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Universidad Politécnica de Madrid Instituto de Sistemas Optoelectrónicos y Microtecnología Nanostructured materials for optical biosensing Optical transducers based on submicro- or nano-structured materials may lead to significant improvements in terms of sensitivity, selectivity, mass transfer and binding kinetics as compared to those based on macro- or non-structured materials. This is due to their unique optical properties at the nanoscale and large surface-to-volume ratio. Additional advantages arise if nanostructured optical transducers are made of Si-based materials and polymers. These benefits include low cost, remarkable engineering possibilities and well-known surface functionalization chemistry. Over the last years, ISOM remarkable achievements on this field have been focused on three subjects that can be summarized as follows. Team Members: Carlos Angulo Barrios, Dr. Eng. Associate Professor Carlos Angulo Barrios received the degree of Ingeniero de Telecomunicación from Universidad Politécnica de Madrid (UPM) in 1998 and the Ph.D. degree from the Royal Institute of Technology (KTH) in 2002. He was a Postdoctoral Associate in the School of Electrical and Computer Engineering at Cornell University from 2002 to 2004, where he developed novel Si-based nanophotonic devices. From 2004 to 2006, he was a Senior Research Associate (“Ramón y Cajal” fellow) at the Nanophotonics Technology Center (NTC), Universidad Politécnica de Valencia (UPV). In 2006, Dr. Barrios returned to UPM where he is a tenured Associate Professor leading research on Nanophotonic Biosensors at the Instituto de Sistemas Optoelectrónicos y Microtecnologia (ISOM). Dr. Barrios has been principal investigator for several national projects and an EU project; he holds six awarded patents and has published in top science journals such as Nature. Dr. Barrios is a reviewer for high impact-factor journals in the fields of Photonics, Materials Science and Sensors and he is an evaluator for the Spanish Agency for Evaluation and Prospective (ANEP). In 2009, he was awarded the UPM Young Investigator Award. Email: carlos.angulo.barrios@upm.es Universidad Politécnica de Madrid Instituto de Sistemas Optoelectrónicos y Microtecnología Victor Canalejas Tejero Victor Canalejas received MSc degrees in Chemical Sciences and Advanced Materials and Nanotechnology in 2006 and 2009, respectively, from Universidad Autónoma de Madrid (UAM) Spain. Between 2008 and 2011, he worked in the Photonic Materials Department at Instituto de Ciencia de Materiales de Madrid (ICMM), dealing with magnetophotonic tunable crystals and 2D-3D self-assembled highly ordered colloidal crystals growing. Since 2011, he is currently a PhD student at ISOM-UPM, where he joined the research group of Dr. Barrios. His work is mainly focused on molecularly imprinted polymers (MIPs) based optical biosensors. Email: victorcanalejas@isom.upm.es Past members Paula Yurrita. Ph.D. Student Martina Francesca. M.Sc. Erasmus Program Student from Politecnico di Milano (Italy) Alyssa Bellingham. M.Sc. EAGLES Program Student from Drexel University (USA) Research: 1.- Slot-waveguide microring resonator based biochemical sensors Slot-waveguides allow light to be guided and strongly confined inside a nanometer-scale region of low refractive index. Thus stronger light-analyte interaction can be obtained as compared to that achievable by a conventional waveguide, in which the propagating beam is confined to the highrefractive-index core of the waveguide. In addition, slot-waveguides can be fabricated by employing CMOS compatible materials and technology, enabling miniaturization, integration with electronic, photonic and fluidic components in a chip, and mass production. These advantages have made the use of slot-waveguides for highly sensitive biochemical optical integrated sensors an emerging field. Nanostructured Materials for Optical Biosensing (a) UPM-ISOM (b) Figure 1. (a) Schematic view of a slot-waveguide. (b) Calculated Ex profile of the quasi-TE eigenmode in a Si (nH = 3.45)/SiO2 (nS = nC = 1.44) slot-waveguide at a wavelength of 1.55 m. E-field is enhanced in the nanoscale slot-region of refractive index nS. ISOM participated as a partner in a European Project entitled “Ultrahigh sensitivity slotwaveguide biosensor on a highly integrated chip for simultaneous diagnosis of multiple diseases” (SABIO). This was a multidisciplinary project involving the emerging fields of micro-nano technology, photonics, fluidics and bio-chemistry, targeting to contribute to the development of intelligent diagnostic equipment for the healthcare of the future. SABIO addressed this objective through the demonstration of a compact polymer-based and silicon-based CMOS-compatible micro-nano system. It integrated optical biosensors for label-free biomolecular recognition based on a novel photonic structure named slot-waveguide with immobilised biomolecular receptors on its surface. This structure offers the possibility of confinement and guidance of light in a nanometer-size void channel enhancing the interaction between an optical probe and biomolecular complexes (antibody-antigen). Figure 2. Left: Top view photograph of a 70-m-radius Si3N4 slot-waveguide microring resonator. Right: Scanning electron microscope image of the coupling region. Nanostructured Materials for Optical Biosensing UPM-ISOM The first experimental demonstration of a slot-waveguide based biochemical sensor was achieved at ISOM by C.A. Barrios et al. for both liquid (bulk) and biomolecule (surface) sensing. These authors employed a vertical (slot/rail interface is normal to the substrate) slot-waveguide ring resonator made of Si3N4 on silicon dioxide. The use of Si3N4 as high-index material (instead of higherindex Si) enables the definition of a wider slot region while maintaining single-mode operation. The main purpose of having a wider slot region is to facilitate filling it with liquids for sensing and optofluidic applications. The device sensor was probed at a wavelength around 1.3 m, which is typically used in telecomm applications (O-band) and leads to lower water optical absorption than that at the other common telecom wavelength, 1.55 m. A spatial discriminating chemical treatment to selectively Si3N4 nitride versus SiO2 was designed for the attachment of biomolecules only to the nitride area of the sensing surface. The effectiveness of the selective surface modification procedure was supported by comparing experimental and numerical calculations of the optical performance of a label-free Si3N4/SiO2 slotwaveguide ring resonator. A Si3N4 microring array with a PDMS microfluidic network was integrated on a Si single chip, enabling accurate multiplexed assays in labs-on-chip. Details on the implementation and characterization of the final SABIO biochip were published in Lab on a Chip and featured on the inside front cover of the printed edition (Fig. 3). A Si3N4 slot-waveguide microring resonator was also used to demonstrate optofluidic device reconfigurability. It was observed that small amounts of organic liquids were trapped, due to capillary and wetting forces, inside the slot-nanochannel, modifying dramatically the microring resonator optical response. Potential optofluidic applications of this effect include permanent and rewriteable photonic configurations, process monitoring, in-situ chemical detection, and study of liquid-solid interfacial forces at the nanoscale. Figure. 3. The SABIO device is featured on the inside front cover of the printed edition of the journal Lab on a Chip. Nanostructured Materials for Optical Biosensing 2.- UPM-ISOM Nanopillar array biosensors We have fabricated periodic lattices of SU-8 resist nano-pillars on both SiO2/Si and ITO/SiO2 substrates. These lattices were used as interrogation platforms for BSA/antiBSA in buffer solution and gestrinone/antigestrinone in whole serum immunoassays. Affinity reactions on the pillar surfaces produce optical thickness changes that are detected by monitoring the surface platform reflectance (SiO2/Si substrates) and/or transmittance (ITO/SiO2 substrates) interference spectra variations. Detection limits below 1 ng/ml were measured. Figure 4. (a) Optical image of SU-8 nanopillar lattices on an ITO/glass substrate in comparison with 1euro cent coin. (b) SEM image of a SU-8 nanopillar lattice. (c) Schematic diagram of the optical interrogation set-up. (d) HRP-h-G immobilization and anti-gestrinone antibody recognition. 3.- Submicron patterning of molecularly imprinted polymers (MIPs) Recognition elements in biochips and biosensors are typically biomacromolecules such as enzymes, antibodies or DNA. These molecules specifically recognize and bind target molecules offering high selectivity against non-specific binding. However, they require low-temperature conservation and restricted operating conditions, which limits their usefulness, particularly for portable systems. Biomimetic receptors like molecularly imprinted polymers (MIPs) are an attractive alternative as Nanostructured Materials for Optical Biosensing UPM-ISOM synthetic receptors. MIPs are created by a templating process at the molecular level. They are able to bind target molecules with similar affinity and specificity to those of their natural counterparts. MIPs offer a number of advantages such as superior stability when exposed to solvents and temperature extremes, the feasibility of creating receptors for a variety of molecular structures, and good engineering possibilities. Electron beam lithography (EBL) is the most popular nanolithography technique. EBL can generate arbitrary patterns with nanometer resolution without the need of moulds or contact masks. Therefore, EBL is a non-contact technique that avoids contamination of the surface to be patterned. Motivated by these remarkable advantages, we have demonstrated for the first time electron-beam direct patterning of MIP films. For this, we synthesized a linear copolymer, poly(methacrylic acid-co2-methacrylamidoethylmethacrylate (P(MAA-co-MAAEMA)), containing both, specific recognition groups (-COOH), for non-covalent binding interactions with template molecule bearing complementary functionalities, and triggerable cross-linkable groups for electron irradiation sensitivity. Thin films of this material, spun on Si substrates, exhibited positive-tone behavior for both EBL (in the dose range 0.1 – 8 mC/cm2) and DUV (255 nm wavelength) photolithography, and high sensitivity and selectivity towards the template molecule R123. The two-fold functionality of the developed linear copolymer as a recognition material and an EBL resist opens new opportunities in the implementation of innovative, nanostructured MIP film-based arrays for multiple target detection. Figure 5. A nanopattern is directly written by EBL on a film of the synthesized polymer mixture, spun on a Si substrate. The mixture behaves as a positive tone EBL resist and the resulting feature acts as MIP capable to recognize the template molecule, R123, with high sensitivity and selectivity. In addition, we carried out patterning of MIP films with minimum features of approximately 1 m via microtransfer molding based on SiO2/Si molds, fabricated by using standard microfabrication techniques. MIP patterns consisted of two-dimensional diffraction gratings that were successfully used as label-free optical bio(mimetic)sensors for specific recognition of a fluoroquinolone antimicrobial, enrofloxacin, broadly applied in human and veterinary medicine. Nanostructured Materials for Optical Biosensing UPM-ISOM Figure 6. AFM image and cross-section scan of a representative portion of a MIP 2D grating. Left inset: optical microscope photograph of the micropatterned MIP film. Right inset: far-field diffraction pattern of a MIP 2D-DG immersed in water. 4.- Aluminum nanohole arrays for label-free optical biosensing Optical techniques based on surface plasmon resonance (SPR) have been shown to be of great success for label-free biosensing applications. SPR systems based on prism-coupling are commercially available. Metallic nanostructures provide new design and integration possibilities which can lead to improvements in key SPR-based biosensor issues such as sensitivity, resolution, multiplexing and biological interfacing. In particular, nanohole arrays based chemical and biological sensors offer numerous applications and display great performance, and recent progresses aim to cheaper fabrication methods. Noble metals Au and Ag are typically used in the fabrication of nanohole array SPR devices because of their low optical losses in the visible and near-infrared ranges. In addition, Au is highly chemically stable making it convenient for biological media. However, the high cost of these metals limits large scale commercialization of these sensors. Al is a more cost-effective plasmonic material: approximately 25 000 times and 425 times cheaper than Au and Ag, respectively. However, Al has been scarcely considered for the implementation of SPR biosensors mainly because of challenges from oxidation and material degradation We have shown that Al can be made a suitable and reliable plasmonic material for implementing nanohole array SPR biosensors. The issue of Al oxidation is tackled by a passivation process consisting of exposing Al nanohole array films to O2 plasma. This treatment produces a robust protecting oxide layer that is more resistant than native oxide against oxidizing agents like aqueous solutions or buffers usually employed in biosensing tests, thus avoiding corrosion and pitting issues. Thus we have fabricated Al nanohole arrays fabricated on both glass and polycarbonate (PC) (for compact disc based biosensing platforms) substrates and demonstrated their workability as labelfree optically interrogated biosensors via bioassays based on biotin analysis through biotinfunctionalized dextran-lipase conjugates immobilized on the biosensor passivated surface in aqueous media. Nanostructured Materials for Optical Biosensing UPM-ISOM Figure 7. (a) SEM photograph of a 500 nm pitch Al nanohole array on a PC substrate. (b) Experimental and calculated spectra of the Al nanohole array. Surface plasmon polariton features are observed (S,P and Q). (c) Swavelength shift for direct and competitive assays carried out on the Al nanohole arrays. (d) Simulated transmitted intensity spectra of a 630 nm period Al nanohole array on PC substrate with a 10 nm thick biofilm (red line) and without a biofilm (black line). Inset shows a schematic cross-section of the simulated nanohole array along the middle of a hole row. SELECTED PUBLICATIONS 1. C.A. Barrios, V. Canalejas-Tejero, S. Herranz, M.C. Moreno-Bondi, M. Avella-Oliver, R. Puchades and A. Maquieira, “Aluminum nanohole arrays fabricated on polycarbonate for compact disc -based label-free optical biosensing,” Plasmonics, Accepted for publication, 2014. 2. Víctor Canalejas-Tejero, Sonia Herranz, Alyssa Bellingham, María Cruz Moreno-Bondi and Carlos Angulo Barrios, “Passivated aluminum nanohole arrays for label-free biosensing applications,” ACS Applied Materials and Interfaces, 6, pp. 1005-1010, 2014. 3. S. Carrasco, V. Canalejas-Tejero, F. Navarro-Villoslada, C.A. Barrios and M.C. Moreno-Bondi, “Cross-linkable linear copolymer with double functionality: resist for electron beam nanolithography and molecular imprinting,” J. Materials Chemistry C, 2014, DOI: 10.1039/C3TC31499E. Nanostructured Materials for Optical Biosensing UPM-ISOM 4. V. Canalejas-Tejero, S. Carrasco, F. Navarro-Villoslada, J.L.G. Fierro, M.C. Capel-Sánchez, M.C. Moreno-Bondi and C.A. Barrios, “Ultrasensitive non-chemically amplified low-contrast negative electron beam lithography resist with dual-tone behaviour,” J. Materials Chemistry C, 1, pp. 1392-1398, 2013. 5. F.J. Ortega, M.J. Bañuls, F.J. Sanza, M.F. Laguna, M. Holgado, R. Casquel, C.A. Barrios, D. López-Romero, A. Maquieira and R. Puchades, “Development of a versatile biotinylated material based on SU-8 ,” J. Materials Chemistry B, 1, pp. 2750-2756, 2013. 6. C.A. Barrios, S. Carrasco, V. Canalejas-Tejero, D. López-Romero, F. Navarro-Villoslada, M.C. Moreno-Bondi, J.L.G. Fierro, M.C. Capel-Sánchez, “Fabrication of luminescent nanostructures by electron-beam direct writing of PMMA resist,” Materials Letters, 88, pp. 93–96, 2012. 7. Carlos Angulo Barrios, “Integrated microring resonator sensor arrays for labs-on-chips,” Analytical and Bioanalytical Chemistry, 403, pp. 1467-1475, 2012. 8. F.J. Ortega, M-J. Bañuls, F.J. Sanza, R. Casquel, M.F. Laguna, M. Holgado, D. López-Romero, C.A. Barrios, Á. Maquieira and R. Puchades, “Biomolecular Interaction Analysis of Gestrinoneanti-Gestrinone Using Arrays of High Aspect Ratio SU-8 Nanopillars,” Biosensors, 2, pp. 291304, 2012. 9. C.A. Barrios, S. Carrasco, M. Francesca, P. Yurrita, F. Navarro-Villoslada, and M.C. MorenoBondi, “Molecularly imprinted polymer for label-free integrated optical waveguide bio(mimetic)sensors,” Sensors and Actuators B, 161, pp. 607– 614, 2012. 10. C.A. Barrios, C. Zhenhe, F. Navarro-Villoslada, D. López-Romero, and M.C. Moreno-Bondi, “Molecularly imprinted polymer diffraction grating as label-free optical bio(mimetic)sensor,” Biosensors and Bioelectronics, 26, pp. 2801–2804, 2011. 11. F.J. Sanza, M. Holgado, F.J. Ortega, R. Casquel, D. López-Romero, M.J. Bañuls, M.F. Laguna, C.A. Barrios, R. Puchades, and A. Maquieira, “Bio-Photonic Sensing Cells over transparent substrates for anti-gestrinone antibodies biosensing,” Biosensors and Biolectronics, 26, pp. 4842-4847, 2011. 12. F.J. Sanza, M.F. Laguna, R. Casquel, M. Holgado, C.A. Barrios, F. Hortigüela, D. López-Romero, J.J. García-Ballesteros, M.J. Bañuls, A. Maquieira, R. Puchades, “Cost-effective SU-8 microstructures by DUV excimer laser lithography for label-free biosensing," Applied Surface Science, 257, pp. 5403-5407, 2011. 13. F.J. Aparicio, M. Holgado, I. Blaszczyk-Lezak, A. Borras, A. Griol, C.A. Barrios, R. Casquel, F. J. Sanza, H. Solhstrom, M. Antelius, A.R. González-Elipe and A. Barranco, "Transparent nanometric organic luminescent films as UV active components in photonic structures," Advanced Materials, 23, pp. 761-765, 2011. 14. C. F. Carlborg, K. B. Gylfason, A. Ka mierczak, F. Dortou, M. J. Bañuls Polo, A. Maquieira Catala, G. M. Kresbach, H. Sohlström, T. Moh, L. Vivien, J. Popplewell, G. Ronan, C.A. Barrios, G. Stemme and W. van der Wijngaart, “A packaged optical slot-waveguide ring resonator sensor array for multiplex label-free assays in labs-on-chips,” Lab Chip, 10, pp. 281–290, 2010. 15. Kristinn B. Gylfason, Carl Fredrik Carlborg, Andrzej Kazmierczak, Fabian Dortu, Hans Sohlström, Laurent Vivien, Carlos A. Barrios, Wouter van der Wijngaart and Göran Stemme, “On-chip temperature compensation in an integrated slot-waveguide ring resonator refractive index sensor array,” Optics Express, 28, pp. 3226-3237, 2010. 16. M. Holgado, C.A. Barrios, F.J. Ortega, F.J. Sanza, R. Casquel, M.F. Laguna, M.J. Bañuls, D. López-Romero, R. Puchades, A. Maquieira, “Label-free biosensing by means of periodic lattices of high aspect ratioSU-8 nano-pillars, “ Biosensors and Bioelectronics, 25, pp. 25532558, 2010. 17. María-José Bañuls, Victoria González-Pedro, Carlos A. Barrios, Rosa Puchades and Ángel Maquieira “Selective chemical modification of silicon nitride/silicon oxide nanostructures to develop label-free biosensors,” Biosensors and Bioelectronics, 25, pp. 1460–1466, 2010. 18. D. López-Romero, C.A. Barrios, M. Holgado, M.F. Laguna, and R. Casquel, “High aspect-ratio SU-8 resist nano-pillar lattice by e-beam direct writing and its application for liquid trapping” Microelectronic Engineering, 87, pp. 663–667, 2010. Nanostructured Materials for Optical Biosensing UPM-ISOM 19. Carlos Angulo Barrios, “Optical Slot-Waveguide based Biochemical Sensors,” Sensors, vol. 9, no. 6, pp. 4751-4765, 2009. 20. Carlos Angulo Barrios “Analysis and modeling of a silicon nitride slot-waveguide microring resonator biochemical sensor,” Proc. of SPIE, vol. 7356, 735605(1-9), 2009. 21. C.A. Barrios, M.J. Bañuls, V. Gonzalez-Pedro, K.B. Gylfason, B. Sánchez, A. Griol, A. Maquieira, H. Sohlström, M. Holgado, and R. Casquel, “Label-free optical biosensing with slotwaveguides,” Optics Letters, 33,7, pp. 708-710, 2008. 22. C.A. Barrios, M. Holgado, O. Guarneros, B. Sánchez, K.B. Gylfason, R. Casquel, and H. Sohlström, “Reconfiguration of microring resonators by liquid adhesion,” Applied Physics Letters, 93, 203114, 2008. 23. C.A. Barrios, K.B. Gylfason, B. Sánchez, A. Griol, H. Sohlström, M. Holgado, and R. Casquel, “Slot-waveguide biochemical sensor,” Optics Letters, 32, pp. 3080-3082, 2007. 24. C.A. Barrios, B. Sánchez, K.B. Gylfason, A. Griol, H. Sohlström, M. Holgado, and R. Casquel, “Demonstration of slot-waveguide structures on silicon nitride/silicon oxide platform,” Optics Express, 15, pp.6846-6856, 2007. 25. Carlos Angulo Barrios, “Ultrasensitive Nanomechanical Photonic Sensor based on Horizontal Slot-Waveguide Resonator,” IEEE Photonics Technology Letters, 18, pp. 2419-2421, 2006.