Spontaneously reactive plasma polymer micropatterns

Polymer 52 (2011) 1882e1890

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Spontaneously reactive plasma polymer micropatterns Gautam Mishra a,1, Christopher D. Easton b, 2, Gregory J.S. Fowler a, 3, Sally L. McArthur b, * a b

Department of Engineering Materials, Kroto Research Institute, University of Sheffield, Broad Lane, Sheffield S3 7HQ, UK Biointerface Engineering, IRIS, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn VIC 3122, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 October 2010 Received in revised form 15 February 2011 Accepted 1 March 2011 Available online 9 March 2011

A combination of spontaneous reactive chemical domains bounded by non-fouling zones provides a means to covalently immobilize biomolecules in structured, spatially defined arrays. These arrays have application in a wide range of biotechnologies including tissue engineering, proteomics, and diagnostics. In this paper, we describe the fabrication of multi-chemistry micropatterns from plasma polymers. X-ray photoelectron spectroscopy (XPS), together with Time-of-Flight Static Secondary Ion Mass Spectrometry (ToF-SSIMS) and confocal imaging has been utilized to confirm the reactivity and integrity of micropatterns fabricated from amine-reactive maleic anhydride (ppMA) on non-fouling tetraglyme (ppTg). The covalent immobilization of antibodies via the formation of amide linkages with the anhydride groups occurs only in the ppMA domains, while antibody activity is confirmed via their ability to attract specific fluorescent antigens. These micropatterns therefore provide a convenient and effective platform for covalently immobilizing biomolecules in spatially defined areas without the need for multiple step wet chemical immobilization strategies. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

Keywords: Photolithographic patterning Plasma polymer Bioarray

1. Introduction A wide range of strategies exist for the immobilization of biomolecules onto surfaces. Covalent immobilization offers several advantages for the immobilization of functional biomolecules, including the generation of surfaces with a higher density of biomolecules and greater stability, i.e. less desorption relative to passively adsorbed biomolecules [1]. Therefore, covalent, spatially defined protein immobilization procedures are highly sought after. Covalent attachment of proteins to microarrays is performed using a variety of chemically activated surfaces, e.g. aldehyde [2], epoxy [3] and active esters [4]. A number of techniques are available to chemically modify a solid surface in order to generate reactive groups for covalent immobilization of biomolecules; for example, ionizing radiation, graft co-polymerization, plasma gas discharge, photochemical grafting, chemical modification, and chemical derivatization [5e8]. Self-assembled monolayers provide yet another technique for the development of biocompatible surfaces conducive for biomolecule immobilization, while recent developments in lipid * Corresponding author. Tel.: þ61 (0) 3 9214 8452. E-mail address: [email protected] (S.L. McArthur). 1 Present address: Kratos Analytical, Wharfside, Trafford Wharf Road, Manchester M17 1GP, UK. 2 Present address: CSIRO Molecular and Health Technologies, Bag 10, Clayton South VIC 3169, Australia. 3 Present address: Department of Chemical and Process Engineering, University of Sheffield, Broad Lane, Sheffield S3 7HQ, UK.

monolayer and bilayer immobilization may improve capabilities for functional membrane protein immobilization [9e12]. Plasma polymerization offers a single step, dry approach for the fabrication of thin functional coatings on bulk materials. Interest in the use of plasma polymers for biomedical applications has increased in recent years [13,14]. Applications include the fabrication of bio-reactive [15,16] and non-bio-reactive coatings for microfluidics, tissue engineering and cell culture applications [17,18]. This technique provides an attractive method for the production of coatings with controllable thickness (<100 nm) with a high degree of chemical functionality retained from the monomeric precursor [19]. A number of monomers have been employed in the past for biotechnology applications, including acids (acrylic acid) [20], amines (allylamine) [21], siloxanes (hexamethyldisiloxane) [22], anhydrides (maleic anhydride) [23] and more unconventional precursors, including plant extracts (1e8 cineole) [24]. One of the greatest challenges when patterning biomolecules, is to separate the bioactive regions with regions of low or no bioactivity. This requires the surface chemistry to be patterned. In traditional bioarrays, this is achieved by spotting the biomolecules on the surface using automated spotters and subsequently capping the free reactive groups on the surface to prevent non-specific binding of other biomolecules. While this has been shown to be effective, it is a relatively complex process and can result in artifacts and poor pattern resolution. With the wide range of monomers available for plasma polymerization, it is possible to create both non-fouling and biomolecule-reactive plasma polymers. A number

0032-3861/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2011.03.004

G. Mishra et al. / Polymer 52 (2011) 1882e1890

of techniques are available for micropatterning of plasma polymers, including physical [25] and lithographic [26] patterning. We recently demonstrated the spatial and chemical resolution advantages of photolithographic techniques over physical techniques for the patterning of plasma polymers [27]. In this paper, we have examined spontaneous reactive micropatterns fabricated from plasma polymers for the immobilization of immunologically active antibodies. Micropatterns were fabricated from plasma polymers of maleic anhydride (ppMA) and tetraglyme (ppTg). The challenge here was to retain the functional specificity of both the coatings during the patterning process. A combination of X-ray photoelectron spectroscopy (XPS), Time-of-Flight Static Secondary Ion Mass Spectrometry (ToF-SSIMS) and confocal imaging was employed to characterize the coatings. These techniques allowed for the visualization of the spatial specificity of reactive zones via derivatization, hydrolysis, and the immobilization of biomolecules. The ToF-SSIMS images clearly demonstrate that the spatial resolution obtained by the photolithographic patterning technique was maintained during the surface activation process. Antibodies were immobilized to the ppMA regions, and exposure of the surfaces to antigen-containing solutions illustrated that the ppTg regions prevented protein adsorption, whilst the ppMA-antibody regions retained their antigen specificity. The results demonstrated that spontaneously reactive micropatterns can be produced from photolithographically patterned plasma polymers while maintaining the specific chemical- and bio-functionality of the different polymers.

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has been explained in detail previously in Ref. [28]. Briefly, substrates were placed in the reactor and the vessel was pumped down to a base pressure of 1  103 mbar. A low-temperature thermally non-equilibrium plasma was ignited and maintained via a radio frequency power source (13.56 MHz, Coaxial Power Systems Ltd., UK) coupled to the reactor via an impedance matching network. Deposition parameters including deposition power, time and monomer flow rate were fixed at the values given in Table 1. 2.3. Photolithographic patterning

2. Experimental

The photolithographic patterning process for plasma polymers was adapted from Goessl et al. [26], and has been described elsewhere [29]. Briefly, Positive photoresist (AZ 1512, MicroChemicals GmbH) was spin coated at 4000 r.p.m (thickness w 1e1.5 mm) onto the base plasma polymer (ppTg). Substrates were then heated to 90  C for 120 s to harden the resist and remove any excess solvent, and subsequently brought into intimate contact with an optical photomask and exposed to ultraviolet radiation (Electronic Vision A16-2 mask aligner) for 5 s. The light-exposed photoresist was removed by immersing the sample in an alkaline developer (AZ 351B, MicroChemicals, GmbH; 1:4 (developer: water)) for 30 s. After immersion, samples were rinsed several times with MiliQ grade water and dried in a stream of nitrogen, ready for the deposition of the second plasma polymer layer. After deposition of the second polymer, the remaining photoresist was dissolved from between the films by immersing the samples in a solution of acetone and sonicating them for 3  20 s. The samples were then extensively washed with MiliQ and dried under a stream of nitrogen.

2.1. Materials

2.4. Derivatization reactions of ppMA

Maleic anhydride (MA), tetraglyme (tetraethylene glycol dimethyl ether, Tg), and trifluoroethylamine were purchased from Aldrich UK at purity >98%. Human fibrinogen (Sigma Aldrich, UK), Alexa Fluor 546-succinimidyl ester protein-labeling kit (Invitrogen, UK), fluorescein-labeled bovine serum albumin protein (Sigma Aldrich, UK), Alexa Fluor AF-546 labeled anti-fibrinogen antibody (Invitrogen, UK), anti-bovine serum albumin (ab34119, Abcam, UK) and antifibrinogen (ab8844, Abcam, UK) polyclonal primary antibodies were employed in this study. Silicon wafers (Orientation: <100>, Thickness; 525 mm, Dopants: P.Boron, Resistivity: 1e10 Ohm cm, Polish: one side) were purchased from Compart Technology Ltd UK. AZ 1512 Photoresist and AZ 351B Developer were both purchased from MicroChemicals, GmbH. All other chemicals were purchased from Aldrich UK and used as supplied.

0.2 mM solution of trifluoroethylamine (99.5% Aldrich, UK) mixed in methanol was allowed to react with freshly ppMA films for 1 h at room temperature (RT). On completion of reaction, the samples were washed several times with methanol, dried under a stream of nitrogen and were immediately used for characterization. Freshly deposited ppMA coatings were immersed in a mixture of deionized water and hydrochloric acid (pH 2.0, w0.3 mM) held at a temperature of 90  C. The hydrolysis reaction was allowed to proceed for 3e4 h. The samples were thoroughly washed several times with fresh deionized water, dried under a stream of nitrogen and immediately used for characterization or protein immobilization experiments.

2.2. Plasma polymerization

X-ray photoelectron spectroscopy (XPS) spectra were acquired using an Axis Ultra DLD spectrometer (Kratos Analytical, UK) with a monochromated Al Ka source. Photoelectrons were transferred to the 165 mm hemispherical analyzer using a combined magnetic and electrostatic lens system. In the spectroscopy mode the samples were irradiated with monochromatic Al Ka source (hn ¼ 1486.6 eV, spot size 300 mm  700 mm) and photoelectrons were collected using eight channeltrons. The sample was isolated electrically in order to eliminate vertical differential charging, and a low-energy electron flood gun was used for charge compensation. The data were

Prior to each deposition, the monomer was degassed several times using freeze thaw cycles. Si substrates were diced to the required size and placed under a stream of air to remove any dust or silicon particles. Substrates were then sonicated several times in isopropyl alcohol (IPA), and then stored in IPA prior to use. Substrates were dried with high purity nitrogen gas before plasma deposition. All polymers were fabricated in a stainless steel T-piece reactor with an internal aluminum disc electrode. The experimental setup

2.5. Surface characterization with XPS

Table 1 Plasma polymerization settings employed in this study (Tg e Tetraglyme, MA e Maleic anhydride, RT e Room temperature). Precursor

Monomer Temp [ C]

Chamber Temp [ C]

PPeak [W]

ton [ms]

toff [ms]

PEffective [W]

Flow Rate [sccm]

Deposition time [min]

Tg MA

95 RT

55 30

10 10

e 80

e 800

10 0.09

2.7 2.5

25 20

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O O

O n

CF3CH2NH2 Trifluoroethylamine

OH O

NHCH2CF3 O n

514 nm) 30 mW, HeNe laser (543 nm) 1 mW, and HeNe laser (633 nm) 5 mW. An Achroplan 10X/0.30W Ph1 water immersion objective lens was used to collect all the images of the patterned samples which were placed in 6 or 12 well tissue culture plates filled with distilled water.

Scheme 1. Derivatisation of plasma polymerized maleic anhydride films with trifluoroethylamine showing the anhydride ring opening and formation of amide linkage (eCNO) and carboxylic groups (eCOOH).

2.8. Antigen and antibody immobilization onto micropatterns

converted to VAMAS format and processed using CasaXPS, version 2.2.37. Survey spectra were obtained from the surface at 160 eV pass energy, 1 eV step size, from 1200 eV to 0 eV, and quantified using empirically derived sensitivity factors. To confirm the coating chemistries, high resolution C1s spectra were collected at pass energy of 20 eV and step size of 0.1 eV for a minimum 300 s.

2.8.1. Direct immobilization of protein for ToF-SSIMS analysis Micropatterns of ppMA on a ppTg background were incubated in a solution of human fibrinogen (1 mg/ml, phosphate-buffered saline (PBS), pH 7.2) for 1 h at RT. The samples were thoroughly washed with PBS followed by distilled water to remove loosely bound protein molecules and salts. Samples were then dried in a stream of pure nitrogen and analyzed immediately using ToFSSIMS.

2.6. ToF-SSIMS imaging The experimental procedure employed for ToF-SSIMS imaging has been detailed previously in Ref. [32]. Briefly, images were obtained using a ToF-SIMS V instrument (ION TOF Inc. Munster, Germany), where the analysis chamber was held at w1  109 mbar during experiments. The primary ion beam was generated using a liquid metal ion gun fitted with a pure bismuth ion source capable of producing Bipþ n (n ¼ 1e6, p ¼ 1-2) ions. The kinetic energy of these ions was 25 keV for singly charged ions and 50 keV for doubly charged ions. The angle of incidence of the primary ion beam relative to the sample surface was 45 . Bi3þþ primary ion source with a kinetic energy of 50 keV was used for all image and spectral data acquisition. All data was collected in high mass resolution bunched mode. Spectra and images from multiple points (3) on each sample were acquired and care was taken to use a new sample area for each analysis. All images contained 128  128 pixels. The field of view is given in figure legends. 2.7. Confocal microscopy Confocal Laser Scanning Microscopy was performed using a ZEISS LSM 510M upright microscope in reflectance mode. The microscope is equipped with three lasers: Ar laser (458, 477, 488,

2.8.2. Blocking protocol for control samples employed in fluorescence study Samples were blocked with TBSTM (Tris-buffered saline Tween 20 Milk) solution for 4 h at RT. The samples were then washed with PBS and distilled water several times before being incubated in 1:500 AF546 labeled anti-fibrinogen antibody solution (PBS, pH 7.2) overnight. The samples were washed several times in PBS and distilled water before fluorescence microscopic analysis. 2.8.3. Direct immobilization of fluorescent antibodies Samples were incubated in antibody solution (1:500, phosphate-buffered saline, pH 7.2) for 4 h tagged with an Alexa Fluor 546 fluorophore (AF-546, lexcitation ¼ 543 nm, lemission ¼ 575 nm, anti-human fibrinogen) and then washed several times in PBS and distilled water before analysis. Fluorescent areas were imaged using 543 nm laser line excitation, and emission imaged via a 560 nm high pass filter. Prior to fluorescent protein micropattern analysis, individual background images of ppMA patterns on ppTg samples (without fluorescent protein) were acquired to measure the autofluorescence of plasma polymers. The patterned samples were left submerged in PBS and a water-dipping objective lens was used. The instrument settings (pinhole, gain and laser intensity) were adjusted to a threshold value where the signals from

Fig. 1. Negative ion ToF-SIMS images (Field of view e 250  250 mm2) of trifluoroethylamine derivatization of ppMA photolithographically patterned on to ppTg coated Si wafer. 1a e 1c collectively show the reaction of anhydride group with trifluoroethylamine while 1d e 1f show regions of ppTg film respectively. The total ion image has been shown in 1g.

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background autofluorescence were negligible; all further fluorescent images were acquired at these settings, thus effectively subtracting each image from its individual autofluorescence background.

3. Results

2.8.4. Assessing the activity of immobilized antibodies Anti-bovine serum albumin and anti-fibrinogen polyclonal antibodies were first immobilized onto the surface (1: 500 dilution, PBS, pH 7.2, 4 h, RT). The samples were washed several times in PBS and distilled water and were blocked overnight in TBSTM solution (RT). After several more washing steps, the patterned surfaces were incubated in the appropriate protein solution (0.01 mg/ml, TBSTM) to determine whether the immobilized antibody could still be recognized by its antigen. The proteins (fibrinogen and bovine serum albumin) used in the experiment were covalently labeled with the fluorescent labels AF-546 (lexcitation ¼ 543 nm, lemission ¼ 575 nm) and flourescein (FITC) (lexcitation ¼ 490 nm, lemission ¼ 525 nm) respectively.

3.1.1. Trifluoroethylamine derivatization of ppMA The chemical functionality of micropatterns derived from ppMA and ppTg was investigated employing ToF-SSIMS imaging and XPS. Derivatisation using trifluoroethylamine (TFEA) provides a means to confirm the spontaneous reactivity of the anhydride groups of ppMA via the formation of amide linkages with amines (Scheme 1). Fig. 1aec shows the F, CN and CNO secondary ion fragments originating from the plasma patterned surfaces and indicates that TFEA compound reacted specifically with the anhydride regions. Images formed from the C2H3O, C3H7O and C2H3O2 secondary ion fragments show that the regions between the TFEA reactive zones are ppTg (Fig. 1def). The total ion image is presented in Fig. 1g.

Relative Atomic Concentration

O 1s

Intensity (arb. unit)

Intensity (arb. unit)

a

3.1. Confirmation of plasma polymerized micropatterned chemistry using ToF-SSIMS imaging and XPS

C 1s - 70.1% O 1s - 29.9%

C 1s

C 1s Peak Assignment

1000

800

600

400

200

0

(1) C-C (285.0 eV) - 36.7% (2) C-C-(O)=O (285.7eV) - 24.8% (3) C-O (286.6 eV) - 8.5% (4) C=O/O-C-O (287.9 eV) - 5.3% (5) C-(O)=O (289.2 eV) - 24.7%

(1)

Binding Energy (eV)

(5) (2) (3) (4)

294

292

284

(1)

N 1s

800

600

280

C 1s - 67.7% N 1s - 2.9% O 1s - 23.3% F 1s - 6.1%

C 1s

F 1s

1000

282

Relative Atomic Concentration

O 1s Intensity (arb. unit)

Intensity (arb. unit)

b

290 288 286 Binding Energy (eV)

400

200

C 1s Peak Assignment (1) C-C (2) C-C-(O)=O / C-N (3) C-O / N-CH2-CF3 (4) C=O / O-C-O / N-C=O (5) C-(O)=O (6) CF3

0

Binding Energy (eV)

(285.0 eV) - 42.7% (285.7eV) - 16.4% (286.6 eV) - 12.8% (287.9 eV) - 9.60% (289.0 eV) - 16.4% (292.8 eV) - 2.1%

(5) (3)

294

(2)

(4)

(6)

292

290

288

286

284

282

280

Binding Energy (eV) Fig. 2. a. C 1s XPS spectra of ppMA as deposited and after derivatization. Inset figure shows the survey scan of ppMA field deposited on Si wafer. 2b shows the C 1s scan after derivatization with TFEA. Note the presence of CF3 peak centered at binding energy 292.8 eV after reaction with TFEA. The presence of F 1s and N 1s signal detected by survey scans after reaction with TFEA can also be observed in inset 2b.

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Fig. 3. Negative ion ToF-SIMS images (Field of view e 500  500 mm2) of hydrolyzed ppMA patterns on pp-TG coated Si wafer. 3a e 3c show the reaction of anhydride group with trifluoroethylamine while 3d e 3f show regions of ppTg film respectively. The total ion image has been shown in 3g.

Fig. 2a presents the C1s spectra of ppMA as deposited and Fig. 2b after derivatization with TFEA, where the introduction of a CF3 peak can be observed for the derivatized ppMA. The survey scan demonstrates the introduction of nitrogen and fluorine peaks (Fig. 2b), further providing evidence of reaction between ppMA and TFEA. It should be noted that no peaks associated with nitrogen or fluorine were observed in the case of freshly deposited ppMA films (Fig. 2a). 3.1.2. Hydrolysis of pp-MA Fig. 3 presents ppMA patterns on ppTg after the sample was hydrolyzed and then derivatized with TFEA (Scheme 2). The low yield (compare Figs. 1 and 3aec) of secondary ion fragments derived from TFEA and amide bonds strongly suggests that loss of anhydride ring structure after hydrolysis inhibits the plasma polymer film from covalently attaching amine groups. Fig. 3def demonstrates the presence of the ppTg plasma polymer. These images clearly show that the non-fouling plasma polymer layer is not only unaffected by hydrolysis process carried out at 90  C, but also remains non-reactive to amine groups. The total ion image is shown in Fig. 3g. Previous XPS studies from the group have shown that hydrolyzed ppMA does not react with TFEA [30]. 3.2. ToF-SIMS imaging of fibrinogen immobilization on reactive micropatterns Fig. 4 shows the single ion images of ppMA and ppTg plasma polymer micropatterns, after incubation in a solution of the protein fibrinogen. The images in Fig. 4a show the presence of amino acid fragments e CH4Nþ (glycine), C2H6Nþ (alanine) and C2H5Sþ (methionine) e co-localized with the ppMA regions in the pattern. OH

O O

O n

Water + HCl 90o C, 2 hours

OH

O

O n

Scheme 2. Simplified reaction schematic showing the hydrolysis of plasma polymerized maleic anhydride films when soaked in 0.1 mM HCl þ Water (pH 1.9) solution for 4 h at 90  C.

The presence of C3H7Oþ (Fig. 4a), in addition to C2H5Oþ and C5H11O2þ (data not shown) secondary ion fragments confirms the presence of the ppTg regions on the micropatterned sample. The selective attachment of fibrinogen on the ppMA film can be observed in the images with negligible ion intensities detected from the amino acid fragments in the areas associated with ppTg. This is clearly demonstrated in Fig. 4b, with the line scan comparing the raw secondary ion intensities of C3H7Oþ indicative of ppTg regions and CH4Nþ ions indicative of region where biomolecules are localized, i.e. ppMA region on the image. 3.3. Confocal imaging of fluorescent antibodies immobilized on protein-reactive micropatterns The localization, specificity and activity of proteins immobilized on the ppMA/ppTg micropatterned patterned surfaces were investigated by fluorescence microscopy after the surfaces were exposed to the AF-546-labeled anti-human fibrinogen solution. Prior to acquisition, background images were obtained of the micropatterns in order to take into account autofluorescence observed from the plasma polymers. An example of the autofluorescence observed is presented in Fig. S1 (Supplementary information). Inaccuracies in quantification of immobilized biomolecules by fluorescence measurement can arise due to fluorophore quenching and photo-bleaching effects during experimental measurements [31]. Though these effects have not been quantified in the current study, care was taken to minimize the exposure of fluorescently labeled biomolecules to confocal laser source in order to minimize photobleaching. Plasma polymer films investigated in the current study were found to be susceptible to photobleaching upon prolonged exposure to confocal laser light source (>5 min), possibly due to scission of unsaturated carbon bonds present in the polymer chains. The effects of photobleaching of fluorophores and plasma polymer films were minimized by analyzing fresh sample areas during each experimental measurement. Fig. 5 shows that the ppMA regions bound the fluorescently labeled antibody, whilst ppTg retained its non-fouling properties and was thus dark in the image. Intensity profiles formed from line scans in specific regions of the sample confirmed low fluorescent signal intensity (<10 (arbitrary unit)) originating from regions

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Fig. 4. a. Positive ion ToF-SIMS single ion images of fragments originating from amino acid groups on ppMA regions (CH4Nþ, C2H6Nþ, and C2H5Sþ), and non-fouling ppTg film (C3H7Oþ). 4b. Intensity line scan across amino acid and pp-TG related secondary ion fragments. The image resolution was calculated to be w3 mm, based on 80:20 definition.

where ppTg chemistry is present (i.e. regions inside pattern features). The line scans show an increase in signal intensity of w200 units between regions where fluorescent antibody is present (ppMA) when compared to the non-fouling (ppTg) areas.

Control surfaces were prepared by exposed the patterned surfaces to the blocking solution (tris-buffered saline containing 0.1 vol% Tween 20 and 5 wt.% skim milk, TBSTM) prior to exposure to the labeled antibody. The results shown in Fig. S2a clearly indicate

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for fluorescently labeled fibrinogen and w90e100 intensity unit increase for fluorescently labeled BSA). 4. Discussion

Fig. 5. Confocal laser scanning microscopy image and corresponding line scan of AF546-labeled-antibody covalently attached to ppMA patterns on a ppTg film background. (The image resolution was calculated to be w2 mm for both the images, based on 80:20 definition).

that blocking process inhibits the antibody immobilization to the surface. This may be due to a combination of effects, including binding of the milk proteins to the ppMA and hydrolysis of anhydride groups in the aqueous environment. To individually assess each of these factors, two ppMA pattern samples were hydrolyzed. One sample was blocked with TBSTM before being incubated with antibody solution (Fig. S2b), while the other was incubated with antibody solution directly after hydrolysis (Fig. S2c). Both samples clearly showed very low levels of antibody binding, with fluorescence intensities not varying significantly across the sample surfaces, indicating that both methods were capable of preventing non-specific binding of the antibody to the ppMA surfaces. 3.4. Activity of antibodies immobilized on plasma polymer micropatterns A two-stage bioassay was used to investigate the ability of ppMA immobilized antibodies to selectively detect their specific antigen. Each antigen was labeled with a specific fluorescent marker and Fig. 6 shows the resulting fluorescence microscopy images. Two separate antibody/antigen systems were investigated, specifically fibrinogen (Fig. 6a) and BSA (Fig. 6b). The pseudo-color red (Fig. 6a) shows the presence of fibrinogen-AF-546 while the pseudo-color green (Fig. 6b) indicates the presence of FITC-BSA. The corresponding intensity profiles determined by the line scans are also shown. The results clearly demonstrate that the anti-fibrinogen and anti-BSA were immunologically active on the micropatterned surfaces. The line scans across the pattern features shown in Fig. 6 clearly show higher fluorescence signal recorded from ppMA when compared to ppTg regions (w40 intensity unit increase recorded

ToF-SSIMS images of ppMA micropatterns on a ppTg background after derivatization with trifluoroethylamine (Fig. 1) showed the presence of amide linkages in the ppMA regions, while the chemical functionality of the ppTg regions was maintained. Following Scheme 2, hydrolysis of a micropattern resulted in the formation of carboxylic acid groups on the ppMA areas, which prevented any subsequent linkages with amine groups as confirmed via ToF-SSIMS images (Fig. 3). Again, the chemical functionality of the ppTg was unchanged. This was verified by XPS analysis, where the surface chemistry of the freshly deposited ppTg film was compared to a ppTg sample which had been soaked for 2 h in hydrolyzing solution (Fig. S3). These results clearly demonstrated that functionality of both the spontaneous reactive and non-fouling chemistries was successfully maintained during the patterning procedure. The biomedical relevance of these micropatterns was demonstrated in Fig. 4. Similar to the concept employed in the derivatization process, it was shown that fibrinogen selectively immobilized onto the ppMA forming amide linkages, while the ppTg retained its non-fouling characteristics. These results were confirmed by the ToF-SSIMS line scan in Fig. 4b The preferential adsorption and localization of proteins on reactive ppMA micropatterned on a ppTg non-fouling base layer coating further provides the evidence of functional group retention by the respective plasma polymer chemistries. Due to abundance of CNO and other similar molecular species present in the complex protein structures, it is difficult to determine the mode of protein attachment (covalent, physical etc.) to the surface by observing the ToF-SIMS secondary ion yields. However, in view of the derivatization reaction of anhydride functional groups discussed earlier, it is likely that protein immobilization via covalent attachment would dominate. The confocal control samples presented in Fig. S2 provided some insight in regards to the speed at which protein immobilization proceeds on these surfaces. Blocking of the surface via milk protein or hydrolysis of the ppMA resulted in very low levels of antibody binding. Considering that incubation proceeds in an aqueous solution, if hydrolysis effectively blocks the surface from immobilization, then the protein immobilization observed throughout this study must therefore progress at a sufficiently fast pace. It is likely that some of the active sites on the surface become hydrolyzed during incubation; however there is a sufficient density of active sites to ensure covalent immobilization of a significant amount of protein. Visually comparing the control images in Fig. S2 to those obtained for the auto-fluorescent plasma polymer film patterns in Fig. S1, it can be observed that the image intensities are very similar (also apparent from intensity line scans for the respective images). The comparison suggests that the signal observed for these samples are very close to the threshold detection limits set for the confocal instrument. Hence, a major component of the intensity observed for these particular samples can be assigned to noise in the acquisition. The results from the bioassay experiments (Figs. 5 and 6) not only confirm the efficacy of the method for producing patterned surfaces of predictable chemical specificity, but also give us an insight into the immunoactivity of antibody after covalent attachment to anhydride groups. Direct immobilization of fluorescently labeled primary antibody on ppMA further confirms the ToF-SSIMS results where the proteins were found to localize on the ppMA regions. Negligible fluorescence signal from ppTg films present on the patterned surfaces also confirm that the non-fouling behavior of the coating is retained.

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Fig. 6. Recognition of fluorescent antigens by antibodies covalently-immobilized on ppMA micropatterns on a ppTg background. The corresponding intensity profiles are also presented. 6a represents AF-546-fibrinogen, while 6b presents FITC-BSA. (The scale bar represent 200 mm scale length and the image resolution was calculated to be w3 mm, based on 80:20 definition.)

The covalent attachment of immunoglobulin to anhydride (ppMA) functional surfaces could proceed via the terminal amine groups present at the Fab end or (much more likely) via numerous other amine groups (present on amino acids) which make up the antibody structure. The data suggests that a significant proportion of the covalently immobilized antibody has some receptor sites that are still both immunologically active and accessible to the bulk solution and subsequently allowing antigen recognition. While other well established plasma polymerization strategies for biomolecule immobilization, such as allylamine (ppAAm), could have been employed to generate similar micropatterns, these systems would require a significant amount of additional processing to achieve similar levels of functionality. Simply incubating a ppAAm surface in a solution of proteins in the same manner that ppMA was in this study would result in physical adsorption of protein rather than covalent immobilization. To achieve covalent immobilization, multi-step immobilization schemes are necessary to ensure that crosslinking of the protein on the surface does not occur and that the protein remains active [32], thereby expanding the methodology and the reagents required. The most common approach for arraying proteins on or in polymer surface has been to pattern areas with ethylene oxide-based polymers via either polymer grafting or SAMS and then simply adsorbs or stamps proteins into the intervening spaces or on top of the polymer layer [33]. In these cases there is little control on the protein orientation or density as proteins interact with the surface via physical rather than chemical bonds. In the instances where proteins have been covalently coupled to surfaces, the same issues that occur with acid and amine based plasma polymers occur, that is, the need for additional wet chemical processing steps. In addition many of these approaches rely on PDMS stamping to produce the array, limiting the spatial resolution of the patterns to >20 mm and typically 50 mm patterns and creating the added complication of silicone contamination at the surfaces [10]. Our previous studies and the present work have clearly demonstrated that it is possible to produce chemically resolved patterns to <3 mm, a significant

improvement on the resolution regularly possible with a stamping processes. The application of spontaneous reactive ppMA with non-fouling ppTg in the generation of micropatterns has therefore resulted in a platform that provides a simplistic method for covalently immobilizing significant amounts of active protein in a spatially defined manner. 5. Conclusions ToF-SSIMS analysis and confocal imaging have been employed to access the functionality of spontaneous reactive micropatterns fabricated from plasma polymers using a photolithographic technique. The ToF-SSIMS images clearly demonstrated that the reactivity of the anhydride groups and the chemical functionality of the non-fouling tetraglyme micropatterns were maintained during the patterning process. Derivatization with trifluoroethylamine and hydrolysis of the micropatterned surfaces was successfully undertaken while maintaining the integrity of the pattern structure. The bio-functionality of these micropatterns was confirmed using both ToF-SSIMS and confocal imaging. It was shown that fibrinogen selectively immobilized onto the ppMA sections of the micropatterns, most likely via the formation of amide linkages, while no antibody was detected on the ppTg sections. The activity of the covalently-immobilized antibodies was confirmed via confocal imaging of fluorescently tagged antigens bound to the antibodies. The ability to maintain the activity of the immobilized antibodies is an important attribute, and when paired with the spatial resolution obtained from these micropatterns, provides a promising platform for the development of surfaces for bioarray technology. Acknowledgments The authors thank the EPSRC for the financial support of this project. We acknowledge NESAC/Bio for use of the NESAC/Bio toolbox which is funded by NIH grant EB-002027.

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