molecules onto the Au(110) surface.
It has been shown that the formation of both monolayer and bilayer coverage is dependent not only on concentration but also on pH as shown in table 5.1. These results also show that there is no simple relationship between the pH and concentration at which a monolayer is formed but that, apart from the results at pH 7.0 and 7.8, the maximum concentration at which adsorption is limited to a monolayer increases with increasing pH.
The conditions for producing a monolayer of the mutant full length CPR and isolated FAD CPR have been established. All future experiments were carried out using a pH 7.2 phosphate buffer solution and a protein concentration less than 0.4 μM for full length CPR and 0.6 μM for the isolated FAD variant of CPR was used.
5.9 References
[1] M. C. Dixon, Journal of Biomolecular Techniques 19, 151 (2008)
[2] M. Wang, D. L. Roberts, R. Paschke, T. M. Shea, B. S. S. Masters and J-J. P. Kim, Proc. Natl. Acad. Sci. 94, 8411 (1997)
[3] M. Rodahl, F. Hook, C. Fredriksson, C. A. Keller, A. Krozer, P. Brzezinski, M. Voinova and B. Kasemo, Faraday Discuss. 107, 229, (1997)
[4] F. Hook, Thesis. Development of a novel QCM technique for protein adsorption studies. Chalmers University of Technology, Goteborg University (2004)
[5] F. Hook, T. Nylander, C. Fant, K. Sott, H. Elwig and B. Kasemo, Anal. Chem. 73, 5796 (2001)
[6] P. Weightman, G. J. Dolan, C. I. Smith, M. C. Cuquerella, N. J. Almond, T. Farrell, D. G. Fernig, C. Edwards and D. S. Martin, Phys. Rev. Lett. 96, 086102 (2006)
[7] A. Bowfield, C. I. Smith, M. C. Cuquerella, T. Farrell, D. G. Fernig, C. Edwards and P. Weightman, Phys. Status Solidi C 5, 2600 (2008)
[8] C. I. Smith, A. Bowfield, G. J. Dolan, M. C. Cuquerella, C. P. Mansley, D. G. Fernig, C. Edwards and P. Weightman, J. Chem. Phys. 130, 044702 (2009)
[9] A. Bowfield, C. I. Smith, G. J. Dolan, M. C. Cuquerella, C. P. Mansley and P. Weightman, e-J. Surf. Sci. Nanotech. 7, 225 (2009)
[10] A. Bowfield, C. I. Smith, C. P. Mansley and P. Weightman, Phys Status Solidi B 247, 1937 (2010)
[11] C. P Mansley, C. I. Smith, A. Bowfield, D. G. Fernig, C. Edwards and P. Weightman, J. Chem. Phys. 132, 214708 (2010)
[12] G. Sauerbrey, Zeitschift Fut Physik, 155 206 (1959)
[13] Image analysis carried out using Image SXM, S. D. Barrett [http://www.ImageSXM.org.uk ] (2011)
[14] Image Analysis in Earth Science, Renée Heilbronner and Steve Barrett, Springer Berlin Heidelberg (2013)
Chapter 6
RA Spectra of Monolayer and
Bilayer
Adsorption
of
P499C
Truncated FAD and Full Length
CPR Molecules
This chapter provides the RA spectral signatures of both monolayer and bilayer coverage of P499C truncated FAD and full length CPR molecules adsorbed on the Au(110) surface. Conditions under which a monolayer and bilayer adsorb were provided from QCM-D experiments discussed in the previous chapter.
6.1 Introduction
Having established the conditions under which monolayers and bilayers of the mutant P499C variants of CPR adsorb on polycrystalline Au in QCM-D experiments, it was necessary to reproduce these conditions in the electrochemical cell to enable RAS studies of monolayer adsorption of variant CPR molecules. The RAS experiments presented in this chapter closely followed the procedures used in preliminary RAS experiments in chapter 4, relating to the preparation of both the protein samples and the Au(110) surface. However slight changes to the experimental procedure were made to accommodate the conditions for monolayer adsorption. These were, to firstly ensure that buffer solutions of appropriate pH were prepared and secondly the concentration of protein in each sample was carefully controlled. The pH of the buffer solution was checked prior to each experiment and the concentration of protein in solution was monitored using the UV-Vis spectrometer. During QCM-D experiments the sensors were rinsed with fresh buffer solution after the adsorption of protein, which was done to try to prevent protein aggregation and to rinse off any additional weakly bonded protein molecules. To replicate this procedure in RAS experiments the electrochemical cell was rinsed with fresh buffer solution, immediately after the adsorption of protein onto the Au(110) surface.