Grafting of Poly(ethylene glycol) on Click Chemistry Modified Si(100

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Grafting of poly(ethylene glycol) on click chemistry modified Si(100) surfaces Benjamin S. Flavel, Marek Jasieniak, Leonora Velleman, Simone Ciampi, Erwann Luais, Joshua Peterson, Hans Griesser, Joe George Shapter, and J. Justin Gooding Langmuir, Just Accepted Manuscript • DOI: 10.1021/la400721c • Publication Date (Web): 07 Jun 2013 Downloaded from http://pubs.acs.org on June 12, 2013

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Grafting of poly(ethylene glycol) on click chemistry modified Si(100) surfaces Benjamin S. Flavel1*, Marek Jasieniak2, Leonora Velleman3, Simone Ciampi4, Erwann Luais4, Joshua Peterson, Hans J. Griesser2, Joe G. Shapter3, and Justin Gooding4*. 1

Institute of Nanotechnology, Karlsruhe Institute of Technology, 76021, Karlsruhe, Germany

2

University of South Australia, Ian Wark Research Institute, Mawson Lakes Campus, Adelaide, SA 5095, Australia

3

School of Chemical and Physical Sciences, Flinders University, Bedford park, Adelaide, SA 5042, Australia

4

School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia

*Corresponding author: [email protected] & [email protected]

Abstract Poly(ethylene glycol) (PEG) is one of the most extensively studied anti-fouling coatings due to its ability to reduce protein adsorption and improve biocompatibility. Although the use of PEG for antifouling coatings is well established, the stability and density of PEG layers are often inadequate to provide optimum antifouling properties. To improve on these shortcomings we employed the stepwise construction of PEG layers onto a silicon surface. Acetylene-terminated alkyl monolayers were attached to non-oxidised crystalline silicon surfaces via a one-step hydrosilylation procedure with 1,8-nonadiyne. The acetylene-terminated surfaces were functionalised via a copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction of the surface-bound alkynes with an azide to produce an amine terminated layer. The amine terminated layer was then further conjugated with PEG to produce an antifouling surface. The antifouling surface properties were investigated by testing adsorption of human serum albumin (HSA) and lysozyme (Lys) onto PEG layers from phosphate buffer solutions. Detailed characterisation of protein fouling was carried out with X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) combined with principal component analysis (PCA). The results revealed no fouling of albumin onto PEG coatings whereas the smaller protein lysozyme adsorbed to very low amounts.

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Introduction Biofunctionalisation of materials is an important aspect in the advancement of biosensors and biomedical devices. Functionalisation of surfaces for biotechnological applications predominantly involves increasing the hydrophilicity of the surface to decrease fouling and improve biocompatibility [1-3]. Electrostatic repulsion and the hydrophilicity of the surface are the main factors that contribute to reducing the adsorption of biomolecules [4]. The modification of surfaces with anti-fouling layers to inhibit non-specific bio-interactions is important for the development of sensors [5], tissue engineering [6], implant materials [7, 8], microfluidics [9, 10], protein purification and diagnostics [11]. Silicon surfaces are widely used in biosensors and biocompatible materials [1214] and therefore it is particularly important to produce silicon surfaces resistant to non-specific biomolecular and cellular adhesion. Numerous surface modification methods have been implemented to attain anti-fouling surfaces including self-assembly [15, 16], polymer grafting [17, 18] and plasma modification [19, 20]. Various types of polymers have been investigated for their antifouling properties including polyacrylates, oligosaccharides and poly(ethylene glycol) (PEG). Much effort has been applied in the modification of surfaces with PEG to reduce protein adsorption and improve biocompatibility. PEG is one of the most extensively studied anti-fouling polymers due to its ability to resist protein adsorption [21]. Studies have determined that its hydrophilicity, entropy, large exclusion volume and coordination with surrounding water molecules in an aqueous environment are main contributing factors in reducing bio-adhesion [22]. Improved surface functionalisation and coating technologies that impart enhanced antifouling performance in addition to high stability are greatly sought after [23-25]. Many of the surface modification techniques used to coat surfaces with PEG are simple but have long-term instability complications due to desorption [26-28]. In addition to improved stability, the PEG layer must be of high enough density and uniformity to provide an optimal barrier against bio-adhesion. An integral factor in the development of antifouling surfaces with long-term stability is the formation of robust chemical bonds between the polymer and the surface. Organic monolayers bound directly to non-oxidised crystalline silicon surfaces through a silicon – carbon bond produce highly stable monolayers in comparison to conventional silane films on oxidised silicon [29-32]. Such monolayers are easily used as platforms for further derivatisation and have therefore become an advantageous approach in the fabrication of complex molecular architectures on surfaces. The stepwise construction of functional layers is also appealing due to the commercial availability of structural constituents and the synthetic versatility of established modular coupling techniques. Monolayers terminated with alkynyl groups have been shown to be easily 3 ACS Paragon Plus Environment

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conjugated via a copper-catalysed azide-alkyne cycloaddition (CuAAC), a commonly used click reaction [33]. Consequently, click chemistry and in particular the CuAAC of alkynes and azides has generated considerable interest as a technique for the stepwise construction of highly organised functional layers on surfaces [34, 35]. Several examples in the literature have already demonstrated the successful use of CuAAC grafting in the fabrication of polymer brushes on 2D solid surfaces. For example, Yameen et al. [36] utilised ‘click chemistry’ in that attachment of polyelectrolyte brushes on planar silicon surfaces and nanochannels. Ostaci et al. [24, 37] grafted poly(ethylene glycol) brushes to alkyne functionalised pseudobrushes and silicon substrates by ‘click chemistry’, and Britcher et al. [38] demonstrated a two step PEGylation ‘click chemistry’ process on porous silicon. Furthermore, these monolayers have been shown to provide unprecedented stabilisation of the underlying silicon surface against oxidation which is important for electrically based biosensing devices [39-41]. The purpose of this work is to investigate the scope and performance of step-wise derivatisation of surfaces in the context of improving control on the density of tethered antifouling species. We report the direct attachment of acetylene-terminated monolayers to non-oxidised silicon surfaces and further derivatisation by a CuAAC reaction to produce an amine terminated layer. The amine terminated layer was then further conjugated with PEG to produce an antifouling surface. Previous attempts to construct antifouling surfaces through direct CuAAC attachment of an antifouling azide (derived from tetra(ethylene oxide)) resulted in an insufficient reduction in fouling most likely due to a low density of tetra(ethylene oxide) attachment [34]. Consequently for this study we employed the CuAAC reaction to create a uniform platform to which the PEG could be later attached through simple coupling techniques in the hopes of achieving a higher density of PEG on the surface.

2. Experimental details 2.1. Materials Hydrofluoric acid (48 wt% solid in water) was purchased from Riedel-de Haen. Dimethyl sulfoxide, dichloromethane,

1,8-nonadiyne,

N,N,N’,N’-tetramethylethylenediamine

(TMEDA)

and

4-

azidoaniline hydrochloride were of the highest purity grade and purchased from Sigma-Aldrich. Copper (I) bromide was purchased from Alfa Aesar (>99%). Albumin from human serum (>99% protein), lysozyme from chicken egg white (more than 90% protein) and phosphate buffer saline (PBS) were purchased from Sigma-Aldrich; mPEG-propionaldehyde, 5 kDa was supplied by Laysan Bio, Inc.; potassium sulfate (AR) and sodium cyanoborohydride (purum, > 95%) were from Chem Supply Pty Ltd.


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2.2. Assembly of monolayers of 1,8-nonadiyne The preparation of 1,8-nonadiyne layers on silicon and subsequent chemical derivatisations are schematically represented in Figure 1. Silicon wafers were rinsed in dichloromethane and dried under argon. The wafers were immersed for 30 min in a piranha solution (2:1 H2SO4/H2O2) maintained at 100 oC. The silicon wafers were then immersed in an aqueous fluoride solution for 1.5 min (2.5% hydrofluoric acid). The samples were placed in a custom-made Schlenk flask containing a sample of 1,8-nonadiyne under an argon atmosphere (H2O < 10 ppb, O2 < 5 ppb). The 1,8-nonadiyne sample was degassed through a minimum of 4 freeze-pump-thaw cycles. The sample was kept under a stream of argon while the reaction vessel was immersed in an oil bath set to 165 °C for 3 h. The reaction vessel was allowed to cool to room temperature and the flask was then opened to the atmosphere. The sample was rinsed thoroughly in dichloromethane then stored in dichloromethane under argon before being either analysed or further reacted with substituted azide species. 2.3. CuAAC derivatisation of acetylene-terminated monolayers with an azide species A 10 mM solution of 4-azidoaniline hydrochloride was prepared by dissolving 8.6 mg 4-azidoaniline hydrochloride in 5 mL DMSO. A solution consisting of 11.6 mg TMEDA, 7.1 mg CuBr and 1.5 mL DMSO was added to a reaction vial containing the alkyne functionalised silicon surfaces. 1 mL of the 4-azidoaniline hydrochloride solution and 2.5 mL DMSO was then added to the reaction vial and the reaction was allowed to proceed for 1 h in the dark at room temperature, without excluding air from the reaction environment. The prepared samples were then rinsed consecutively with copious amounts of DMSO and water, dried under air then stored under argon until analysis or further reaction. 2.4. PEG attachment PEG grafts were fabricated via reductive amination under marginal solvation conditions [42]. The amine functionalised samples of silicon were treated with a solution containing 2.5 mg/ml mPEG aldehyde, 3.0 mg/ml sodium cyanoborohydride and 110 mg/ml potassium sulphate in 0.01 M PBS. The reaction was carried out at 60 oC (lower critical solution temperature of 5 kDa mPEG, LCST) for 18 hours. Grafting PEG at the LCST has been shown previously to produce higher graft densities [42]. After the treatment the samples were removed from the solution, washed sequentially 5x with water and finally dried under a stream of nitrogen. A thorough washing aimed to remove trace elements from the modified surfaces, like sodium or potassium which, when present, would interfere with ToF-SIMS analysis and obscure the results.


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2.5. Protein fouling protocol For the laboratory protein fouling tests, human serum albumin (HSA) or lysozyme was dissolved in 0.01 M phosphate buffer saline (pH 7.4) to a concentration of 0.1 mg ml-1. Tested samples were immersed in the protein solution at room temperature. The adsorption was allowed to progress for 3 h. Then samples were immersed in a 0.01M PBS solution for 2h followed by a thorough washing in 0.01 M PBS solution to remove loosely bound protein. Finally samples were rinsed with Milli-Q water and blown dry in a nitrogen stream. The fouling tendency of samples was determined by XPS, from the surface atomic concentration of nitrogen, and by ToF-SIMS with the aid of principle component analysis. 2.6. ToF-SIMS analysis ToF-SIMS measurements were performed with a PHI TRIFT V nanoTOF instrument (PHI Electronics Ltd, USA). A 30 keV, pulsed primary

197

Au+ ion beam was used to sputter and ionise species from

each sample surface. PHI’s patented dual beam charge neutralisation system using a combination of low energy argon ions (up to 10 eV) and electrons (up to 25 eV) was employed to provide an excellent charge neutralisation performance. Positive mass axis calibration was done with CH3+, C2H5+ and C3H7+. Spectra were acquired in the bunched mode for 60 seconds from an area of 100 μm x 100 μm. The corresponding total primary ion dose was less than 1 x 1012 ions cm-2, and thus met the static SIMS regime [43]. A mass resolution m/∆m of > 7000 at nominal m/z = 27 amu (C2H3+) was typically achieved. Each sample was characterised by ten positive ion mass spectra, which were collected from sample areas that did not overlap. All recognisable, clear (i.e. unobscured by overlaps) immonium ions from 2 up to 160 amu range were used in calculations. The peaks were normalised to the total intensity of all selected peaks. Multiple mass spectra were processed with the aid of principal component analysis, PCA [44]. PCA was performed using PLS_Toolbox version 3.0 (Eigenvector Research, Inc., Manson, WA) along with MATLAB software v. 6.5 (MathWorks Inc., Natick, MA). 2.7 XPS analysis X-ray photoelectron spectroscopy was performed with a Kratos AXIS Ultra DLD spectrometer, using monochromatic AlKα radiation (hν = 1486.7 eV). The system is equipped with a magnetically confined charge compensation system (low energy electrons are confined and transported to the sample surface by magnetic field). Spectra were recorded using an acceleration voltage of 15 keV at a power of 225 W. Survey spectra were collected with a pass energy of 160 eV and an analysis area of


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300 × 700 μm. High-resolution spectra were obtained using a 20 eV pass energy and an analysis area of

300 × 700 μm. Data analysis was performed with CasaXPS software (Casa Software Ltd). All

binding energies were referenced to the low energy C 1s peak at 285.0 eV. Core level envelopes were curve-fitted with the minimum number of mixed Gaussian–Lorentzian component profiles. The Gaussian–Lorentzian mixing ratio (typically 30% Lorentzian and 70% Gaussian functions); the full width at half maximum, and the positions and intensities of peaks were left unconstrained to result in a best fit. 3. Results 3.1 Preparation of amine functionalised silicon wafers via hydrosilylation and click chemistry

A three-step process was used to construct a PEG surface of optimal surface coverage on silicon. The method of attachment involved the hydrosilylation of silicon surfaces with an acetylene terminated layer followed by a CuAAC click reaction to produce an amine terminated layer then subsequent attachment of aldehyde PEG via reductive amination. The attachment of 1,8-nonadyine to a silicon surface resulted in the surface shown in Figure 1b. Characterisation of this surface has been previously reported [34]. Briefly, XPS revealed the presence of a densely packed monolayer of 1,8nonadiyne. The Si 2p region was dominated by a doublet, with the Si 2p3/2 at BE of around 100.0 eV. A low intensity peak was also observed at 103.4 eV, indicating traces of silica contaminants. The survey spectrum for the amine-terminated surface formed upon “clicking” 4-azidoaniline to the alkyne-terminated substrate surface showed the presence of silicon, carbon, oxygen and nitrogen, with atomic concentrations summarised in Table 1. The initial oxygen levels of the amine terminated substrate appear fairly high (13.4 %) which may be attributed to surface bound water [45]. Figure 2a shows the C 1s core level spectra for the amine functionalised silicon surface where a peak centred around 285.0 eV can be seen, which is characteristic of carbon-bound carbons (C‒C). Additionally, a contribution on the low binding energy (BE) side (BE of ~ 284.0 eV) can be seen, which could be attributed to carbon atoms in the C‒Si environment. However, we did not try to fit this component as its low intensity would produce fits with poor S/N. As shown in Table 1, following PEG immobilisation (aldehyde-terminated, 5 kDa) onto the amine terminated substrate an increase in the atomic concentration of oxygen and carbon is observed accompanied by a decrease of the silicon exposure. However, as the silicon signal did not completely diminish after PEG grafting this would suggest the PEG layer is thinner than the sampling depth of the XPS (5 nm) or the PEG layer is patchy. Given the use of a low molecular weight PEG a thickness of less than 5 nm is probable. Atomic force microscopy can be used to analyse the integrity of the PEG layer but was not explored


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in this study. A decrease in the nitrogen exposure is also evident after grafting PEG. In the C 1s core level spectra of the PEG immobilised surface (Figure 2b) two distinct peaks can be seen at 285.0 eV and 286.5 eV, where the high BE component corresponds to the carbon atoms chemically associated with oxygen via a single bond (C‒O) originating from the PEG and the low BE component corresponds once again to hydrocarbons (C‒C/C‒H). The PEG terminated surface exhibits an oxygen concentration of 23.87% (Table 1) which does not correspond to what is expected of a pure PEG layer (33% oxygen), but nevertheless indicates a high density of PEG grafts [46]. The PEG and grafting conditions used in our study was consistent with that used by Kingshott et al which achieved PEG layers containing 19.6 – 21.3% oxygen when grafting onto heptylamine and allylamine polymer layers [46]. The improvement in the PEG grafting suggests a higher density of amine groups present on our click-generated amine layers in comparison with the heptylamine and allylamine layers. The high density of amine groups reinforces the application of click chemistry for the pre-modification of surfaces to improve the yield of subsequent grafting.

Figure 1. Hydride terminated Si(100) surface (a). Assembly of monolayers of 1,8-nonadiyne (b) and subsequent derivatisation with an azide species (Cu(I) catalysed Huisgen 1,3-dipolar cycloaddition reaction with 4-azidoaniline hydrochloride) (b). Immobilisation of PEG via reductive amination (c).

(a)

C-C/C-H

(b)

C-O

C-C/C-H

8 ACS Paragon Plus Environment Figure 2. C 1s core level spectra of amine-functionalised surface (a) and its PEG modified derivative (b)

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Table 1 Atomic concentrations for amine-functionalised surface and its PEG derivative by XPS

Concentration, atomic % Sample O

N

Amine-terminated

13.43

0.92

PEG-terminated

23.87

0.59

*

C (% CC/CH; %C-O)* 36.88 (100 ; 0) 47.45 (39.5 ; 60.5)

Si 48.36 28.09

composition of the C1 s envelope

To determine the potential for the PEG modified silicon surfaces in the prevention of nonspecific adsorption (bio-fouling), the surfaces were exposed to two test proteins HSA and lysozyme and their interaction measured by XPS and ToF-SIMS.

The high resolution C 1s XPS spectra of the PEG-modified surfaces after contact with the solutions of lysozyme and HSA are shown in Figure 3. It can be seen that following exposure to the two test proteins the C 1s core-level spectrum remains identical and indistinguishable from the control PEG graft in Figure 2b. Most importantly no evidence of amide C (BE=288.2 eV) can be seen which would be indicative of significant protein fouling [46].

(a)

C-O

C-O

(b) C-C/C-H

C-C/C-H

Figure 3 C 1s core level spectra of PEG modified surfaces after contact with lysozyme (a) and HSA (b)

The quantitative data derived from the survey and C 1s spectra for the PEG modified surfaces after incubation in the protein solutions are listed in Table 2. Differences in the nitrogen concentration among the samples (0.59, 0.70 and 0.83 for [PEG], [PEG+lys] and [PEG+HSA] respectively) are too small to be used in the evaluation of fouling. The detection limit of XPS is 10-2 of a monolayer [47] which equates to the detection of a few ng/cm2 adsorbed protein. However, this detection limit


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varies greatly depending on the substrate analysed. Clearly, XPS does not indicate any detectable fouling of lysozyme and HSA onto the PEG grafted surfaces. Table 2 Atomic concentrations for PEG modified surfaces after contact with lysozyme and human serum albumin

Concentration, atomic % Sample O

N

PEG + lys

22.71

0.70

PEG + HSA

23.34

0.83

*

C (% CC/CH; %C-O)* 48.05 (39.5 ; 60.5) 49.13 (41.3 ; 58.7)

Si 28.54 26.70

composition of the C1 s envelope

To provide greater information on the extent of the antifouling capabilities of these PEG coated samples, ToF-SIMS analysis was performed on the PEG surfaces after exposure to the protein solutions. The PEG surfaces were tested for the adsorption of HSA and lysozyme. ToF-SIMS is a highly specific analytical tool that has been shown to detect extremely low amounts of adsorbed proteins on PEG surfaces which were undetectable by XPS [46]. ToF-SIMs has been shown to achieve detection limits in the attomole range [48-51]. The sensitivity of ToF-SIMS for the detection of proteins arises from the identification of characteristic amino acid mass fragments (immonium ions) generated from the amino acids that are present in the backbone of the proteins [52].

Figure 4 displays the positive survey ToF-SIMS spectra (m/z 0 – 200) for the amine terminated surface, PEG grafted surface and PEG grafted surface after contact with lysozyme and HSA. For the amine terminated surface (Figure 4a) the intense ion signals at nominal m/z 28, 29, 43, 73, and 147 amu can be attributed mainly to Si+, SiH+, CH3Si+, (CH3)3Si+ and C5H15OSi2+ fragments, which reflect covalently immobilised nonadiyne onto silicon. Most of the individual nominal masses contain multiple fragments (e.g. there are 5 components within the nominal mass of 42 amu as shown in Error! Reference source not found.a) but in this initial assignment we refer to the most prominent component within the group. The ToF-SIMS positive mass spectrum of the PEG modified surface is shown in Figure 4b. The major peaks at nominal m/z 29, 31, 43, 45, 57, 59, 73, and 87, amu correspond to CHO+, CH3O+, C2H3O+, C2H5O+, C3H5O+, C3H7O+, C4H7O+, C3H5O2+/C4H9O+, and C4H7O2+ respectively. These fragments are the PEG fingerprints since they are present in the spectrum due to the fragmentation of the PEG backbone. The spectrum indicates high density of PEG on the surface, though low levels of the 10 ACS Paragon Plus Environment

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aminated substrate characteristics are still detectable in the high resolution mass spectra (numerous silicon containing positive fragment ions such as Si+, SiH+, CH2Si+, CH3Si+), consistent with the use of relatively low molecular weight PEG and hence a thickness of the vacuum-dried grafted layer not significantly exceeding the probing depth of ToF-SIMS.


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Figure 4 Positive survey mass spectra for: (a) amine terminated Si surface; (b) PEG modified surface; (c) PEG modification after laboratory fouling test with lysozyme; (d) PEG modification after laboratory fouling test with human serum albumin. Insets: High mass resolution of static ToF-SIMs in the 42 amu region.

Traces (c) and (d) in Figure 4 present the ToF-SIMS spectra for the PEG coated surface after exposure to lysozyme and HSA respectively. Both spectra appear very similar to the spectrum of the initial PEG surface (Figure 4b). Survey spectra show only intensities at nominal masses, which do not always


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provide sufficient discrimination between the examined samples. Differences, however, emerge upon close inspection of each nominal mass range utilising high mass resolution of static ToF-SIMS. This is illustrated in the insets in Figure 4Error! Reference source not found., which presents the magnified spectra in the mass region m/z 41.85 – 42.15 amu for the amine terminated surface, PEG coated surface and PEG coated surfaces after contact with HSA and lysozyme, a region that is hardly visible in the survey spectra in Figure 4. Interestingly, there appears to be a slight increase in the intensity of the C2H4N+ fragment after exposure to lysozyme (Figure 4c inset) but not to HSA (Figure 4Figure 4Error! Reference source not found.d inset). As the C2H4N+ immonium ion reflects the alanine residue present in both proteins, it is likely that lysozyme has slightly adsorbed onto the PEG surface whereas HSA did not adsorb. The differences between the samples shown by the positive mass spectra in Figure 4 are based only on a few peaks selected from a single positive mass spectrum. Although this univariate analysis is useful, it disregards information contained in the remaining peaks that can be important in proper evaluation of the anti-fouling potential of these surfaces. Therefore in this study PCA was also used to extract information from the complex ToF-SIMS data and to aid in the data interpretation. In our attempt to classify the spectra by PCA we used a set of positive fragment ions that are listed in Table 3.

Table 3 Positive fragment ions used in PCA

No

Fragment

m/z

No

1

H3 N+

17.026 16

C3H8NO+

74.056

2

H4 N+

18.036 17

C5H10N+

84.082

3

CH2N+

28.018 18

C4H8NO+

86.070

4

CH3N+

29.026 19

C5H12N+

86.096

5

CH4N+

30.034 20

C4H4NO2+

98.028

6

C2H4N+

42.033 21

C4H10N3+

100.082

7

C2H6N+

44.051 22

C4H8NO2+

102.059

8

C3H4N+

54.033 23

C5H8N3+

110.055

9

C3H6N+

56.049 24

C6H10NO+

112.070

10

C3H8N+

58.066 25

C8H10N+

120.081


Fragment

m/z

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11

C3H4NO+

70.038 26

C9H8N+

130.069

12

C4H8N+

70.067 27

C8H10NO+

136.078

13

C4H10N+

72.082 28

C10H11N2+

159.081

Our focus was only on the nitrogen-containing peaks, which are characteristic of amino acid residues present in proteins so they could be used to indicate nonspecific protein adsorption. In the initial selection, which was based on the assignments taken from literature [53-56], 39 peaks were considered, however we had to eliminate some immonium ions as other spectral lines interfered with them (e.g. CH3N2+, which is related to arginine, overlaps with C2H3O+, which originates from the PEG).


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Figure 5 Scores on PC1 and PC2 (a) and loadings on PC1 (b) of immonium ions from static positive mass spectra of PEG modification and after its exposure to lysozyme and HSA.

Figure 5a shows the scores plot of immonium ions on PC1 and PC2 for the pristine PEG coated samples and for the PEG coated samples after contact with lysozyme (PEG+lysozyme) and HSA (PEG+HSA). Each sample was characterised by 10 groups of immonium ions derived from replicate positive static secondary ion mass spectra. PC1 and PC2 capture around 90 % of the total variance, which indicates that most of the original information is retained in this two-PC’s model. The PEG and PEG+HSA groups overlap along both PC’s, which reflects their similar surface chemistries in terms of nitrogen species. HSA appears not to foul onto the PEG modification upon contact. This cluster has positive scores on PC1. A high positive loading of C3H8N+ on PC1 indicates its association with the PEG surface prior to contact with the proteins (Figure 5b). In contrast, the PEG+lysozyme cluster has negative scores on PC1 and it is well-separated from its PEG precursor signifying that the PEG surfaces before and after exposure to lysozyme are different. The loadings of immonium ions on PC1 point at the peaks that discriminate the groups. The spectra for PEG+lysozyme have more intense peaks at nominal m/z 28, 30, 44, 70, 84, 86 and 100 amu. The highest intensity at m/z 70 amu (C 4H8N+) reflects the proline residue, which is present in the lysozyme structure. In summary, PCA on the ToF-SIMS data reveals some fouling of lysozyme onto the PEG modified surface. These PCA results indicate that the PEG surface is fully antifouling to large proteins only. However, it 15 ACS Paragon Plus Environment

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should be noted that the amount of lysozyme adsorbed onto the surface is small. Lysozyme is a lowmolecular weight protein (15 kDa) and is most likely too small to be completely repelled by the hydrated chains of the PEG layer and can therefore diffuse, to a limited extent, through the PEG layer and absorb onto the surface or become trapped in the PEG layer [17, 57, 58]. This molecular weight dependence is evident from the comparison of the adsorption of a comparatively large protein (HSA, 67 kDa) onto the PEG coated surface (Error! Reference source not found.d), where ToF-SIMS was unable to detect any presence of HSA. These results therefore indicate that the surface is completely resistant to fouling by large proteins while smaller proteins may be able to adsorb to a small extent.

4. Conclusion The stepwise construction of an anti-fouling layer was achieved through the conjugation of PEG to an amine terminated silicon surface which was prepared by covalent attachment of an acetylene terminated layer and subsequent CuAAC derivatisation with an amine terminated azide. This method to attach PEG onto surfaces proved an excellent method for preparing antifouling surfaces. A high surface density of PEG was suggested by XPS results, where characteristic peaks from the acetylene/azide monolayer disappear after PEG grafting and a characteristic peak from PEG coupling (C-O) is observed. Characterisation of the antifouling properties of the PEG layer was carried out with ToF-SIMS as XPS was not sensitive enough to suggest any fouling of lysozyme and HSA onto the PEG surfaces. The ToF-SIMs data after exposure of the PEG surfaces to two proteins; lysozyme (15 kDa) and HSA (67 kDa) revealed minute amounts of fouling by lysozyme and no fouling by HSA. PCA was used to further extract and clarify the information from the ToF-SIMS data and confirmed the adsorption of lysozyme onto the PEG modified surface due to discrepancies in the scores plot between the PEG surfaces before and after lysozyme exposure. In summary, although a high surface density of grafted PEG was achieved, the surface is completely antifouling to larger proteins while smaller biological species are significantly but not completely repelled. With the smaller biological species, it is hypothesized these may be able to penetrate into the PEG layer and adsorb and hence are less effectively repelled. In conclusion the step-wise construction of PEG reported here produced a surface where fouling was completely undetectable via XPS techniques and ToF-SIMS, a more sensitive technique, revealed complete antifouling against larger proteins and detected only slight amounts of smaller proteins adsorbed.


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