Kent Academic Repository Volcke, C., Gandhiraman, R.P., Gubala, V., Raj, J., Cummins, Th., Fonder, G., Nooney, R.I., Mekhalif, Z., Herzog, G., Daniels, S. and others (2010) Reactive amine surfaces for biosensor applications, prepared by plasma-enhanced chemical vapour modification of polyolefin materials. Biosensors and Bioelectronics, 25 (8). pp. 1875-1880. ISSN 0956-5663. Downloaded from https://kar.kent.ac.uk/45241/ The University of Kent's Academic Repository KAR The version of record is available from https://doi.org/10.1016/j.bios.2009.12.034 This document version Author's Accepted Manuscript DOI for this version Licence for this version CC BY-NC-ND (Attribution-NonCommercial-NoDerivatives) Additional information Unmapped bibliographic data:(cid:13)(cid:10)LA - English Field not mapped to EPrints(cid:13)(cid:10)J2 - Biosens. Bioelectron. Field not mapped to EPrints(cid:13)(cid:10)C2 - 20117925 Field not mapped to EPrints(cid:13)(cid:10)AD - Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland Field not mapped to EPrints(cid:13)(cid:10)AD - Research Centre in Physics of Matter and Radiation, University of Namur (FUNDP), 61 rue de Bruxelles, 5000 Namur, Belgium Field not mapped to EPrints(cid:13)(cid:10)AD - Biomedical Diagnostics Institute Programme, Tyndall National Institute, University College Cork, Cork, Ireland Field not mapped to EPrints(cid:13)(cid:10)AD - School of Physical Sciences, Dubl... Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version. Author Accepted Manuscripts If this document is identified as the Author Accepted Manuscript it is the version after peer review but before type setting, copy editing or publisher branding. Cite as Surname, Initial. (Year) 'Title of article'. To be published in Title of Journal , Volume and issue numbers peer-reviewed accepted version. Available at: DOI or URL (Accessed: date). Enquiries If you have questions about this document contact [email protected]. Please include the URL of the record in KAR. If you believe that your, or a third party's rights have been compromised through this document please see our Take Down policy (available from https://www.kent.ac.uk/guides/kar-the-kent-academic-repository#policies). Reactive amine surfaces for biosensor applications, prepared by plasma-enhanced chemical vapour modification of polyolefin materials C. Volcke1, 2, *, §, R.P. Gandhiraman1, *, V. Gubala1, J. Raj3, Th. Cummins1, 4, G. Fonder5, R. Nooney1, Z. Mekhalif5, G. Herzog3, S. Daniels1, 6, D.W.M. Arrigan3, A.A. Cafolla1, 4 and D.E. Williams1, 7 1 Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland. 2 Research Centre in Physics of Matter and Radiation (PMR), University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium. 3 Biomedical Diagnostics Institute Programme, Tyndall National Institute, University College Cork, Cork, Ireland. 4 School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. 5 Laboratory of Chemistry and Electrochemistry of Surfaces, University of Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, Belgium. 6 National Center for Plasma Science and Technology, Dublin City University, Glasnevin, Dublin 9, Ireland. 7 MacDiarmid Institute for Advanced Materials and Nanotechnology, Department of Chemistry, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Abstract Functionalization of the plastic chips for selective immobilization of biomolecules is one of the key challenges to be addressed in commercialization of the next-generation point-of-care (POC) diagnostics devices. Multistep liquid-phase deposition process requires a large quantity of solvent to be used in anhydrous conditions, providing quantity of industrial liquid waste. We, in this work, have demonstrated a solventless plasma-based process that integrates low-cost, high throughput, high reproducibility and ecofriendly process for the functionalization of POC device platforms. Amine functionalities have been deposited by plasma-enhanced chemical vapour deposition 1 (PECVD) using a new precursor. For a successful and efficient plasma functionalization process, an understanding of the influence of plasma process parameters on the surface characteristics is essential. The influence of the plasma RF power and the deposition time on the deposited amount of amino functionalities and on their capacity to immobilize nano-objects (i.e., nanoparticles) and biomolecules (i.e. DNA) was examined. Surfacial properties were related to the binding capacity of the films and to the amino content, as revealed by the “nanoparticle approach” and DNA attachment experiments. The key process determinants were to have a sufficient power in the plasma to activate and partially fragment the monomer but not too much as to lose the reactive amine functionality, and sufficient deposition time to develop a reactive layer but not to consume or erode the amine reactivity. An immunoassay performed using human immunoglobulin (IgG) as a model analyte shows an improvement of the detection limit by two orders of magnitude beyond that obtained using devices activated by liquid-phase reaction. Keywords: biosensors, polymer, plasma enhanced chemical vapour deposition, nanoparticle, DNA, immunoassay. * Those authors contributed equally § To whom correspondance should be addressed. Phone: +32 81 72 54 30. Fax: +32 81 72 47 18. E-mail : [email protected] 2 1. Introduction Immobilization of biomolecules onto surfaces, and particularly onto polymeric substrates, is a key issue for the fabrication of next-generation biosensors and biomedical diagnostic devices. Zeonor®, a type of cycloolefin polymer (COP), is one such polymer presenting excellent optical properties, good chemical resistance, ease of fabrication and cost effectiveness (Diaz-Quijada 2007, Kameoka 2001). Though several biomolecule immobilization techniques have been intensively reported in the literature during the last decade, only a few recent communications address the issue of activation of cycloolefin polymers (Jönsson 2008, Laib and McCraith 2007, Raj 2009). In general, biomolecular immobilization is classified as chemical (Kwon 2006, Mateo-Marti 2005) or physical (LaGraff and Chu-LaGraff 2006, Sethuraman 2004) immobilization. Chemical immobilization, through covalent linkage of biomolecules onto surfaces has shown good reproducibility and coverage (compared to physical adsorption) and therefore has become predominant in the research literature. In such cases, liquid-phase deposition of functional groups, further attaching proteins or nucleic acids, on activated surfaces is a routinely used procedure. For example, one of the most studied methods is the self-assembly of aminosilanes on oxidized silicon surfaces (Choi 2006, Oh 2002, Song 2008, Zhang and Srinivasan 2004). These modification procedures have still not been extensively investigated for polymers, for which physical immobilization is the dominant methodology. Liquid-phase deposition chemistry for attachment of aminosilanes onto surfaces, as broadly studied, is demanding (water-free environment, time-consuming, hazardous materials) and hardly applicable to large industrial production requirements (Kurth and Bein 1995). Gas-phase deposition of amine reactive groups, using plasma 3 enhanced chemical vapour deposition (PECVD) for example, overcomes the drawbacks of liquid-phase deposition (Foose 2007, White and Tripp 2000). PECVD, a versatile surface engineering technique, has proven to be an excellent tool for surface modification at low temperatures (Dudek 2009a, Favia 2001, Gandhiraman 2007, Muguruma and Kase 2006). In principle, it offers significant advantages: uniformly diverse functional coating deposition on various surfaces (even on complex 3D structures); precise control of thickness; and adaptability for large mass production processing. Surface amination by PECVD has previously been studied using, e.g., ethylene diamine (EDA) as precursor on different substrates (Gomathi 2008, Jung 2006, Slocik 2006). Unfortunately, the adhesion and stability of these PECVD deposited EDA films onto cycloolefin polymers were very poor (unpublished data). The results were interpreted to imply merely physisorption and not reactive chemisorption of EDA molecules onto the polymeric surface. Therefore new precursors, potentially presenting good adhesion and stability on polymeric surfaces, and also offering amine groups for subsequent reaction, are needed. In this context, 3-(aminopropyl) triethoxysilane (APTES), commonly used for liquid-phase deposition of amine functional groups (Moon 1997, Qian 1999, Gu and Cheng 2008, Simon 2002, Sridharan 2008) is expected to present all the requirements needed. Previous investigations of chemical vapour deposition (CVD) on silicon substrate at elevated temperatures highlighted its suitability (Arroyo-Hernandez 2006). However, to be appropriate for polymeric surfaces, PECVD deposition has to be used. PECVD deposition of APTES is expected to result in reproducible silanized surfaces containing reactive amine groups, with good adhesion to cycloolefin polymer through a siloxane network. The characteristics of PECVD coatings are highly 4 dependent on the nature of the substrate, the precursor and the plasma characteristics including input power and deposition time (Di Mundo 2007). In this paper, we present a detailed investigation on the optimization of the reactive-amine content of a deposited amino-film on a COP surface, for improved biomolecule attachment by appropriate choice of experimental parameters. Plasma conditions, for example, the plasma electron density, were shown to have significant effects on the performance of the coating. Aminated coatings have been prepared by PECVD of APTES onto a Zeonor® substrate. The influence of the plasma RF power and the deposition time on the deposited amount of amino functionalities and on their capacity for immobilizing nano-size objects (nanoparticles) and biomolecules (ssDNA) was investigated. Water contact angles, atomic force microscopy (AFM), ellipsometry and polarization modulation infrared reflection-absorption spectroscopy (PMIRRAS) have been used for physical and chemical characterization. Fluorescence microscopy has been performed to quantify the binding of an amine-reactive fluorophore and a labelled ssDNA. Reactive amine coverage on the surface has been evaluated by attachment of nanoparticles, visualised by atomic force microscope (AFM). This was a particularly effective and simple evaluation tool. An immunoassay was performed using human immunoglobulin G (IgG) as a model analyte and the detection limit was compared to that of the liquid-phase deposition process. We describe overall a procedure for determining the optimal deposition condition to create a surface presenting a high amount of amino groups, capable of attaching the highest amount of biomolecules. 5 2. Experimental Part 2.1. Materials Plain Zeonor® slides (Zeonor 1060R, Zeon, Japan) were obtained from Åmic AB (Uppsala, Sweden). 3-aminopropyltriethoxysilane (APTES), Lissamine® rhodamine B sulfonyl chloride (l = 532 nm excitation and l = 550 nm emission) and exc. em acetonitrile were purchased from Sigma Aldrich and used without further treatment. 2.2. PECVD system The plasma deposition was carried out using a computer controlled Europlasma CD 300 PECVD system. The aluminum vacuum chamber was connected to a Dressler’s CESAR 136 RF power source operating at 13.56 MHz, capable of generating a maximum of 600 W RF power, supplied with an automated impedance match-box for effective RF energy input to the plasma. In the PECVD process, the input radio frequency (RF) current/ voltage was supplied to the powered electrode, a 24 cm x 21 cm plate with a 6 cm diameter hole in the middle, placed slightly below the top of the chamber. The powered electrode was cooled with running water. A 24 cm x 21 cm x 1.2 cm electrically isolated (floating potential), water cooled hollow metallic setup placed 10 cm below the powered electrode was used as a substrate holder. The detailed description and a schematic of the system used is shown elsewhere (Dudek 2009b, Gandhiraman 2009). 2.3. Surface preparation Zeonor® slides were first cleaned with dry air before being loaded in the plasma chamber. The system pressure was pumped down to a base pressure of 25 mTorr. APTES precursor was stored in a container (connected to the vacuum chamber). As 6 the vapour pressure of APTES is less than 10 Torr at 100° C, the APTES container and the supply line from source to chamber was heated at 80° C. Plasma pre- treatment for cleaning and activation was carried out using a mix of argon (50 sccm) and oxygen (50 sccm) plasma at 250 watt RF power for 3 minutes. The oxygen supply was then closed and the plasma RF power was decreased to the desired value (7 watt, 14 watt or 25 watt). APTES was then introduced in the chamber for the required deposition time (2 min, 4 min or 8 min). The operating pressure was ~ 70 mTorr. 2.4. Surface characterization 2.4.1. Ellipsometry The thickness of the APTES coating on Zeonor® was characterized using J.A. Woollam Co., Inc EC-400, M-2000UI Spectroscopic Ellipsometer. All layers were modelled as a simple silicon dioxide dispersion layer to extract an effective thickness. 2.4.2. Plasma electron density measurement: Hairpin probe The hair pin probe is a diagnostic technique for measuring absolute electron density in the discharge using a microwave resonance probe (Dudek 2009b). The principle of the probe is based on measuring the dielectric constant of the plasma surrounding the resonant structure. The probe, consisting of two parallel wires, short circuited at one end and open at the other, resembles a hairpin. The typical length of the hairpin was 1-2 cm. The microwave resonance of the hairpin was used to determine electron density. 2.4.3. Water contact angles 7 The film wettability was analyzed by measuring the water contact angle of the film surface (First Ten Angstroms FTA200 contact angle analyser). High purity HPLC grade water (Sigma Aldrich) was used for the measurement. 2.4.4. Roughness measurements - Atomic force microscopy (AFM) AFM examinations were performed in ambient air with a commercial microscope (Dimension 3100 controlled by a Nanoscope IIIa controller, Digital Instruments, Santa-Barbara – CA, USA), in the Tapping-Mode™, using standard silicon cantilevers (BudgetSensors®, Innovative Solutions Bulgaria Ltd, Bulgaria) with a 7 nm radius of curvature and a 42 N.m-1 spring constant (nominal values). Topographic images were recorded at a scanning rate of 1-2 Hz, and a resonance frequency of about 300 kHz (nominal value). The background slope was resolved using a first order polynomial function. No further filtering was performed. The surface roughness of substrates and PECVD deposited APTES coatings was evaluated over 3 images (5 µm x 5 µm) and the standard deviation was then calculated. The root-mean-square roughness (Rrms) was defined as the average of height deviations taken from the mean plane (Haidopoulos 2007). 2.4.5. Polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) Appropriate substrates had to be prepared for infrared reflection-absorption spectroscopy since surfaces highly reflecting to IR light are required for this technique. The substrates were obtained by sputtering gold on cleaned silicon wafer (with piranha solution, followed by extensive rinsing). Zeonor® was dissolved in xylene (0.25 gr/l) by 10 min sonication and the solution was filtered through a 8 millipore 0.2 µm filter. The solution was finally spin coated onto the gold coated silicon substrates (Jönsson 2008). PMIRRAS spectra of the COP-coated gold surfaces, before and after APTES deposition were recorded on a Bruker Equinox 55- PMA37 spectra equipped with liquid nitrogen cooled mercury cadmium-telluride (MCT) detector and a zinc-selenide photoelastic modulator. The infrared light was modulated between s- and p-polarization at a frequency of 50 kHz. The incident angle upon the sample surface was around 85°. Signals generated from each polarization (Rs and Rp) were detected simultaneously by a lock-in amplifier and used to calculate the differential surface reflectivity (DR/R) = (Rp - Rs)/(Rp + Rs). The spectra were an average of 640 scans and were taken at a spectral resolution of 2 cm-1. 2.4.6. Nanoparticle (NP) approach Preparation of silica NPs Silica NP’s were prepared using a microemulsion method (Arriagada and Osseo- Asare 1999). The microemulsion was formed by adding water (0.96 ml) to a mixture of cyclohexane (15 ml), n-hexanol (3.6 ml) and Triton® X-100 (3.788 g). Following this, tetraethylorthosilicate (TEOS) (0.2 ml) and NH OH (0.16 ml) were added to start 4 the growth of the silica NPs. The reaction was stirred for 24 hrs, after which TEOS (0.1 ml) was added with rapid stirring. After 30 minutes 3- trihydroxysilylpropylmethylphosphonate sodium salt (THPMP) (0.08 ml) was added with stirring to prevent aggregation of the nanoparticles. After a further 5 minutes 3- aminopropyltrimethoxysilane (APTMS) (0.02 ml) was added for conjugation to the amine functionalized surfaces. The NPs were separated from the solution with the addition of excess absolute ethanol and centrifuged twice with ethanol and once with deionized water (Heraeus, Biofuge pico). Sonication was used between the washing 9