Abstract—The adsorption of bovine serum albumin (BSA), immunoglobulin G (IgG) and fibrinogen (Fgn) on fluorinated self- assembled monolayers have been studied using time of flight secondary ion mass spectrometry (ToF-SIMS) and Spectroscopic Ellipsometry (SE). The objective of the work has to establish the utility of ToF-SIMS for the determination of the amount of protein adsorbed on the surface. Quantification of surface adsorbed proteins was carried out using SE and a good correlation between ToF-SIMS results and SE was achieved. The surface distribution of proteins were also analysed using Atomic Force Microscopy (AFM). We show that the surface distribution of proteins strongly affect the ToF- SIMS results. Keywords—ToF-SIMS, Spectroscopic Ellipsometry, Protein, Atomic Force Microscopy. I. INTRODUCTION IOMOLECULAR adsorption at solid surfaces is an important and central phenomenon to many medical and technological applications. When a foreign implant is introduced inside the body, a layer of biomolecules, primarily proteins, adsorbs onto the surface of the implant and this influences cell adhesion and behaviour. The rapid adsorption of proteins to almost all surfaces in contact with a biological fluid underlines the importance of controlling protein attachment and this remains a pressing issue in the development of advanced medical and diagnostic devices. The determination of the amount of protein attached to the surface is a critical measurement performed by using a wide range of methods[1]. Time of Flight Secondary Ion Mass Spectrometry (ToF- SIMS) is a promising technique for protein adsorption analysis because of its chemical specificity, sensitivity and the potential to obtain information on the orientation of proteins on surfaces[2]. The analysis of surface adsorbed proteins using SIMS includes three objectives: (i) measurement of the surface density of adsorbed proteins, (ii) the detection of low amounts of proteins at surface and (iii) the determination of the identity and or orientation of adsorbed proteins[3]. Because SIMS is sensitive to a wide range of parameters, it is important to develop analytical strategies that are able to distinguish, for example, the effect of density from that of composition and orientation. In ToF-SIMS, an energetic primary ion impacts the surface of interest, causing the Santanu Ray is with the National Physical Laboratory, Teddington, Middlesex, TW14 0HD, United Kingdom, Boulder, (phone: +44 20 8943 8735; fax: +4420 8943 6453; e-mail: santanu.ray@npl.co.uk). Alexander G. Shard, is with National Physical Laboratory, Teddington, Middlesex, TW14 0HD, United Kingdom, Boulder, (phone: +44 20 8943 6193; fax: +4420 8943 6453; e-mail: alex.shard@npl.co.uk). ejection of secondary species, some of which are ions and these are mass analysed to produce a mass spectrum of atomic and cluster ions originating from the surface. Because of the energetic nature of this process, the spectrum is typically of highly fragmented species and organic fragments with mass < 1000 u are detached. Therefore, small amino acid fragments are used to study proteins in ToF-SIMS. The advantage of ToF-SIMS is that it is very surface sensitive, which means that it is sensitive to the orientation of proteins. Because of the complexity of ToF-SIMS spectra, advanced data analysis techniques are often required to interpret the resulting data. Wagner et. al.[4] showed that combination of ToF-SIMS and Principle Component Analysis (PCA) provides new insights into the composition of adsorbed protein films on biomaterial surfaces. In their work, they used single component adsorbed protein films on three model substrates and multivariate analysis of the ToF-SIMS data to distinguish different protein films. From the work of Wagner et. al.[5] and others[6-8], it is established that the distribution of proteins on the adsorbed surface from a mixed protein solution is better explored by ToF-SIMS because of its extreme surface sensitivity and good spatial resolution[9]. However, quantitative analysis of surface adsorbed proteins using ToF-SIMS is still far from being routine and secondary ion intensities may be strongly affected by their local environment, changes in concentration, dewetting and changes in the vacuum environment. In this present work, we have used ToF-SIMS and Spectroscopic Ellipsometry (SE) to study the adsorption of three different proteins on gold with a perfluorodecanethiol self assembled monolayer (SAM). Quantification of surface adsorbed proteins was carried out using SE and a good correlation between ToF-SIMS results and SE was achieved. The surface distribution of proteins were also analysed using Atomic Force Microscopy (AFM). We discuss the ability of SIMS to quantitatively determine the amount of protein at a surface and the advantages and limitations of the technique. II. MATERIALS AND METHODS A. Materials Bovine serum albumin (BSA) (99%), immunoglobulin G (IgG) (95%) and fibrinogen (Fgn) (96.3%) were purchased from Sigma (Life Science) and were used without further purification. 1H,1H,2H,2H-Perfluorodecanethiol (97%) was acquired from Aldrich (Chemistry) and analytical reagent grade ethanol was purchased from Fisher Scientific. To prepare protein solutions, phosphate buffered saline (PBS) solution (pH 7.4±0.1) was obtained from Fisher BioReagents and was used as received. Gold-coated quartz crystals were Santanu Ray and Alexander G. Shard Secondary Ion Mass Spectrometry of Proteins B World Academy of Science, Engineering and Technology International Journal of Physical and Mathematical Sciences Vol:5, No:4, 2011 552 International Scholarly and Scientific Research & Innovation 5(4) 2011 scholar.waset.org/1307-6892/16011 International Science Index, Physical and Mathematical Sciences Vol:5, No:4, 2011 waset.org/Publication/16011