Combination of nanophotonic biosensors and light-assisted immobilization procedures for the detection of cardiac biomarkers

[ES] El cuidado de la salud es un campo en el que la detección precoz de enfermedades está cobrando cada vez más importancia. Hoy en día, profesionales y ciudadanos demandan que las técnicas de diagnóstico sean de alta calidad, tanto para el sistema de sanidad privado como para el público. Cuando se utilizan técnicas de diagnóstico de manera inadecuada, eso puede acarrear bastantes consecuencias, tales como un serio peligro sobre la salud y la sobrecarga técnica y económica de los servicios de salud. Eso es debido a que las técnicas de diagnóstico disponibles hoy en día son demasiado costosas, centralizadas en laboratorios y necesitan profesionales altamente cualificados para poder llevar a cabo dichas tareas, lo que conllevaría una demora en el tiempo, siendo este muchas veces vital para los enfermos. Es muy necesario, por lo tanto, reflexionar sobre la necesidad y emergencia de tales prácticas preventivas, especialmente para enfermedades de alto riesgo como el cáncer, el Alzheimer o la primera causa de muerte en el mundo, las enfermedades cardiovasculares. En este contexto, el objetivo principal del trabajo realizado durante esta Tesis Doctoral es ayudar a superar estos problemas mediante la exploración de la posibilidad de utilizar tecnología fotónica para el desarrollo de sistemas de análisis que puedan ser utilizados para el diagnóstico y pronóstico de las enfermedades cardiovasculares. Este objetivo se ha abordado mediante la combinación de la tecnología nanofotónica, consistiendo en la nanofabricación de las estructuras PBG de sensado que ofrece varios beneficios, como una alta sensibilidad, una extrema reducción de tamaño y un proceso de fabricación compatible con el de la industria microelectrónica, con un método de biofuncionalización obteniendo una capa de bioreconocimiento estable y selectiva mediante el uso de la reacción TEC asistida por luz capaz de proporcionar unas capas de bio-reconocimiento extremadamente finas con una inmovilización espacialmente selectiva.[CA] L'atenció a la salut és un camp en què la detecció precoç de malalties està cobrant cada vegada més importància. Hui en dia, professionals i ciutadans demanen que les tècniques de diagnòstic siguin d'alta qualitat, tant per al sistema de sanitat privat com per al públic. Quan s'utilitzen tècniques de diagnòstic de manera inadequada, això pot comportar bastants conseqüències, com ara, un seriós perill sobre la salut i la sobrecàrrega tècnica i econòmica dels serveis de salut. Això és degut al fet que les tècniques de diagnòstic disponibles hui en dia són molt costoses, centralitzades en laboratoris i necessiten professionals altament qualificats per poder realitzar aquestes tasques, lo que comportaria a una demora en el temps que moltes vegades es vital pels malalts. És molt necessari, per tant, reflexionar sobre la necessitat i emergència de tals practiques preventives, especialment per a malalties d'alt risc com el càncer, l'Alzheimer o la primera causa de mort al món, les malalties cardiovasculars. En aquest context, l'objectiu principal del treball realitzat durant aquesta Tesi Doctoral és ajudar a superar aquests problemes mitjançant l'exploració de la possibilitat d'utilitzar tecnologia fotònica per al desenvolupament de sistemes d'anàlisis que puguin ser utilitzats per al diagnòstic i pronòstic de les malalties cardiovasculars. Aquest objectiu s'ha abordat mitjançant la combinació de la tecnologia nanofotònica, consistint en la nanofabricació de les estructures de detecció de PBG fotòniques que ofereix diversos beneficis, com una alta sensibilitat, una extrema reducció de mida i un procés de fabricació compatible amb el de la indústria microelectrònica, amb un mètode de biofuncionalització obtenint una capa de bio-reconeixement estable i selectiva mitjançant l'ús de la reacció TEC assistida per llum capaç de proporcionar unes capes de bioreconeixement extremadament fines amb una immobilització espacialment selectiva. preventives, especialment per a malalties d'alt risc com el càncer, l'Alzheimer o la primera causa de mort al món, les malalties cardiovasculars. En aquest context, l'objectiu principal del treball realitzat durant aquesta Tesi Doctoral és ajudar a superar aquests problemes mitjançant l'exploració de la possibilitat d'utilitzar tecnologia fotònica per al desenvolupament de sistemes d'anàlisis que puguin ser utilitzats per al diagnòstic i pronòstic de les malalties cardiovasculars. Aquest objectiu s'ha abordat mitjançant la combinació de la tecnologia nanofotònica, consistint en la nanofabricació de les estructures de detecció de PBG fotòniques que ofereix diversos beneficis, com una alta sensibilitat, una extrema reducció de mida i un procés de fabricació compatible amb el de la indústria microelectrònica, amb un mètode de biofuncionalització obtenint una capa de bio-reconeixement estable i selectiva mitjançant l'ús de la reacció TEC assistida per llum capaç de proporcionar unes capes de bioreconeixement extremadament fines amb una immobilització espacialment selectiva.[EN] Healthcare is a field where the early detection of diseases is becoming more and more important. Nowadays, professionals and citizens demand high quality diagnosis techniques offered by both private and public health systems. When the application of diagnostic tests is not adequate, different consequences can be observed such as health hazard and technical and economic overload of health services. This is due to the fact that the diagnostic techniques available are expensive, centralized in laboratories and with the need for highly qualified professionals to carry out these tasks, what can fundamentally lead to delays in time, being critical for the patient's health. It is very necessary, therefore, to reflect on the need and emergency of such preventive practices, especially for high-risk diseases such as cancer, Alzheimer or the first cause of death in the world, the cardiovascular diseases. Within this context, the main objective of the work done during this PhD Thesis is to help on overcoming these problems by exploring the possibility of using photonic technology for the development of analysis devices which might be used for the early diagnosis and prognosis of cardiovascular diseases. This objective has been addressed by combining nanophotonic technology, by the nanofabrication of the photonic PBG sensing structures, which provides several benefits such as a high sensitivity, an extreme size reduction and a fabrication process being compatible with that from the microelectronics industry, with a light-assisted biofunctionalization method forming a stable and selective biorecognition layer using TEC reaction able to provide extremely thin biorecognition layers with a spatially-selective immobilization.Sabek, J. (2019). Combination of nanophotonic biosensors and light-assisted immobilization procedures for the detection of cardiac biomarkers [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124821TESI

Computational binding study of cardiac troponin I antibody towards cardiac versus skeletal troponin I

[EN] A computational study of the interaction of cardiac troponin I (cTnI) with its specific antibody and of that antibody with skeletal troponin I (sTnI), the principal interferon of cTnI, is carried out. Computational and simulation tools such as FTSite, FTMap, FTDock and pyDock are used to determine the binding sites of these molecules and to study their interactions and molecular docking performance, allowing us to obtain relevant information related with the antigen-antibody interaction for each of the targets. In the context of the development of immunosensing platforms, this type of computational analysis allows performing a preliminary in-silico affinity study of the available bioreceptors for a better selection when moving to the experimental stage, with the subsequent saving in cost and time.Cardiac troponin I (cTnI); Skeletal troponin I (sTnI); Immunosensing; Binding site; Molecular docking; FTSite; FTMap; FTDock; pyDockSabek, J.; Martinez-Perez, P.; García-Rupérez, J. (2019). Computational binding study of cardiac troponin I antibody towards cardiac versus skeletal troponin I. Computational Biology and Chemistry. 80:147-151. https://doi.org/10.1016/j.compbiolchem.2019.04.002S1471518

Thiol-click photochemistry for surface functionalization applied to optical biosensing

[EN] In the field of biosensing, suitable procedures for efficient probes immobilization are of outmost importance. Here we present different light-based strategies to promote the covalent attachment of thiolated capture probes (oligonucleotides and proteins) on different materials and working formats. One strategy employs epoxylated surfaces and uses the light to accomplish the ring opening by a thiol moiety present in a probe. However, most of this work lies on the use of thiol-ene photocoupling chemistry to covalently attach probes to the supports. And thus, both alkenyl and thiol derivatized surfaces are assayed to immobilize thiol or alkene ended probes, respectively, and their performances are compared. Also, the effect of the number of thiols carried by the probe is analyzed comparing single-point and multi-point attachment. The performance of the analogous tethering, but onto alkynylated surfaces is also carried out, and the sensing response is related to the surfaces hydrophobicity. A newly developed reaction is also discussed where a fluorinated surface undergoes the covalent immobilization of thiolated probes activated by light, creating small hydrophilic areas where the probes are attached, and leaving the rest of the surface highly hydrophobic and repellent against protein unspecific adsorption. These mixed surfaces confine the sample (aqueous) uniquely on the hydrophilic spots lowering the background signal and thus increasing the sensitivity. These probe immobilization approaches are applied to fluorescence microarray and label-free nanophotonic biosensing. All the exposed reactions have in common the photoactivation of the thiol moieties, and give rise to quick, clean, versatile, orthogonal and biocompatible reactions. Water is the only solvent used, and light the only catalyzer applied. Thus, all of them can be considered as having the attributes of click-chemistry reactions. For these reasons we named them as thiol-click photochemistry, being a very interesting pool of possibilities when building a biosensor.This work was supported by the European Union program Horizon 2020, projects H2020-PHC-634013 and H2020-ICT-644242, and by the Spanish Ministry of Economy and Competitiveness, project CTQ2016-75749-R. Authors thank the whole "Signal and Measurement" research group, from the IDM, UPV, for sharing space, research and life. Special thanks to Pilar Jimenez-Meneses, Rafael Alonso, Daniel Gonzalez-Lucas, Pilar Aragon and Patricia Noguera for their contribution to the development of thiol photoattaching chemistry and surface wettability modulation.Bañuls Polo, M.; González Martínez, MÁ.; Sabek, J.; García-Rupérez, J.; Maquieira Catala, Á. (2019). Thiol-click photochemistry for surface functionalization applied to optical biosensing. Analytica Chimica Acta. 1060:103-113. https://doi.org/10.1016/j.aca.2019.01.055S103113106

Live Tracking Biofunctionalization and Label-Free Protein Detection Performed by a Nanophotonic Biosensor

[EN] A label-free biosensor based on silicon-on-insulator (SOI) photonic bandgap (PBG) structures is performed for specific protein detection. First, the SOI sensing surface is functionalized using triethoxyvinylsilane (TEVS) organosilane. Then, a UV light photocatalyzed immobilization of polyclonal half anti-bovine serum albumin (haBSA) antibodies is performed. Finally, a direct detection of target BSA antigen is carried out. Both the immobilization and the detection steps are monitored by making a continuous tracking of the PBG edge shift. In order to confirm the recognition of the antigen by the immobilized antibody, a fluorophore-labelled secondary antibody was flowed at the end of the experiment in order to perform a confirmation fluorescence test after the photonic detection.This work was supported by Horizon 2020 Framework Program of the European Union under the project H2020-PHC-634013 (PHOCNOSIS).Sabek, J.; Torrijos-Morán, L.; Díaz-Betancor, Z.; Bañuls Polo, M.; Maquieira Catala, Á.; García-Rupérez, J. (2018). Live Tracking Biofunctionalization and Label-Free Protein Detection Performed by a Nanophotonic Biosensor. Proceedings. 4(1):1-6. https://doi.org/10.3390/ecsa-5-05718S164

Real Time Monitoring of a UV Light-Assisted Biofunctionalization Protocol Using a Nanophotonic Biosensor

[EN] A protocol for the covalent biofunctionalization of silicon-based biosensors using a UV light-induced thiol-ene coupling (TEC) reaction has been developed. This biofunctionalization approach has been used to immobilize half antibodies (hIgG), which have been obtained by means of a tris(2-carboxyethyl)phosphine (TCEP) reduction at the hinge region, to the surface of a vinyl-activated silicon-on-insulator (SOI) nanophotonic sensing chip. The response of the sensing structures within the nanophotonic chip was monitored in real time during the biofunctionalization process, which has allowed us to confirm that the bioconjugation of the thiol-terminated bioreceptors onto the vinyl-activated sensing surface is only initiated upon UV light photocatalysis.This work was supported by the Horizon 2020 Programme of the European Union under the project H2020-PHC-634013 (PHOCNOSIS).Sabek, J.; Torrijos-Morán, L.; Griol Barres, A.; Díaz-Betancor, Z.; Bañuls Polo, M.; Maquieira Catala, Á.; García-Rupérez, J. (2018). Real Time Monitoring of a UV Light-Assisted Biofunctionalization Protocol Using a Nanophotonic Biosensor. Biosensors. 9(1):1-9. https://doi.org/10.3390/bios90100061991Chin, C. D., Linder, V., & Sia, S. K. (2007). Lab-on-a-chip devices for global health: Past studies and future opportunities. Lab Chip, 7(1), 41-57. doi:10.1039/b611455eWu, J., Dong, M., Santos, S., Rigatto, C., Liu, Y., & Lin, F. (2017). Lab-on-a-Chip Platforms for Detection of Cardiovascular Disease and Cancer Biomarkers. Sensors, 17(12), 2934. doi:10.3390/s17122934Estevez, M. C., Alvarez, M., & Lechuga, L. M. (2011). Integrated optical devices for lab-on-a-chip biosensing applications. Laser & Photonics Reviews, 6(4), 463-487. doi:10.1002/lpor.201100025Vestergaard, M., Kerman, K., & Tamiya, E. (2007). An Overview of Label-free Electrochemical Protein Sensors. Sensors, 7(12), 3442-3458. doi:10.3390/s7123442Johnson, B. N., & Mutharasan, R. (2012). Biosensing using dynamic-mode cantilever sensors: A review. Biosensors and Bioelectronics, 32(1), 1-18. doi:10.1016/j.bios.2011.10.054Jonkheijm, P., Weinrich, D., Schröder, H., Niemeyer, C. M., & Waldmann, H. (2008). Chemical Strategies for Generating Protein Biochips. Angewandte Chemie International Edition, 47(50), 9618-9647. doi:10.1002/anie.200801711Phaner-Goutorbe, M., Dugas, V., Chevolot, Y., & Souteyrand, E. (2011). Silanization of silica and glass slides for DNA microarrays by impregnation and gas phase protocols: A comparative study. Materials Science and Engineering: C, 31(2), 384-390. doi:10.1016/j.msec.2010.10.016Escorihuela, J., Bañuls, M.-J., Grijalvo, S., Eritja, R., Puchades, R., & Maquieira, Á. (2014). Direct Covalent Attachment of DNA Microarrays by Rapid Thiol–Ene «Click» Chemistry. Bioconjugate Chemistry, 25(3), 618-627. doi:10.1021/bc500033dEscorihuela, J., Bañuls, M. J., Puchades, R., & Maquieira, Á. (2012). DNA microarrays on silicon surfaces through thiol-ene chemistry. Chemical Communications, 48(15), 2116. doi:10.1039/c2cc17321bGonzález-Lucas, D., Bañuls, M.-J., García-Rupérez, J., & Maquieira, Á. (2017). Covalent attachment of biotinylated molecular beacons via thiol-ene coupling. A study on conformational changes upon hybridization and streptavidin binding. Microchimica Acta, 184(9), 3231-3238. doi:10.1007/s00604-017-2310-4Alonso, R., Jiménez-Meneses, P., García-Rupérez, J., Bañuls, M.-J., & Maquieira, Á. (2018). Thiol–ene click chemistry towards easy microarraying of half-antibodies. Chemical Communications, 54(48), 6144-6147. doi:10.1039/c8cc01369aGonzález-Guerrero, A. B., Alvarez, M., Castaño, A. G., Domínguez, C., & Lechuga, L. M. (2013). A comparative study of in-flow and micro-patterning biofunctionalization protocols for nanophotonic silicon-based biosensors. Journal of Colloid and Interface Science, 393, 402-410. doi:10.1016/j.jcis.2012.10.040Povinelli, M. L., Johnson, S. G., & Joannopoulos, J. D. (2005). Slow-light, band-edge waveguides for tunable time delays. Optics Express, 13(18), 7145. doi:10.1364/opex.13.007145Ruiz-Tórtola, Á., Prats-Quílez, F., González-Lucas, D., Bañuls, M.-J., Maquieira, Á., Wheeler, G., … García-Rupérez, J. (2018). High sensitivity and label-free oligonucleotides detection using photonic bandgap sensing structures biofunctionalized with molecular beacon probes. Biomedical Optics Express, 9(4), 1717. doi:10.1364/boe.9.001717Golas, A., Parhi, P., Dimachkie, Z. O., Siedlecki, C. A., & Vogler, E. A. (2010). Surface-energy dependent contact activation of blood factor XII. Biomaterials, 31(6), 1068-1079. doi:10.1016/j.biomaterials.2009.10.03

Experimental study of an evanescent-field biosensor based on 1D photonic bandgap structures

[EN] A photonic bandgap (PBG) biosensor has been developed for the label-free detection of proteins. As the sensing in this type of structures is governed by the interaction between the evanescent field going into the cladding and the target analytes, scanning near-field optical microscopy has been used to characterize the profile of that evanescent field. The study confirms the strong exponential decrease of the signal as it goes into the cladding. This means that biorecognition events must occur as close to the PBG structure surface as possible in order to obtain the maximum sensing response. Within this context, the PBG biosensor has been biofunctionalized with half-antibodies specific to bovine serum albumin (BSA) using a UV-induced immobilization procedure. The use of half-antibodies allows one to reduce the thickness of the biorecognition volume down to ca. 2.5 nm, thus leading to a higher interaction with the evanescent field, as well as a proper orientation of their binding sites towards the target sample. Then, the biofunctionalized PBG biosensor has been used to perform a direct and real-time detection of the target BSA antigen.This research was funded by the European Commission through the Horizon 2020 Programme (PHC-634013-PHOCNOSIS project) and by the Spanish Ministry of Economy and Competitiveness (TEC2015-63838-C3-1-R-OPTONANOSENS project and FJCI-2015-27228 grant).Sabek, J.; Díaz-Fernández, FJ.; Torrijos-Morán, L.; Díaz-Betancor, Z.; Maquieira Catala, A.; Bañuls Polo, M.; Pinilla-Cienfuegos, E.... (2019). Experimental study of an evanescent-field biosensor based on 1D photonic bandgap structures. Beilstein Journal of Nanotechnology. 10:967-974. https://doi.org/10.3762/bjnano.10.97S96797410Wu, J., Dong, M., Santos, S., Rigatto, C., Liu, Y., & Lin, F. (2017). Lab-on-a-Chip Platforms for Detection of Cardiovascular Disease and Cancer Biomarkers. Sensors, 17(12), 2934. doi:10.3390/s17122934Qavi, A. J., Washburn, A. L., Byeon, J.-Y., & Bailey, R. C. (2009). Label-free technologies for quantitative multiparameter biological analysis. Analytical and Bioanalytical Chemistry, 394(1), 121-135. doi:10.1007/s00216-009-2637-8Luan, E., Shoman, H., Ratner, D., Cheung, K., & Chrostowski, L. (2018). Silicon Photonic Biosensors Using Label-Free Detection. Sensors, 18(10), 3519. doi:10.3390/s18103519Washburn, A. L., & Bailey, R. C. (2011). Photonics-on-a-chip: recent advances in integrated waveguides as enabling detection elements for real-world, lab-on-a-chip biosensing applications. The Analyst, 136(2), 227-236. doi:10.1039/c0an00449aIqbal, M., Gleeson, M. A., Spaugh, B., Tybor, F., Gunn, W. G., Hochberg, M., … Gunn, L. C. (2010). Label-Free Biosensor Arrays Based on Silicon Ring Resonators and High-Speed Optical Scanning Instrumentation. IEEE Journal of Selected Topics in Quantum Electronics, 16(3), 654-661. doi:10.1109/jstqe.2009.2032510Huertas, C. S., Fariña, D., & Lechuga, L. M. (2016). Direct and Label-Free Quantification of Micro-RNA-181a at Attomolar Level in Complex Media Using a Nanophotonic Biosensor. ACS Sensors, 1(6), 748-756. doi:10.1021/acssensors.6b00162Baker, J. E., Sriram, R., & Miller, B. L. (2015). Two-dimensional photonic crystals for sensitive microscale chemical and biochemical sensing. Lab on a Chip, 15(4), 971-990. doi:10.1039/c4lc01208aPhaner-Goutorbe, M., Dugas, V., Chevolot, Y., & Souteyrand, E. (2011). Silanization of silica and glass slides for DNA microarrays by impregnation and gas phase protocols: A comparative study. Materials Science and Engineering: C, 31(2), 384-390. doi:10.1016/j.msec.2010.10.016Díaz-Fernández, F. J., Pinilla-Cienfuegos, E., García-Meca, C., Lechago, S., Griol, A., & Martí, J. (2019). Characterisation of on-chip wireless interconnects based on silicon nanoantennas via near-field scanning optical microscopy. IET Optoelectronics, 13(2), 72-76. doi:10.1049/iet-opt.2018.507

Basic building blocks development for a SiN platform in the visible range

Integrated silicon photonics in the visible range is one of the areas that is emerging and growing in the market for different applications, such as: biosensing, optogenetics, quantum computing, imaging and display, fluorescence microscopy, cytometry, tomography, LIDAR and Lifi. Here, we present the first steps to build a process design kit of components in the visible range centered at 633 nm using a LPCVD silicon nitride platform. The first basic elements are presented (grating couplers, single mode waveguides and a MMI)

Solvent-Controlled Synthesis and Luminescence Properties of Uniform Eu:YVO4 Nanophosphors with Different Morphologies

A facile solvothermal route has been developed for the preparation of tetragonal europium-doped yttrium orthovanadate nanoparticles (Eu:YVO4) and is based on a homogeneous precipitation reaction at 120 °C from solutions of rare earth precursors (yttrium acetylacetonate and europium nitrate) and sodium orthovanadate in ethylene glycol or ethylene glycol/water mixtures. The nature of the solvent has a dramatic effect on the morphology and crystallinity of the resulting nanoparticles. Polycrystalline nanoellipsoids (130¿×¿60 nm) were obtained in pure ethylene glycol, whereas quasispherical nanoparticles (100 nm) with monocrystalline character precipitated in ethylene glycol/water (7:3 by volume) mixtures. To explain these different morphological and structural features, the mechanism of particles formation was investigated. The effects of the doping level on the luminescence properties (emission spectra and luminescence lifetime) were also evaluated to find the optimum nanophosphors. Finally, it is shown that the luminescent efficiency of the quasispherical nanoparticles was higher than that of the nanoellipsoids; this can be related to differences in crystallinity and in impurity contentPeer Reviewe