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Plasma Nanotextured Polystyrene for Intense DNA Microarrays
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Procedia Engineering
Procedia Engineering 00 (2011) 000–000 Procedia Engineering 25 (2011) 1573 – 1576 www.elsevier.com/locate/procedia
Proc. Eurosensors XXV, September 4-7, 2011, Athens, Greece
Plasma Nanotextured Polystyrene for Intense DNA Microarrays K. Tsougenia, P. S. Petroub, A. Tserepia, S. E. Kakabakosb, E. Gogolidesaa* b
a Institute of Microelectronics, NCSR “Demokritos”, PO BOX 60228, 153 10 Aghia Paraskevi, Greece Institute of Radioisotopes and Radiodiagnostic Products, NCSR “Demokritos”, PO BOX 60228, 153 10 Aghia Paraskevi, Greece
Abstract Plasma roughened nanotextured Polystyrene surfaces with high aspect ratio topography were used as substrates for the fabrication of DNA microarrays. To take advantage of the increased protein adsorption properties of the the nanotextured surfaces, the array is formed by depositing conjugates of biotinylated oligonucleotides with streptavidin with a nano-plotter. The fabrication of DNA microarrays with uniform and intense spots is demonstrated. Such substrates are expected to find wide application in lab-on-a-chip devices for a new generation of surface-based microanalysis.
© 2011 Published by Elsevier Ltd. Keywords: PS slides, O2 plasma nanotexturing, protein arrays, DNA arrays
1. Introduction Polystyrene is a commonly used polymer for biochips, because of its optical transparency, and good electrical and mechanical properties [1]. Modification of the surface properties (chemical composition and topography) of polystyrene may alter its wetting behaviour and the adsorption of biomolecules on it. The latter is a very important property for polymeric surfaces in general when used as substrates for fabrication of biomolecule microarrays [2]. Microarrays have become an invaluable tool for large-scale and high-throughput bioanalytical applications. They consist of immobilized biomolecules spatially addressed on solid substrates such as planar surfaces, microchannels and microwells. They allow fast, easy, and parallel detection of thousands of addressable elements in a single experiment using minimum
* Corresponding author. Tel.: +30-210-6503237; fax: +30-210-6511723. E-mail address: [email protected]
1877-7058 © 2011 Published by Elsevier Ltd. doi:10.1016/j.proeng.2011.12.389
1574 2
K. Tsougeni al. / Procedia Engineering 25 (2011) 1573 – 1576 Author name et / Procedia Engineering 00 (2011) 000–000
sample volumes. Biomolecules commonly immobilized on microarrays include oligonucleotides, polymerase chain reaction (PCR) products and proteins. The last years, substrates on which biomicroarrays can be fabricated shift from Si [1] and glass to polymeric materials [2], including polystyrene. In our previous works we have shown [3-6] the formation and the control of surface roughness (texture) on polymers as a result of plasma processing. In addition, we have demonstrated the ability of plasma-induced rough polymeric open surfaces to adsorb significantly higher amounts of proteins, compared with the flat (untreated) surfaces [7], paving the way for highly sensitive microarrays. In the present work, we demonstrate the use of random plasma nanotextured polystyrene (PS) as substrate for the fabrication of intense DNA arrays. 2. Experimental Oligonucleotides were immobilized onto the surface in the form of biotinylated oligonucleotide/streptavidin conjugates. The microarrays were created using a BioOdyssey Caligrapher MiniArrayer (Bio-Rad Laboratories Inc., USA), kept in the spotter under controlled humidity conditions (average humidity 65%) to allow for coupling onto the surface and after hybridization with fluorescently labelled oligonucleotides were evaluated using a Microarray scanner (ScanArray Gx). The process of oligonucleotide immobilization and hybridization is schematically depicted in Fig.1.
Fig 1. Schematic representation of process followed for immobilization of biotinylated oligonucleotides/streptavidin conjugates on PS surfaces and subsequent hybridization with fluorescently labelled oligonucleotides: Deposition of conjugates in 50 μM phosphate buffer, pH 7.0, on PS substrates, incubation for 1h at room temperature in a humidity chamber, blocking with 10 g/l BSA solution in the same buffer for 2h, hybridization and washing with serially diluted HEN and scanning using a Perkin Elmer Gx microarray scanner.
3. Results Optically transparent 1-mm thick PS slides were purchased from Nalgene Inc. PS slides were plasma treated under highly anisotropic conditions (O2 pressure 0.75 Pa, plasma power 1900 W, and bias voltage -80 V), generated in a helicon plasma reactor (Micromachining Etching Tool, MET, from Alcatel). O2 plasma etched and nanotextured the PS surfaces, leading to high aspect ratio (HAR) topography. Fig. 2 illustrates PS surfaces, (a) flat and (b) plasma-treated for 5 min, viewed at 80o tilt under scanning electron microscopy. Fig. 2 (b) reveals the formation of densely packed micro- and nano-columns (height >1 μm), after 5 min O2 plasma treatment. Both the width and height of the surface texture depends on the process duration. Column formation on PS surfaces is the result of alumina sputtering from the reactor dome and of impurities existing in the commercial PS material causing micromasking on the polymer surface [8].
a)
b)
K. Tsougeni et al. // Procedia Procedia Engineering – 1576 Author name Engineering 25 00 (2011) (20111)1573 000–000 Fig 2. Tilted (by 80o) view SEM images of (a) an untreated and (b) of plasma etched PS slides after 5min plasma etching. (Conditions: O2 plasma, 1900 W, 0.75 Pa, 100 sccm, -80 V, -20 oC).
Fluorescence Intensity (RFU)
At first the PS nanostructured surfaces have been evaluated with respect to their protein adsorption capacity. In Fig. 3 the fluorescence intensity of protein coated surfaces as a function of b-BSA concentration is presented for untreated and for 20 min O2 plasma-treated PS surfaces. A 2-5x increase, depending on the protein concentration, was achieved for surfaces treated in O2 plasma for 20 min compared to the untreated surface. For the plasma nanotextured surfaces, saturation with protein was not achieved even for b-BSA concentration as high as 500 μg/ml, while for the untreated surfaces plateau values were obtained at concentrations equal to or greater than 100 μg/ml. However, these plateau values were about 5 times lower than those obtained from the nanotextured surfaces under conditions of nonsaturation. 120 100 80 60 40 20 0
0
100
200
300
400
500
b-BSA Concentration (μg/ml) Fig 3. Fluoroscence intensity of b-BSA spots for untreated ({) and 20 min ( ) O2 plasma treated surfaces as a function of b-BSA concentration.
In Figure 4, images from a microarray consisting of b-BSA spots deposited by an automatic spotter on untreated and 20-min O2 plasma treated surfaces after reaction with AF546-labeled streptavidin are presented, confirming the 2-5x increase in spot intensity shown in Fig. 3. 200 μg/ml
500 μg/ml
200 μg/ml
500 μg/ml
(b) (a) Fig 4. Fluorescence images of b-BSA spot microarray deposited by a nanoplotter on (a) untreated and (b) 20-min O2 plasma treated PS surfaces.
The nanostructured PS surfaces have been then used to immobilize oligonucleotides in form of biotinylated oligonucleotide/streptavidin conjugates. A picture of AlexaFluor 546 labelled biotinylated oligonucleotide spots deposited by an automatic microspotter on a 5-min O2-treated PS surface is presented in Fig. 5. Oligonucleotide adsorption via the oligonucleotide/streptavidin conjugates on PS nano-columnar surfaces increases with plasma treatment time compared to flat untreated surfaces. This increase is due to modified surface chemistry as well as to increased surface area. In addition, the images
1575 3
1576 4
K. Tsougeni al. / Procedia Engineering 25 (2011) 1573 – 1576 Author name et / Procedia Engineering 00 (2011) 000–000
indicate excellent spot homogeneity as compared to untreated surface. Furthermore, oligonucleotides corresponding to wild type sequence of 5382insC BRCA1 mutation were immobilized onto nanotextured PS slides and the hybridization with fluorescently labelled complementary sequences was determined. The hybridization signal was also increased by 2-4 times as a result of higher immobilized oligonucleotide concentration.
(a)
(b)
Fig 5. Spots of AlexaFluor 546 labelled biotinylated oligonucleotides/streptavidin conjugate deposited by a nanoplotter on (a) flat PS surface and (b) 5-min treated PS surface. Intensity of spots (b) is 5 times higher than spots (a).
4. Conclusions We have thus shown that optimized polymeric surfaces with plasma-induced high aspect ratio topography for maximum oligonucleotide adsorption could be used for fabrication of DNA microarrays with uniform and intense spots. Such substrates are expected to find wide application in lab-on-a-chip devices for a new generation of surface-based micro-analysis. Our results for PS are complementary to those for Polydimethylsiloxane (PDMS) nanotextured in SF6 plasmas [9] showing the generality of the concept. References [1] Verpoorte E, DeRooij NJ. Microfluidics meets MEMS. Proceedings of IEEE 2003;91:930. [2] Heyries KA, Marquette CA, Blum LJ. Straightforward Protein Immobilization on Sylgard 184 PDMS Microarray Surface. Langmuir 2007;23:4523. [3] Tsougeni K, Vourdas N, Cardinaud C, Tserepi A, Gogolides E. Mechanisms of Oxygen Plasma Nanotexturing of Organic Polymer Surfaces: From Stable Super Hydrophilic to Super Hydrophobic Surfaces. Langmuir 2009;25(19) :11748-11759. [4] Tserepi A, Gogolides E, Misiakos K, Vlachopoulou ME, Vourdas N. Greek Patent Application 20050100473; PCT Application Number GR2006/000011. [5] Tserepi A, Vlachopoulou ME, Gogolides E. Nanotexturing of poly(dimethylsiloxane) in plasmas for creating robust superhydrophobic surfaces. Nanotechnology 2006;17:3977. [6] Vourdas N, Tserepi A, Gogolides E. Nanotextured super-hydrophobic transparent poly(methyl methacrylate) surfaces using high-density plasma processing. Nanotechnology 2007;18:125304. [7] Tsougeni K, Petrou PS, Tserepi A, Kakabakos SE, Gogolides E. Plasma Nanotextured PMMA Surfaces for Protein Arrays: Increased Protein Binding and Enhanced Detection Sensitivity. Langmuir 2010;26(17):13883-13891. [8] Gogolides E, Constantoudis V, Kokkoris G, Kontziampasis D, Tsougeni K, Boulousis G, Vlachopoulou M, Tserepi A. Controlling roughness: From etching to nanotexturing and plasma-directed organization on organic and inorganic materials. J Physics D: Applied Physics 2011;44 (17):174021. [9] Vlachopoulou ME, Tserepi A, Petrou PS, Gogolides E, Kakabakos SE. Protein arrays on high-surface-area plasma-nanotextured poly(dimethylsiloxane)-coated glass slides. Colloids and Surfaces B: Biointerfaces 2011;83 (2):270-276.
Procedia Engineering
Procedia Engineering 00 (2011) 000–000 Procedia Engineering 25 (2011) 1573 – 1576 www.elsevier.com/locate/procedia
Proc. Eurosensors XXV, September 4-7, 2011, Athens, Greece
Plasma Nanotextured Polystyrene for Intense DNA Microarrays K. Tsougenia, P. S. Petroub, A. Tserepia, S. E. Kakabakosb, E. Gogolidesaa* b
a Institute of Microelectronics, NCSR “Demokritos”, PO BOX 60228, 153 10 Aghia Paraskevi, Greece Institute of Radioisotopes and Radiodiagnostic Products, NCSR “Demokritos”, PO BOX 60228, 153 10 Aghia Paraskevi, Greece
Abstract Plasma roughened nanotextured Polystyrene surfaces with high aspect ratio topography were used as substrates for the fabrication of DNA microarrays. To take advantage of the increased protein adsorption properties of the the nanotextured surfaces, the array is formed by depositing conjugates of biotinylated oligonucleotides with streptavidin with a nano-plotter. The fabrication of DNA microarrays with uniform and intense spots is demonstrated. Such substrates are expected to find wide application in lab-on-a-chip devices for a new generation of surface-based microanalysis.
© 2011 Published by Elsevier Ltd. Keywords: PS slides, O2 plasma nanotexturing, protein arrays, DNA arrays
1. Introduction Polystyrene is a commonly used polymer for biochips, because of its optical transparency, and good electrical and mechanical properties [1]. Modification of the surface properties (chemical composition and topography) of polystyrene may alter its wetting behaviour and the adsorption of biomolecules on it. The latter is a very important property for polymeric surfaces in general when used as substrates for fabrication of biomolecule microarrays [2]. Microarrays have become an invaluable tool for large-scale and high-throughput bioanalytical applications. They consist of immobilized biomolecules spatially addressed on solid substrates such as planar surfaces, microchannels and microwells. They allow fast, easy, and parallel detection of thousands of addressable elements in a single experiment using minimum
* Corresponding author. Tel.: +30-210-6503237; fax: +30-210-6511723. E-mail address: [email protected]
1877-7058 © 2011 Published by Elsevier Ltd. doi:10.1016/j.proeng.2011.12.389
1574 2
K. Tsougeni al. / Procedia Engineering 25 (2011) 1573 – 1576 Author name et / Procedia Engineering 00 (2011) 000–000
sample volumes. Biomolecules commonly immobilized on microarrays include oligonucleotides, polymerase chain reaction (PCR) products and proteins. The last years, substrates on which biomicroarrays can be fabricated shift from Si [1] and glass to polymeric materials [2], including polystyrene. In our previous works we have shown [3-6] the formation and the control of surface roughness (texture) on polymers as a result of plasma processing. In addition, we have demonstrated the ability of plasma-induced rough polymeric open surfaces to adsorb significantly higher amounts of proteins, compared with the flat (untreated) surfaces [7], paving the way for highly sensitive microarrays. In the present work, we demonstrate the use of random plasma nanotextured polystyrene (PS) as substrate for the fabrication of intense DNA arrays. 2. Experimental Oligonucleotides were immobilized onto the surface in the form of biotinylated oligonucleotide/streptavidin conjugates. The microarrays were created using a BioOdyssey Caligrapher MiniArrayer (Bio-Rad Laboratories Inc., USA), kept in the spotter under controlled humidity conditions (average humidity 65%) to allow for coupling onto the surface and after hybridization with fluorescently labelled oligonucleotides were evaluated using a Microarray scanner (ScanArray Gx). The process of oligonucleotide immobilization and hybridization is schematically depicted in Fig.1.
Fig 1. Schematic representation of process followed for immobilization of biotinylated oligonucleotides/streptavidin conjugates on PS surfaces and subsequent hybridization with fluorescently labelled oligonucleotides: Deposition of conjugates in 50 μM phosphate buffer, pH 7.0, on PS substrates, incubation for 1h at room temperature in a humidity chamber, blocking with 10 g/l BSA solution in the same buffer for 2h, hybridization and washing with serially diluted HEN and scanning using a Perkin Elmer Gx microarray scanner.
3. Results Optically transparent 1-mm thick PS slides were purchased from Nalgene Inc. PS slides were plasma treated under highly anisotropic conditions (O2 pressure 0.75 Pa, plasma power 1900 W, and bias voltage -80 V), generated in a helicon plasma reactor (Micromachining Etching Tool, MET, from Alcatel). O2 plasma etched and nanotextured the PS surfaces, leading to high aspect ratio (HAR) topography. Fig. 2 illustrates PS surfaces, (a) flat and (b) plasma-treated for 5 min, viewed at 80o tilt under scanning electron microscopy. Fig. 2 (b) reveals the formation of densely packed micro- and nano-columns (height >1 μm), after 5 min O2 plasma treatment. Both the width and height of the surface texture depends on the process duration. Column formation on PS surfaces is the result of alumina sputtering from the reactor dome and of impurities existing in the commercial PS material causing micromasking on the polymer surface [8].
a)
b)
K. Tsougeni et al. // Procedia Procedia Engineering – 1576 Author name Engineering 25 00 (2011) (20111)1573 000–000 Fig 2. Tilted (by 80o) view SEM images of (a) an untreated and (b) of plasma etched PS slides after 5min plasma etching. (Conditions: O2 plasma, 1900 W, 0.75 Pa, 100 sccm, -80 V, -20 oC).
Fluorescence Intensity (RFU)
At first the PS nanostructured surfaces have been evaluated with respect to their protein adsorption capacity. In Fig. 3 the fluorescence intensity of protein coated surfaces as a function of b-BSA concentration is presented for untreated and for 20 min O2 plasma-treated PS surfaces. A 2-5x increase, depending on the protein concentration, was achieved for surfaces treated in O2 plasma for 20 min compared to the untreated surface. For the plasma nanotextured surfaces, saturation with protein was not achieved even for b-BSA concentration as high as 500 μg/ml, while for the untreated surfaces plateau values were obtained at concentrations equal to or greater than 100 μg/ml. However, these plateau values were about 5 times lower than those obtained from the nanotextured surfaces under conditions of nonsaturation. 120 100 80 60 40 20 0
0
100
200
300
400
500
b-BSA Concentration (μg/ml) Fig 3. Fluoroscence intensity of b-BSA spots for untreated ({) and 20 min ( ) O2 plasma treated surfaces as a function of b-BSA concentration.
In Figure 4, images from a microarray consisting of b-BSA spots deposited by an automatic spotter on untreated and 20-min O2 plasma treated surfaces after reaction with AF546-labeled streptavidin are presented, confirming the 2-5x increase in spot intensity shown in Fig. 3. 200 μg/ml
500 μg/ml
200 μg/ml
500 μg/ml
(b) (a) Fig 4. Fluorescence images of b-BSA spot microarray deposited by a nanoplotter on (a) untreated and (b) 20-min O2 plasma treated PS surfaces.
The nanostructured PS surfaces have been then used to immobilize oligonucleotides in form of biotinylated oligonucleotide/streptavidin conjugates. A picture of AlexaFluor 546 labelled biotinylated oligonucleotide spots deposited by an automatic microspotter on a 5-min O2-treated PS surface is presented in Fig. 5. Oligonucleotide adsorption via the oligonucleotide/streptavidin conjugates on PS nano-columnar surfaces increases with plasma treatment time compared to flat untreated surfaces. This increase is due to modified surface chemistry as well as to increased surface area. In addition, the images
1575 3
1576 4
K. Tsougeni al. / Procedia Engineering 25 (2011) 1573 – 1576 Author name et / Procedia Engineering 00 (2011) 000–000
indicate excellent spot homogeneity as compared to untreated surface. Furthermore, oligonucleotides corresponding to wild type sequence of 5382insC BRCA1 mutation were immobilized onto nanotextured PS slides and the hybridization with fluorescently labelled complementary sequences was determined. The hybridization signal was also increased by 2-4 times as a result of higher immobilized oligonucleotide concentration.
(a)
(b)
Fig 5. Spots of AlexaFluor 546 labelled biotinylated oligonucleotides/streptavidin conjugate deposited by a nanoplotter on (a) flat PS surface and (b) 5-min treated PS surface. Intensity of spots (b) is 5 times higher than spots (a).
4. Conclusions We have thus shown that optimized polymeric surfaces with plasma-induced high aspect ratio topography for maximum oligonucleotide adsorption could be used for fabrication of DNA microarrays with uniform and intense spots. Such substrates are expected to find wide application in lab-on-a-chip devices for a new generation of surface-based micro-analysis. Our results for PS are complementary to those for Polydimethylsiloxane (PDMS) nanotextured in SF6 plasmas [9] showing the generality of the concept. References [1] Verpoorte E, DeRooij NJ. Microfluidics meets MEMS. Proceedings of IEEE 2003;91:930. [2] Heyries KA, Marquette CA, Blum LJ. Straightforward Protein Immobilization on Sylgard 184 PDMS Microarray Surface. Langmuir 2007;23:4523. [3] Tsougeni K, Vourdas N, Cardinaud C, Tserepi A, Gogolides E. Mechanisms of Oxygen Plasma Nanotexturing of Organic Polymer Surfaces: From Stable Super Hydrophilic to Super Hydrophobic Surfaces. Langmuir 2009;25(19) :11748-11759. [4] Tserepi A, Gogolides E, Misiakos K, Vlachopoulou ME, Vourdas N. Greek Patent Application 20050100473; PCT Application Number GR2006/000011. [5] Tserepi A, Vlachopoulou ME, Gogolides E. Nanotexturing of poly(dimethylsiloxane) in plasmas for creating robust superhydrophobic surfaces. Nanotechnology 2006;17:3977. [6] Vourdas N, Tserepi A, Gogolides E. Nanotextured super-hydrophobic transparent poly(methyl methacrylate) surfaces using high-density plasma processing. Nanotechnology 2007;18:125304. [7] Tsougeni K, Petrou PS, Tserepi A, Kakabakos SE, Gogolides E. Plasma Nanotextured PMMA Surfaces for Protein Arrays: Increased Protein Binding and Enhanced Detection Sensitivity. Langmuir 2010;26(17):13883-13891. [8] Gogolides E, Constantoudis V, Kokkoris G, Kontziampasis D, Tsougeni K, Boulousis G, Vlachopoulou M, Tserepi A. Controlling roughness: From etching to nanotexturing and plasma-directed organization on organic and inorganic materials. J Physics D: Applied Physics 2011;44 (17):174021. [9] Vlachopoulou ME, Tserepi A, Petrou PS, Gogolides E, Kakabakos SE. Protein arrays on high-surface-area plasma-nanotextured poly(dimethylsiloxane)-coated glass slides. Colloids and Surfaces B: Biointerfaces 2011;83 (2):270-276.