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Surface biofunctionalization of nanostructured GeSbSe chalcogenide glass thin films
Journal of Non-Crystalline Solids 355 (2009) 208–212
Contents lists available at ScienceDirect
Journal of Non-Crystalline Solids journal homepage: www.elsevier.com/locate/jnoncrysol
Surface biofunctionalization of nanostructured GeSbSe chalcogenide glass thin films R.J. Martín-Palma a,c,*, M.C. Demirel a,b, H. Wang b, C.G. Pantano a,c a b c
Materials Research Institute, The Pennsylvania State University, University Park, PA 16802, USA Department of Engineering Science and Mechanics, The Pennsylvania State University, University Park, PA 16802, USA Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA
a r t i c l e
i n f o
Article history: Received 18 June 2008 Received in revised form 6 October 2008 Available online 13 December 2008 PACS: 87.85.fk 81.15.kk 77.84.Bw 47.20.Km 77.84.Jd 36.20.Ng
a b s t r a c t Thin nanostructured chalcogenide films were grown using the oblique angle deposition (OAD) technique and subsequently polymerized with thin poly(amino-p-xylylene) (PPX) films. Our objective was twofold, i.e., to use deposited polymeric thin films to allow the attachment of biomolecules to chalcogenide glass thin films, and at the same time, to increase surface area by OAD to enhance surface functionality. The effectiveness of this approach was evaluated by Fourier transform infrared spectroscopy (FTIR), together with a combination of fluorescent protein immobilization and confocal microscopy characterization. It is shown that the presence of amine groups on the surface of the polymer coated chalcogenide thin films yield a notable increment of surface coverage with proteins at large evaporation oblique angles which is expected to enhance detection performance of the film in biosensor applications. Ó 2008 Elsevier B.V. All rights reserved.
Keywords: Biosensors Vapor phase deposition Chalcogenides FTIR measurements Polymers and organics
1. Introduction Although many different materials have been grown using the oblique angle deposition (OAD) technique [1–6], very few have been synthesized using infrared transparent or glass forming materials. Among these materials, chalcogenide glasses have been successfully deposited by OAD [7], thus adding extra functionality to their unique properties as to expand their range of optical and morphological properties [8,9] and, in turn, their applications. Chalcogenide glasses are of great technological importance, since their high transmission in the infrared (IR) region [10], together with the possibility of modifying their optical band gap and index of refraction by illumination [11], make them very attractive materials in the areas of infrared optics, optical signal imaging, and data storage. Moreover, optical materials transparent in the mid-IR spectral region offer access to fundamental fingerprint absorptions of organic molecules and biomolecules [12,13]. In this respect, mid-IR sensors are currently receiving increased attention because of their inherent molecular selectivity. * Corresponding author. Address: Materials Research Institute, The Pennsylvania State University, University Park, PA 16802, USA. E-mail address: [email protected] (R.J. Martín-Palma). 0022-3093/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jnoncrysol.2008.10.013
However, since functionalization of chalcogenide glasses remains a challenge, the use of thin polymer coatings may provide an alternative solution. In this regard, polymer coating are of interest as interfaces for biomedical applications given their potential for the incorporation of functional groups, which can be used to conjugate biomolecules including proteins, antigens or cell receptors to implant surfaces [14]. The resulting biomimetic coatings provide interfaces that may allow control of the interactions between biomaterials and organisms. In particular, poly(p-xylylene) (PPX) can be used as a conformal thin film coating with high dielectric strength, very low permeability to chemicals, and high physical strength at elevated temperatures. Additionally, polymers of the PPX type show an extraordinarily high biostability compared to common polymer coatings [15]. Thin films of PPX can be grown by combining pyrolysis and evaporation of [2.2]paracyclophane under low vacuum. Paracyclophane was first prepared in 1949 by Brown and Farthing [16] and systematically investigated by Cram and coworkers from 1951 onward [17]. Chemically, paracyclophane is a dimer of two p-xylylene molecules that have an unusual three-dimensional aromatic structure compared to the planar benzene ring. In the present paper, deposition of PPX polymeric layers followed by functionalization with 1,6-hexamethylene diisocyanate (HMDI) is explored as a method to link protein biomolecules to
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209
2.2. Poly(amino-p-xylylene) deposition The precursors for the subsequent deposition of poly(amino-pxylylene) (PPX) thin films were prepared according to a previously reported method [18,19]. Accordingly, fabrication of the PPX thin film starts with an amino-paracyclophane dimer, which is placed in an evacuated chamber and converted to a reactive vapor of monomers by pyrolysis. The deposition rate and the deposition pressure are controlled by the evaporation temperature (175 °C) of the dimer and the pyrolysis temperature (690 °C). 2.3. Surface functionalization The PPX-coated chalcogenide films were placed in a dry 50 mL round bottom flask with a surface coupling reagent (5 mL of anhydrous toluene, 30 lL of 1,6-hexamethylene diisocyanate, HMDI, a small amount of the catalyst, di-(n-butyl)tin dilaurate and the flask was sealed. After incubation at room temperature for 4 h, the film was taken out, washed subsequently with anhydrous toluene and dried under vacuum. A detailed description of the immobilization process onto PPX-coatings via HMDI coupling can be found elsewhere (see for instance Ref. [15]).
Fig. 1. Schematics of the process used to link protein biomolecules to the surface of GeSbSe thin films grown at two different angles: (a) normal incidence and (b) 85°. Deposition at increased oblique angles result in thin films of increased surface area.
the surface of GeSbSe chalcogenide glasses, and thereby provide selective adsorption sites for biosensors. In particular, GeSbSe thin films of very different morphology, i.e. planar and columnar, were coated with PPX layers and subsequently functionalized with HDMI as shown in Fig. 1. Finally, green fluorescent protein (GFP) immobilization was used to evaluate the effectiveness of the overall process. 2. Techniques and materials 2.1. Deposition of GeSbSe chalcogenide glasses Amorphous GeSbSe thin films were grown on silicon substrates from commercially available bulk chalcogenide glasses with nominal composition Ge28Sb12Se60. The silicon substrates were cleaned with acetone and ethanol, dried with nitrogen, and immediately loaded into the vacuum chamber. The GeSbSe was thermally evaporated using a current of 80 A with substrates placed at normal incidence (0°) and at an oblique angle of 85° with respect to the substrate normal.
Fig. 2. Characteristic surface morphologies of GeSbSe thin films grown at (a) normal incidence (0°) and (b) at 85°.
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2.4. GFP expression The expression of GFP has been described elsewhere [19]. Briefly, the cDNA of the GFP is transferred into a phagemid vector and expressed in Escherichia coli DH5aFT cells in our lab. Single clones were picked for expression, determination of fluorescence levels, and sequencing. GFP is purified using a 6xHis column (C-terminal). Superfolder GFP is stable at 85 °C, in 10 M urea, and pH 4– 11. 2.5. Protein coupling to PPX surface After PPX-coating and functionalization with the selected surface reagent (HMDI), they were dried in the desiccator for 20 min. Then, films are taken out and placed in a flask with a 2 lg/ml solution of GFP, and capped overnight at room temperature. Films are finally removed from solution and placed in the desiccator to dry for 20 min and subsequently rinsed in deionized water. 2.6. Surface characterization Scanning electron microscope (SEM) characterization was performed by the use of a FEI Quanta 200 equipment. Attenuated total reflectance infrared (ATR-IR, Thermo Nicolet IR, diamond crystal) data were collected with respect to a silicon wafer reference in air and recorded using Norton-Beer apodization with 4 cm 1 resolution. For each spectrum 100 scans are co-added. 2.7. Confocal laser microscopy The Olympus Fluoview 300 confocal laser scanning microscope with a single-line 488 nm blue laser (10% transmissivity) was used
with the 40 oil 1.30 NA objective for imaging. A variable bandpass filter 500–600 nm was used and the intensity per field analysis was performed in ImagePro Plus 5.0 (Media Cybernetics, Inc). 3. Results Thin chalcogenide films were grown at two different evaporation angles with respect to the substrate normal: 0° (normal incidence) and at 85°. Figs. 2(a) and (b) show their differentiated characteristic surface morphologies. Subsequent surface modification of GeSbSe chalcogenide structures was performed by the growth of poly(p-xylylene) (PPX) thin films. Figs. 3 (bottom) and 4 (bottom) show the ATR-IR spectra in the 3600–700 cm 1 wavenumber range of GeSbSe thin films grown at 0° and 85°, coated with PPX thin films. After surface modification, functionalization with HDMI was performed. The corresponding ATR-IR spectra are presented in Figs. 3 (middle) and 4 (middle). GeSbSe layers coated with PPX thin films were incubated with green fluorescent protein (GFP), which is known to react with HMDI linker groups under mild conditions. GFP is a monomeric protein with a molecular weight of 28 kDa and it has a beta-can structure with 11 antiparallel b-strands and three a-helices. The wavelength of excitation and emission of GFP are, respectively, in the blue (480 nm) and green (509 nm) portion of the visible spectrum. A 2 lg/ml solution of GFP is incubated overnight on the nanostructured PPX surface. Thin films are subsequently rinsed after incubation to ensure that only covalently bonded molecules would remain attached to the surface, thus eliminating GFP molecules adsorbed. Figs. 3 (top) and 4 (top) show the corresponding infrared spectra. Finally, confocal microscopy characterization was used to prove the presence of proteins immobilized on the surface of PPX-coated GeSbSe thin films. In this regard, Fig. 5 shows the measured fluo-
Fig. 3. Characteristic ATR-IR spectra of GeSbSe chalcogenide thin film grown at 0° polymerized with PPX (bottom), after HMDI coupling (middle) and after GFP immobilization (top).
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211
Fig. 4. Characteristic ATR-IR spectra of GeSbSe chalcogenide thin film grown at an oblique angle of 85° polymerized with PPX (bottom), after HMDI coupling (middle) and after GFP immobilization (top).
rescent intensity of the two GeSbSe surfaces of very different roughness after GFP incubation. From confocal characterization, larger fluorescence intensity is measured from thin films of larger surface area, i.e., those in which the GeSbSe layers were grown at large oblique evaporation angles. 4. Discussion From Fig. 2(a) and (b) it is observed that thin film growth at large evaporation angles (close to 90°) results in surface texturing at the nanoscale, leading to increased surface area. As previously
Fig. 5. Fluorescence intensity determined by confocal microscopy after GFP immobilization. GeSbSe thin films grown at normal incidence (0°) and 85° were used as substrates.
demonstrated by performing quantitative microstructural analysis of GeSbSe chalcogenide glasses, rms roughness increases exponentially with increasing evaporation angle from around 1.15 nm at 0° to over 20 nm at 85° [8]. In addition, the surface area increment for films grown at 0° is close to 2% when compared to that of an atomically flat surface, while this increment is over 30% for films grown at 85°. From the spectra shown in Figs. 3 (bottom) and 4 (bottom), peaks corresponding to PPX are clearly identified in the 3010– 2800 cm 1 wavenumber range, associated to CH stretching modes. Additionally, absorption peaks found in the region 3450– 3160 cm 1 are associated to amine N–H stretching vibrations, while these observed in the 1650–1580 cm 1 are associated to amine N–H deformation vibrations [20]. Moreover, peaks located at 1620 cm 1 and 1578 cm 1 are assigned to the C@O stretch (amide I) and the N–H bend (amide II) respectively. Consequently, the presence of PPX thin films containing a significant amount of amine groups is consistent with the infrared spectra shown in Figs. 3 (top) and 4 (top). The spectra of HMDI-functionalized films (Figs. 3 (middle) and 4 (middle)) show the absorption of amide groups, thus indicating the binding between the amine (–NH2) groups on the surface of the nanostructured PPX film and the HMDI. In addition, the absorption at 2200 cm 1 indicates the free isocyanate groups on the surface of the PPX film. This absorption of free isocyanate groups decreases significantly and eventually disappears after the GFP-coupling to the surface, as shown in Figs. 3 (top) and 4 (top). It is worth pointing out that infrared characterization does not allow quantifying the fraction of free isocyanate groups that are attached to the polymer backbone and the fraction that are free to attach to the proteins. According to our estimations, the fraction of free isocyanate groups should be below 0.5 after carefully washing, since the possibility that both of the isocyanate groups are attached to the polymer cannot be prevented.
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As discussed in the previous paragraph, infrared characterization of PPX-coated GeSbSe thin films clearly indicates the presence of amine groups. However, definitive confirmation of the presence of active amine groups requires the assessment of their reactivity with bioorganic molecules. Thus, from fluorescence intensity measurements (Fig. 5) it was determined that the growth of GeSbSe thin films at large oblique angles results in large surface areas leading to increased bioactivity. Finally, it should be noted that fast substrate rotation combined with tilting did not produce any significant improvement in fluorescence intensity. 5. Summary and conclusions Poly-(p-xylylene) (PPX) thin films were deposited onto GeSbSe chalcogenide glasses grown at two different angles (normal incidence, 0°, and 85°) with the objective of nanostructuring their surface, and was followed by functionalization with PPX and HMDI. In this state, the presence of amine groups was determined by infrared spectroscopy. Subsequently, fluorescent proteins were shown to be immobilized onto these nanostructured films by confocal microscopy. The protein coverage, as shown by the fluorescence intensity, was increased for the GeSbSe chalcogenide layers grown at large angles. Accordingly, large evaporation angle leads to higher transparency and greater potential performance of the sensor. This is a key factor for the further development of optical biosensors with increased sensitivity. Acknowledgments The authors would like to acknowledge support from the NSF IMI on New Functionality in Glass, and the Penn State University
Materials Research Institute. R.J.M.-P. acknowledges support from the Ministerio de Educación y Ciencia (MEC, Spain) through Grant No. PR2007-0085. M.C.D. and H.W are supported by a Young Investigator Program Award from the Office of Naval Research (N000140710801), References [1] K. Robbie, M.J. Brett, A. Lakhtakia, Nature 384 (1996) 616. [2] R. Messier, V.C. Venugopal, P.D. Sunal, J. Vac. Sci. Technol. A 18 (4) (2000) 1538. [3] A. Lakhtakia, R. Messier, Sculptured Thin Films. Nanoengineered Morphology and Optics, SPIE, Bellingham, 2005. [4] M.H. Hawkeye, M.J. Brett, J. Vac. Sci. Technol. A 25 (5) (2007) 1317. [5] M.C. Demirel, E. So, T.M. Ritty, S. Naidu, A. Lakhtakia, J. Biomed. Mat. Res. B 81B (2007) 219. [6] S. Boduroglu, M. Cetinkaya, W.J. Dressick, A. Singh, M.C. Demirel, Langmuir 23 (2007) 11391. [7] J.V. Ryan, M. Horn, A. Lakhtakia, C.G. Pantano, Proc. SPIE 5593 (2004) 643. [8] R.J. Martín-Palma, J.V. Ryan, C.G. Pantano, J. Appl. Phys. 101 (2007) 083513. [9] R.J. Martín-Palma, J.V. Ryan, C.G. Pantano, J. Vac. Sci. Technol. A 25 (3) (2007) 587. [10] K.A. Cerqua-Richardson, J.M. McKinley, B. Lawrence, S. Joshi, A. Villeneuve, Opt. Mat. 10 (1998) 155. [11] A. Ganjoo, H. Jain, C. Yu, R. Song, J.V. Ryan, J. Irudayaraj, Y.J. Ding, C.G. Pantano, J. Non-Cryst. Solids 352 (2006) 584. [12] B. Mizaikoff, Anal. Chem. A 75 (11) (2003) 258A. [13] C. Yu, A. Ganjoo, H. Jain, C.G. Pantano, J. Irudayaraj, Anal. Chem. 78 (8) (2006) 2500. [14] J. Lahann, H. Höcker, R. Langer, Angew. Chem. Int. Ed. 40 (4) (2001) 726. [15] J. Lahann, D. Klee, H. Höcker, Macromol. Rapid Commun. 19 (1998) 441. [16] C.J. Brown, A.C. Farthing, Nature 164 (1949) 915. [17] D.J. Cram, H. Steinberg, J. Am. Chem. Soc. 73 (12) (1951) 5691. [18] Y. Elkasabi, H.-Y. Chen, J. Lahann, Adv. Mater. 18 (12) (2006) 1521. [19] M. Cetinkaya, A. Zeytun, J. Sofo, M.C. Demirel, Chem. Phys. Lett. 419 (1–3) (2006) 48. [20] G. Socrates, Infrared and Raman Characteristic Group Frequencies. Tables and Charts, 3rd Ed., Wiley, Chichester, 2001.
Contents lists available at ScienceDirect
Journal of Non-Crystalline Solids journal homepage: www.elsevier.com/locate/jnoncrysol
Surface biofunctionalization of nanostructured GeSbSe chalcogenide glass thin films R.J. Martín-Palma a,c,*, M.C. Demirel a,b, H. Wang b, C.G. Pantano a,c a b c
Materials Research Institute, The Pennsylvania State University, University Park, PA 16802, USA Department of Engineering Science and Mechanics, The Pennsylvania State University, University Park, PA 16802, USA Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA
a r t i c l e
i n f o
Article history: Received 18 June 2008 Received in revised form 6 October 2008 Available online 13 December 2008 PACS: 87.85.fk 81.15.kk 77.84.Bw 47.20.Km 77.84.Jd 36.20.Ng
a b s t r a c t Thin nanostructured chalcogenide films were grown using the oblique angle deposition (OAD) technique and subsequently polymerized with thin poly(amino-p-xylylene) (PPX) films. Our objective was twofold, i.e., to use deposited polymeric thin films to allow the attachment of biomolecules to chalcogenide glass thin films, and at the same time, to increase surface area by OAD to enhance surface functionality. The effectiveness of this approach was evaluated by Fourier transform infrared spectroscopy (FTIR), together with a combination of fluorescent protein immobilization and confocal microscopy characterization. It is shown that the presence of amine groups on the surface of the polymer coated chalcogenide thin films yield a notable increment of surface coverage with proteins at large evaporation oblique angles which is expected to enhance detection performance of the film in biosensor applications. Ó 2008 Elsevier B.V. All rights reserved.
Keywords: Biosensors Vapor phase deposition Chalcogenides FTIR measurements Polymers and organics
1. Introduction Although many different materials have been grown using the oblique angle deposition (OAD) technique [1–6], very few have been synthesized using infrared transparent or glass forming materials. Among these materials, chalcogenide glasses have been successfully deposited by OAD [7], thus adding extra functionality to their unique properties as to expand their range of optical and morphological properties [8,9] and, in turn, their applications. Chalcogenide glasses are of great technological importance, since their high transmission in the infrared (IR) region [10], together with the possibility of modifying their optical band gap and index of refraction by illumination [11], make them very attractive materials in the areas of infrared optics, optical signal imaging, and data storage. Moreover, optical materials transparent in the mid-IR spectral region offer access to fundamental fingerprint absorptions of organic molecules and biomolecules [12,13]. In this respect, mid-IR sensors are currently receiving increased attention because of their inherent molecular selectivity. * Corresponding author. Address: Materials Research Institute, The Pennsylvania State University, University Park, PA 16802, USA. E-mail address: [email protected] (R.J. Martín-Palma). 0022-3093/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jnoncrysol.2008.10.013
However, since functionalization of chalcogenide glasses remains a challenge, the use of thin polymer coatings may provide an alternative solution. In this regard, polymer coating are of interest as interfaces for biomedical applications given their potential for the incorporation of functional groups, which can be used to conjugate biomolecules including proteins, antigens or cell receptors to implant surfaces [14]. The resulting biomimetic coatings provide interfaces that may allow control of the interactions between biomaterials and organisms. In particular, poly(p-xylylene) (PPX) can be used as a conformal thin film coating with high dielectric strength, very low permeability to chemicals, and high physical strength at elevated temperatures. Additionally, polymers of the PPX type show an extraordinarily high biostability compared to common polymer coatings [15]. Thin films of PPX can be grown by combining pyrolysis and evaporation of [2.2]paracyclophane under low vacuum. Paracyclophane was first prepared in 1949 by Brown and Farthing [16] and systematically investigated by Cram and coworkers from 1951 onward [17]. Chemically, paracyclophane is a dimer of two p-xylylene molecules that have an unusual three-dimensional aromatic structure compared to the planar benzene ring. In the present paper, deposition of PPX polymeric layers followed by functionalization with 1,6-hexamethylene diisocyanate (HMDI) is explored as a method to link protein biomolecules to
R.J. Martín-Palma et al. / Journal of Non-Crystalline Solids 355 (2009) 208–212
209
2.2. Poly(amino-p-xylylene) deposition The precursors for the subsequent deposition of poly(amino-pxylylene) (PPX) thin films were prepared according to a previously reported method [18,19]. Accordingly, fabrication of the PPX thin film starts with an amino-paracyclophane dimer, which is placed in an evacuated chamber and converted to a reactive vapor of monomers by pyrolysis. The deposition rate and the deposition pressure are controlled by the evaporation temperature (175 °C) of the dimer and the pyrolysis temperature (690 °C). 2.3. Surface functionalization The PPX-coated chalcogenide films were placed in a dry 50 mL round bottom flask with a surface coupling reagent (5 mL of anhydrous toluene, 30 lL of 1,6-hexamethylene diisocyanate, HMDI, a small amount of the catalyst, di-(n-butyl)tin dilaurate and the flask was sealed. After incubation at room temperature for 4 h, the film was taken out, washed subsequently with anhydrous toluene and dried under vacuum. A detailed description of the immobilization process onto PPX-coatings via HMDI coupling can be found elsewhere (see for instance Ref. [15]).
Fig. 1. Schematics of the process used to link protein biomolecules to the surface of GeSbSe thin films grown at two different angles: (a) normal incidence and (b) 85°. Deposition at increased oblique angles result in thin films of increased surface area.
the surface of GeSbSe chalcogenide glasses, and thereby provide selective adsorption sites for biosensors. In particular, GeSbSe thin films of very different morphology, i.e. planar and columnar, were coated with PPX layers and subsequently functionalized with HDMI as shown in Fig. 1. Finally, green fluorescent protein (GFP) immobilization was used to evaluate the effectiveness of the overall process. 2. Techniques and materials 2.1. Deposition of GeSbSe chalcogenide glasses Amorphous GeSbSe thin films were grown on silicon substrates from commercially available bulk chalcogenide glasses with nominal composition Ge28Sb12Se60. The silicon substrates were cleaned with acetone and ethanol, dried with nitrogen, and immediately loaded into the vacuum chamber. The GeSbSe was thermally evaporated using a current of 80 A with substrates placed at normal incidence (0°) and at an oblique angle of 85° with respect to the substrate normal.
Fig. 2. Characteristic surface morphologies of GeSbSe thin films grown at (a) normal incidence (0°) and (b) at 85°.
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2.4. GFP expression The expression of GFP has been described elsewhere [19]. Briefly, the cDNA of the GFP is transferred into a phagemid vector and expressed in Escherichia coli DH5aFT cells in our lab. Single clones were picked for expression, determination of fluorescence levels, and sequencing. GFP is purified using a 6xHis column (C-terminal). Superfolder GFP is stable at 85 °C, in 10 M urea, and pH 4– 11. 2.5. Protein coupling to PPX surface After PPX-coating and functionalization with the selected surface reagent (HMDI), they were dried in the desiccator for 20 min. Then, films are taken out and placed in a flask with a 2 lg/ml solution of GFP, and capped overnight at room temperature. Films are finally removed from solution and placed in the desiccator to dry for 20 min and subsequently rinsed in deionized water. 2.6. Surface characterization Scanning electron microscope (SEM) characterization was performed by the use of a FEI Quanta 200 equipment. Attenuated total reflectance infrared (ATR-IR, Thermo Nicolet IR, diamond crystal) data were collected with respect to a silicon wafer reference in air and recorded using Norton-Beer apodization with 4 cm 1 resolution. For each spectrum 100 scans are co-added. 2.7. Confocal laser microscopy The Olympus Fluoview 300 confocal laser scanning microscope with a single-line 488 nm blue laser (10% transmissivity) was used
with the 40 oil 1.30 NA objective for imaging. A variable bandpass filter 500–600 nm was used and the intensity per field analysis was performed in ImagePro Plus 5.0 (Media Cybernetics, Inc). 3. Results Thin chalcogenide films were grown at two different evaporation angles with respect to the substrate normal: 0° (normal incidence) and at 85°. Figs. 2(a) and (b) show their differentiated characteristic surface morphologies. Subsequent surface modification of GeSbSe chalcogenide structures was performed by the growth of poly(p-xylylene) (PPX) thin films. Figs. 3 (bottom) and 4 (bottom) show the ATR-IR spectra in the 3600–700 cm 1 wavenumber range of GeSbSe thin films grown at 0° and 85°, coated with PPX thin films. After surface modification, functionalization with HDMI was performed. The corresponding ATR-IR spectra are presented in Figs. 3 (middle) and 4 (middle). GeSbSe layers coated with PPX thin films were incubated with green fluorescent protein (GFP), which is known to react with HMDI linker groups under mild conditions. GFP is a monomeric protein with a molecular weight of 28 kDa and it has a beta-can structure with 11 antiparallel b-strands and three a-helices. The wavelength of excitation and emission of GFP are, respectively, in the blue (480 nm) and green (509 nm) portion of the visible spectrum. A 2 lg/ml solution of GFP is incubated overnight on the nanostructured PPX surface. Thin films are subsequently rinsed after incubation to ensure that only covalently bonded molecules would remain attached to the surface, thus eliminating GFP molecules adsorbed. Figs. 3 (top) and 4 (top) show the corresponding infrared spectra. Finally, confocal microscopy characterization was used to prove the presence of proteins immobilized on the surface of PPX-coated GeSbSe thin films. In this regard, Fig. 5 shows the measured fluo-
Fig. 3. Characteristic ATR-IR spectra of GeSbSe chalcogenide thin film grown at 0° polymerized with PPX (bottom), after HMDI coupling (middle) and after GFP immobilization (top).
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211
Fig. 4. Characteristic ATR-IR spectra of GeSbSe chalcogenide thin film grown at an oblique angle of 85° polymerized with PPX (bottom), after HMDI coupling (middle) and after GFP immobilization (top).
rescent intensity of the two GeSbSe surfaces of very different roughness after GFP incubation. From confocal characterization, larger fluorescence intensity is measured from thin films of larger surface area, i.e., those in which the GeSbSe layers were grown at large oblique evaporation angles. 4. Discussion From Fig. 2(a) and (b) it is observed that thin film growth at large evaporation angles (close to 90°) results in surface texturing at the nanoscale, leading to increased surface area. As previously
Fig. 5. Fluorescence intensity determined by confocal microscopy after GFP immobilization. GeSbSe thin films grown at normal incidence (0°) and 85° were used as substrates.
demonstrated by performing quantitative microstructural analysis of GeSbSe chalcogenide glasses, rms roughness increases exponentially with increasing evaporation angle from around 1.15 nm at 0° to over 20 nm at 85° [8]. In addition, the surface area increment for films grown at 0° is close to 2% when compared to that of an atomically flat surface, while this increment is over 30% for films grown at 85°. From the spectra shown in Figs. 3 (bottom) and 4 (bottom), peaks corresponding to PPX are clearly identified in the 3010– 2800 cm 1 wavenumber range, associated to CH stretching modes. Additionally, absorption peaks found in the region 3450– 3160 cm 1 are associated to amine N–H stretching vibrations, while these observed in the 1650–1580 cm 1 are associated to amine N–H deformation vibrations [20]. Moreover, peaks located at 1620 cm 1 and 1578 cm 1 are assigned to the C@O stretch (amide I) and the N–H bend (amide II) respectively. Consequently, the presence of PPX thin films containing a significant amount of amine groups is consistent with the infrared spectra shown in Figs. 3 (top) and 4 (top). The spectra of HMDI-functionalized films (Figs. 3 (middle) and 4 (middle)) show the absorption of amide groups, thus indicating the binding between the amine (–NH2) groups on the surface of the nanostructured PPX film and the HMDI. In addition, the absorption at 2200 cm 1 indicates the free isocyanate groups on the surface of the PPX film. This absorption of free isocyanate groups decreases significantly and eventually disappears after the GFP-coupling to the surface, as shown in Figs. 3 (top) and 4 (top). It is worth pointing out that infrared characterization does not allow quantifying the fraction of free isocyanate groups that are attached to the polymer backbone and the fraction that are free to attach to the proteins. According to our estimations, the fraction of free isocyanate groups should be below 0.5 after carefully washing, since the possibility that both of the isocyanate groups are attached to the polymer cannot be prevented.
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As discussed in the previous paragraph, infrared characterization of PPX-coated GeSbSe thin films clearly indicates the presence of amine groups. However, definitive confirmation of the presence of active amine groups requires the assessment of their reactivity with bioorganic molecules. Thus, from fluorescence intensity measurements (Fig. 5) it was determined that the growth of GeSbSe thin films at large oblique angles results in large surface areas leading to increased bioactivity. Finally, it should be noted that fast substrate rotation combined with tilting did not produce any significant improvement in fluorescence intensity. 5. Summary and conclusions Poly-(p-xylylene) (PPX) thin films were deposited onto GeSbSe chalcogenide glasses grown at two different angles (normal incidence, 0°, and 85°) with the objective of nanostructuring their surface, and was followed by functionalization with PPX and HMDI. In this state, the presence of amine groups was determined by infrared spectroscopy. Subsequently, fluorescent proteins were shown to be immobilized onto these nanostructured films by confocal microscopy. The protein coverage, as shown by the fluorescence intensity, was increased for the GeSbSe chalcogenide layers grown at large angles. Accordingly, large evaporation angle leads to higher transparency and greater potential performance of the sensor. This is a key factor for the further development of optical biosensors with increased sensitivity. Acknowledgments The authors would like to acknowledge support from the NSF IMI on New Functionality in Glass, and the Penn State University
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