Biogratings: Diffractive Transducers for Biosensing in Photonic Platforms

Tesis por compendio[ES] El desarrollo científico y tecnológico de las últimas décadas ha dado lugar a sistemas sensores capaces de obtener, procesar y transmitir información sobre multitud de aspectos físicos y químicos, y utilizarla para mejorar aspectos clave de multitud de áreas de nuestra sociedad. Los sensores químicos son dispositivos compactos y miniaturizados capaces de ofrecer soluciones alternativas a las técnicas de análisis instrumental convencionales. En especial, los biosensores han adquirido gran relevancia por los avances que han supuesto para sectores estratégicos como el diagnóstico clínico, la industria alimentaria y el medio ambiente. Los biosensores ópticos se basan en interacciones entre la luz y la materia para transducir eventos de bioreconocimiento y presentan prestaciones importantes como la estabilidad, inmunidad a estímulos externos y versatilidad en el desarrollo de aproximaciones sin marcaje (label-free). Este último aspecto suele aprovechar fenómenos nanoscópicos y su desarrollo se encuentra muy ligado al progreso de la nanociencia y nanotecnología. Un aspecto clave en el biosensado sin marcaje consiste en descubrir y desarrollar nuevas estrategias de transducción. En este sentido, aunque se encuentren aun en una etapa temprana de desarrollo, los biosensores difractivos presentan un gran potencial en términos de simplicidad, miniaturización, y capacidad para minimizar señales no deseadas fruto de interacciones no específicas, entre otros aspectos.[CA] El desenvolupament científic i tecnològic de les últimes dècades ha donat lloc a sistemes sensors capaços d'obtindre, processar i transmetre informació sobre multitud d'aspectes físics i químics, i utilizar-la per a millorar aspectes clau de multitud d'arees de la nostra societat. Els sensors químics són dispositius compactes i miniaturitzats capaços d'oferir solucions alternatives a les tècniques d'analisi instrumental convencionals. Especialment, els biosensors han adquirit gran rellevància pels avanços que han suposat per als sectors estratègics com el diagnòstic clínic, la industria alimentària i el medi ambient. Els biosensors òptics es basen en interaccions entre la llum i la matèria per a transduir esdeveniments de bioreconèixement i presenten prestacions importants com estabilitat, immunitat a estímuls externs i versatilitat en el desenvolupament d'aproximacions sense marcatge (label-free). Aquest últim aspecte sol aprofitat fenòmens nanoscòpics i el seu desenvolupament es troba molt lligat al progrés de la nanociència i nanotecnologia. Un aspecte clau en el biosensat sense marcatge consisteix a descobrir i desenvolupar noves estratègies de transducció. En aquest sentit, encara que es troben fins i tot en una etapa primerenca de desenvolupament, els biosensors difractius presenten un gran potencial en termes de simplicitat, miniaturització, i capacitat per a minimitzar senyals no desitjats fruit d'interaccions no específiques, entre altres aspectes.[EN] The scientific and technological progress in recent decades has given rise to sensor systems capable of obtaining, processing, and transmitting information on a multitude of physical and chemical aspects and using it to improve key aspects of many areas of our society. Chemical sensors are compact, miniaturized devices capable of offering alternative solutions to conventional instrumental analysis techniques. In particular, biosensors have become highly relevant due to the progress they have brought to strategic sectors such as clinical diagnostics, the food industry, and the environment. Optical biosensors rely on interactions between light and matter to transduce biosensing events and provide important features such as stability, immunity to external stimuli, and versatility in the development of label-free approaches. This last aspect usually exploits nanoscopic phenomena and its development in closely linked to the progress in nanoscience and nanotechnology. A key aspect of label-free biosensing is the discovery and development of new transduction strategies. In this regard, although they are at an early stage of development, diffractive biosensors offer great potential in terms of simplicity, miniaturization, and the ability to minimize unwanted signals from non-specific interactions, among other aspects.This work was financially supported by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (MCIN/AEI/10.13039/501100011033) co-funded by the European Union “ERDF A way of making Europe” (PID2019-110713RB-I00, TED2021-132584B-C21, PID2019-110877GB-I00), Ministerio de Economía y Competitividad (TEC2016-80385-P), Generalitat Valenciana (PROMETEO/2019/048 PROMETEO/2020/094, PROMETEO/2021/015, IDIFEDER/2021/046). A.J.D. ackowledges the FPI-UPV 2017 grant program. The authors acknowledge Instituto de Microelectrónica de Barcelona CNM-CSIC for the support in the fabrication of the measured chip samples on the Multiproject CNM-VLC silicon nitride technology platform.Juste Dolz, AM. (2023). Biogratings: Diffractive Transducers for Biosensing in Photonic Platforms [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/194251Compendi

Indirect Microcontact Printing to Create Functional Patterns of Physisorbed Antibodies

[EN] Microcontact printing (mu CP) is a practical and versatile approach to create nanostructured patterns of biomolecular probes, but it involves conformational changes on the patterned bioreceptors that often lead to a loss on the biological activity of the resulting structures. Herein we introduce indirect mu CP to create functional patterns of bioreceptors on solid substrates. This is a simple strategy that relies on physisorbing biomolecular probes of interest in the nanostructured gaps that result after patterning backfilling agents by standard mu CP. This study presents the approach, assesses bovine serum albumin as backfilling agent for indirect mu CP on different materials, reports the limitations of standard mu CP on the functionality of patterned antibodies, and demonstrates the capabilities of indirect mu CP to solve this issue. Bioreceptors were herein structured as diffractive gratings and used to measure biorecognition events in label-free conditions. Besides, as a preliminary approach towards sensing biomarkers, this work also reports the implementation of indirect mu CP in an immunoassay to detect human immunoglobulin E.This research was funded by the Spanish Ministry of Economy and Competitiveness (CTQ2016-75749-R), FEDER, Generalitat Valenciana (PROMETEO II/2014/040), and Universitat Politecnica de Valencia (FPI program).Juste-Dolz, AM.; Avella-Oliver, M.; Puchades, R.; Maquieira Catala, A. (2018). Indirect Microcontact Printing to Create Functional Patterns of Physisorbed Antibodies. Sensors. 18(9). https://doi.org/10.3390/s18093163S316318

Denaturing for nanoarchitectonics: local and periodic UV-laser photodeactivation of protein biolayers to create functional patterns for biosensing

The nanostructuration of biolayers has become a paradigm for exploiting nanoscopic light-matter phenomena for biosensing, among other biomedical purposes. In this work, we present a photopatterning method to create periodic structures of biomacromolecules based on a local and periodic mild denaturation of protein biolayers mediated by UV-laser irradiation. These nanostructures are constituted by a periodic modulation of the protein activity, so they are free of topographic and compositional changes along the pattern. Herein, we introduce the approach, explore the patterning parameters, characterize the resulting structures, and assess their overall homogeneity. This UV-based patterning principle has proven to be an easy, cost-effective, and fast way to fabricate large areas of homogeneous one-dimensional protein patterns (2 min, 15 × 1.2 mm, relative standard deviation ≃ 16%). This work also investigates the implementation of these protein patterns as transducers for diffractive biosensing. Using a model immunoassay, these patterns have demonstrated negligible signal contributions from non-specific bindings and comparable experimental limits of detection in buffer media and in human serum (53 and 36 ng·mL−1 of unlabeled IgG, respectively)

Denaturing for Nanoarchitectonics: Local and Periodic UV-Laser Photodeactivation of Protein Biolayers to Create Functional Patterns for Biosensing

[EN] The nanostructuration of biolayers has become a paradigm for exploiting nanoscopic light-matter phenomena for biosensing, among other biomedical purposes. In this work, we present a photopatterning method to create periodic structures of biomacromolecules based on a local and periodic mild denaturation of protein biolayers mediated by UV-laser irradiation. These nanostructures are constituted by a periodic modulation of the protein activity, so they are free of topographic and compositional changes along the pattern. Herein, we introduce the approach, explore the patterning parameters, characterize the resulting structures, and assess their overall homogeneity. This UV-based patterning principle has proven to be an easy, cost-effective, and fast way to fabricate large areas of homogeneous one-dimensional protein patterns (2 min, 15 x 1.2 mm, relative standard deviation ? 16%). This work also investigates the implementation of these protein patterns as transducers for diffractive biosensing. Using a model immunoassay, these patterns have demonstrated negligible signal contributions from non-specific bindings and comparable experimental limits of detection in buffer media and in human serum (53 and 36 ng & BULL;mL-1 of unlabeled IgG, respectively).This work was financially supported by the Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion (MCIN/AEI/10.13039/501100011033) , co-funded by the European Union "ERDF A way of making Europe", under grants PID2019-110713RB-I00 and PDI2019-104276RB-I00, and Generalitat Valenciana (PROMETEO/2020/094 and PROMETEO/2019/048) . A.J.-D. acknowledges the FPI-UPV 2017 grant program. The authors thank Angel Lopez Munoz for the construction of the scanning system.Juste-Dolz, AM.; Delgado-Pinar, M.; Avella-Oliver, M.; Fernández-Sánchez, ME.; Cruz, JL.; Andrés, MV.; Maquieira Catala, A. (2022). Denaturing for Nanoarchitectonics: Local and Periodic UV-Laser Photodeactivation of Protein Biolayers to Create Functional Patterns for Biosensing. ACS Applied Materials & Interfaces. 14(36):41640-41648. https://doi.org/10.1021/acsami.2c128084164041648143

BIO bragg gratings on microfibers for label-free biosensing

[EN] Discovering nanoscale phenomena to sense biorecognition events introduces new perspectives to exploit nano science and nanotechnology for bioanalytical purposes. Here we present Bio Bragg Gratings (BBGs), a novel biosensing approach that consists of diffractive structures of protein bioreceptors patterned on the surface of optical waveguides, and tailored to transduce the magnitude of biorecognition assays into the intensity of single peaks in the reflection spectrum. This work addresses the design, fabrication, and optimization of this system by both theoretical and experimental studies to explore the fundamental physicochemical parameters involved. Functional biomolecular gratings are fabricated by microcontact printing on the surface of tapered optical microfibers, and their structural features were characterized. The transduction principle is experimentally demonstrated, and its quantitative bioanalytical prospects are assessed in a representative immunoassay, based on patterned protein probes and selective IgG targets, in label-free conditions. This biosensing system involves appealing perspectives to avoid unwanted signal contributions from non-specific binding, herein investigated in human serum samples. The work also proves how the optical response of the system can be easily tuned, and it provides insights into the relevance of this feature to conceive multiplexed BBG systems capable to perform multiple label-free biorecognition assays in a single device.Spanish Ministry of Science and Innovation (CTQ2016-75749-R and TEC2016-80385-P), Agencia Estatal de Investigacion and Fondo Europeo de Desarrollo Regional (PID2019-110877 GB-I00 and PDI2019-104276RB-I00) and Generalitat Valenciana (PROMETEO/2019/048 and PROMETEO/2017/103). A.J.-D. acknowledges the FPI-UPV 2017 grant program, M.A.-O acknowledges the APSOSTD/2019 program of the GVA.Juste-Dolz, AM.; Delgado-Pinar, M.; Avella-Oliver, M.; Fernández-Sánchez, ME.; Pastor Abellán, D.; Andrés, MV.; Maquieira Catala, Á. (2021). BIO bragg gratings on microfibers for label-free biosensing. Biosensors and Bioelectronics. 176:1-8. https://doi.org/10.1016/j.bios.2020.112916S1817

Identification of high-affinity phage-displayed VH fragments by use of a quartz crystal microbalance with dissipation monitoring

[EN] Phage display has become a powerful tool for antibody discovery in a wide variety of fields. This technology allows specific binders for a given antigen to be selected from combinatorial libraries. A key step in the process is characterizing and evaluating antibody clones thus selected to reliably identify the best antigen binders. Novel characterization methods can provide essential insight into the binding mechanism and supplement the information obtained with conventional techniques. In this work, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to determine the kinetic and thermodynamic binding parameters for phagedisplayed VH antibody fragments. Phytohemagglutinin (PHA), a legume lectin of analytical interest, was used as a complex model antigen to select specific VH fragments from a phage-displayed library. Eight VH fragments with a unique amino acid sequence were identified as PHA binders by using the well-established enzyme-linked immunosorbent assay (ELISA). QCM-D measurements, structural analysis and principal component analysis (PCA) were used to evaluate the antibody fragments and identify clone clusters with similar binding characteristics and molecular interaction mechanisms. This unprecedented study has enabled the identification of highaffinity phage-displayed VH antibody fragments for PHA, which could be useful for PHA analysis (apparent association constant ranged from 108 to 1010 M-1). In fact, the proposed methodology provides a useful tool for evaluating and characterizing antibody fragments with capabilities beyond those of conventional techniquesThis work was funded by the Spanish Ministry of Science and Innovation and the Spanish Ministry of Economy and Competitiveness (Projects RTI2018-096410-B-C21 and CTQ2016-75749-R, respectively), GVA's Prometeo program (Grant 2020/094) and the European Regional Development Fund (ERDF).Gómez-Arribas, LN.; Juste-Dolz, AM.; Peltomaa, R.; Giménez-Romero, D.; Morais, S.; Barderas, R.; Cuadrado, C.... (2021). Identification of high-affinity phage-displayed VH fragments by use of a quartz crystal microbalance with dissipation monitoring. Sensors and Actuators B Chemical. 340:1-10. https://doi.org/10.1016/j.snb.2021.12995411034

An all-in-one point-of-care testing device for multiplexed detection of respiratory infections

[EN] The impact of the COVID-19 pandemic has reinforced the need for rapid, cost-effective, and reliable point-of-care testing (POCT) devices for massive population screening. The co-circulation of SARS-CoV-2 with several seasonal respiratory viruses highlights the need for multiplexed biosensing approaches. Herein, we present a fast and robust all-in-one POCT device for parallel viral antigen and serological analysis. The biosensing approach consists of a functionalized polycarbonate disc-shaped surface with microfluidic structures, where specific bioreagents are immobilized in microarray format, and a portable optoelectronic analyzer. The biosensor quantifies the concentration of viral antigens and specific immunoglobulins G and M for SARS-CoV-2, influenza A/B, adenovirus, and respiratory syncytial virus, using 30¿¿L of a sample. The semi-automated analysis of 6 samples is performed in 30¿min. Validation studies performed with 135 serum samples and 147 nasopharyngeal specimens reveal high diagnostic sensitivity (98¿100%) and specificity (84¿98%), achieving an excellent agreement (¿¿=¿0.937) with commercial immunoassays, which complies with the World Health Organization criteria for POC COVID-19 diagnostic tests. The versatility of the POCT device paves the way for the detection of other pathogens and analytes in the incoming post-pandemic world, integrating specific bioreagents against different variants of concerns and interests.W. T and A. J-D acknowledge the financial support for the Ph.D. studies (PAID-19-01-06 and FPI-UPV 2017 grants, respectively). S.M awards grant PID2019-110713RB-I00 funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe." A.M awards grant DOGV no. 8815, May 19, 2020, pp. 16712-16726. The research was also supported by the Generalitat Valenciana (PROMETEO/2020/094, Proyecto "88/2021"). The authors gratefully acknowledge the outstanding contribution of Alicia Parra, Violeta Espada, Roberto Renuncio, and Jose A. Marzal for collecting the nasopharyngeal swab samples from the Health Centre of the Universitat Politecnica de Valencia. We also gratefully thank Dr. L. Mira and G. Rios for carefully reading this manuscript and the four anonymous reviewers for their insightful comments and suggestions.Teixeira, W.; Pallás-Tamarit, Y.; Juste-Dolz, AM.; Sena-Torralba, A.; Gozalbo-Rovira, R.; Rodríguez-Díaz, J.; Navarro, D.... (2022). An all-in-one point-of-care testing device for multiplexed detection of respiratory infections. Biosensors and Bioelectronics. 213:1-7. https://doi.org/10.1016/j.bios.2022.1144541721