Real-time monitoring of fenitrothion in water samples using a silicon nanophotonic biosensor

[EN] Due to the large quantities of pesticides extensively used and their impact on the environment and human health, a prompt and reliable sensing technique could constitute an excellent tool for in-situ monitoring. With this aim, we have applied a highly sensitive photonic biosensor based on a bimodal waveguide interferometer (BiMW) for the rapid, label-free, and speci¿c quanti¿cation of fenitrothion (FN) directly in tap water samples. After an optimization protocol, the biosensor achieved a limit of detection (LOD) of 0.29 ng mL¿¿1 (1.05 nM) and a half-maximal inhibitory concentration (IC50)of 1.71 ng mL¿¿1 (6.09 nM) using a competitive immunoassay and employing diluted tap water. Moreover, the biosensor was successfully employed to determine FN concentration in blind tap water samples obtaining excellent recovery percentages with a time-to-result of only 20 min without any sample pre-treatment. The features of the biosensor suggest its potential application for real time, fast and sensitive screening of FN in water samples as an analytical tool for the monitoring of the water quality.This work received financial support from DIONISOS Project (Retos Colaboracion RTC-2017-6222-5). The ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706)Ramirez-Priego, P.; Estévez, M.; Díaz-Luisravelo, HJ.; Manclus Ciscar, JJ.; Montoya, Á.; Lechuga, LM. (2021). Real-time monitoring of fenitrothion in water samples using a silicon nanophotonic biosensor. Analytica Chimica Acta. 1152:1-9. https://doi.org/10.1016/j.aca.2021.338276S191152Sánchez-Santed, F., Colomina, M. T., & Herrero Hernández, E. (2016). Organophosphate pesticide exposure and neurodegeneration. Cortex, 74, 417-426. doi:10.1016/j.cortex.2015.10.003Chough, S. 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F., Mahboob, M., Danadevi, K., Saleha Banu, B., & Grover, P. (2002). Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 516(1-2), 139-147. doi:10.1016/s1383-5718(02)00033-5Yeh, S.-P., Sung, T.-G., Chang, C.-C., Cheng, W., & Kuo, C.-M. (2005). Effects of an organophosphorus insecticide, trichlorfon, on hematological parameters of the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Aquaculture, 243(1-4), 383-392. doi:10.1016/j.aquaculture.2004.10.017Smith, A. G., & Gangolli, S. D. (2002). Organochlorine chemicals in seafood: occurrence and health concerns. Food and Chemical Toxicology, 40(6), 767-779. doi:10.1016/s0278-6915(02)00046-7Kumar, P., Kim, K.-H., & Deep, A. (2015). Recent advancements in sensing techniques based on functional materials for organophosphate pesticides. 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Sensitive determination of fenitrothion in water samples based on an electrochemical sensor layered reduced graphene oxide, molybdenum sulfide (MoS2)-Au and zirconia films. Electrochimica Acta, 292, 667-675. doi:10.1016/j.electacta.2018.09.187Kant, R. (2019). Surface plasmon resonance based fiber–optic nanosensor for the pesticide fenitrothion utilizing Ta2O5 nanostructures sequestered onto a reduced graphene oxide matrix. Microchimica Acta, 187(1). doi:10.1007/s00604-019-4002-8Zinoviev, K. E., Gonzalez-Guerrero, A. B., Dominguez, C., & Lechuga, L. M. (2011). Integrated Bimodal Waveguide Interferometric Biosensor for Label-Free Analysis. Journal of Lightwave Technology, 29(13), 1926-1930. doi:10.1109/jlt.2011.2150734Fernández Gavela, A., Grajales García, D., Ramirez, J., & Lechuga, L. (2016). Last Advances in Silicon-Based Optical Biosensors. Sensors, 16(3), 285. doi:10.3390/s16030285Maldonado, J., Estévez, M.-C., Fernández-Gavela, A., González-López, J. J., González-Guerrero, A. B., & Lechuga, L. M. (2020). Label-free detection of nosocomial bacteria using a nanophotonic interferometric biosensor. The Analyst, 145(2), 497-506. doi:10.1039/c9an01485cHuertas, 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.6b00162Maldonado, J., González-Guerrero, A. B., Domínguez, C., & Lechuga, L. M. (2016). Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients. Biosensors and Bioelectronics, 85, 310-316. doi:10.1016/j.bios.2016.04.095González-Guerrero, A. B., Maldonado, J., Dante, S., Grajales, D., & Lechuga, L. M. (2016). Direct and label-free detection of the human growth hormone in urine by an ultrasensitive bimodal waveguide biosensor. Journal of Biophotonics, 10(1), 61-67. doi:10.1002/jbio.201600154Chocarro-Ruiz, B., Herranz, S., Fernández Gavela, A., Sanchís, J., Farré, M., Marco, M. P., & Lechuga, L. M. (2018). Interferometric nanoimmunosensor for label-free and real-time monitoring of Irgarol 1051 in seawater. Biosensors and Bioelectronics, 117, 47-52. doi:10.1016/j.bios.2018.05.044Chocarro-Ruiz, B., Pérez-Carvajal, J., Avci, C., Calvo-Lozano, O., Alonso, M. I., Maspoch, D., & Lechuga, L. M. (2018). A CO2optical sensor based on self-assembled metal–organic framework nanoparticles. Journal of Materials Chemistry A, 6(27), 13171-13177. doi:10.1039/c8ta02767fManclús, J. J., Primo, J., & Montoya, A. (1996). Development of Enzyme-Linked Immunosorbent Assays for the Insecticide Chlorpyrifos. 1. Monoclonal Antibody Production and Immunoassay Design. Journal of Agricultural and Food Chemistry, 44(12), 4052-4062. doi:10.1021/jf960144qDante, S., Duval, D., Fariña, D., González-Guerrero, A. B., & Lechuga, L. M. (2015). Linear readout of integrated interferometric biosensors using a periodic wavelength modulation. Laser & Photonics Reviews, 9(2), 248-255. doi:10.1002/lpor.20140021

Novel sensing algorithm for linear read-out of bimodal waveguide interferometric biosensors

Altres ajuts: the ICN2 was supported by the CERCA programme of the Generalitat de Catalunya.Biosensors employing photonics integrated circuits, and specifically those that rely on interferometric evanescent wave working principles, have outstanding performances due to the extreme sensitivity exhibited in one-step and direct assay, without the need of amplification. Within the interferometric configurations, the Bimodal Waveguide (BiMW) interferometric sensor stands out due to its demonstrated sensitivity for real-life applications and the simplicity of its design. To overcome the ambiguities that arise from the periodic nature of interferometric read-outs, a new all-optical modulation and the subsequent trigonometry-based algorithm have been proposed and applied to the BiMW biosensor. This new algorithm has been successfully employed for the selective identification and quantification of the external Spike (S) protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Our biosensing results from this simple, quick, and user-friendly method demonstrate high sensitivity and specificity and pave the way towards a point-of-care device for general use

Low-cost point-of-care biosensor device for clinical diagnosis in developing countries

Actualment el diagnòstic de Tuberculosi (TB) es realitza en laboratoris centralitzats, emprant equips voluminosos, reactius complexos i personal capacitat, augmentant els costos i el temps per obtenir els resultats. Per aquesta raó, l'objectiu d'aquesta tesi doctoral és el desenvolupament d'una plataforma point-of-care (POC) capaç d'oferir una resposta ràpida i fiable en el diagnòstic de TB. Per dur a terme aquest objectiu, la plataforma POC integra un nou sensor fotònic incorporat en un cartutx de micofluídica d'un sol ús. El sensor fotònic consisteix en un conjunt de interferòmetres Mach-Zehnder que ofereixen una alta sensibilitat. En primer lloc, es va dur a terme una caracterització òptica per estudiar el rendiment de la plataforma POC i la seva capacitat per a ser emprada en aplicacions biosensoras. Un cop caracteritzada òpticament, es van avaluar diferents estratègies de biofuncionalització per incorporar anticossos específics com a bioreceptors a la superfície del sensor. Després d'un estudi en profunditat, es va seleccionar i es va emprar l'estratègia de biofuncionalització òptima per l'anàlisi dels biomarcadors de TB. Els biomarcadors de TB es van avaluar tant en solució tampó com en mostres biològiques, particularment en orina humana. El biomarcador més prometedor i conegut de TB és el lipoarabinomanan (LAM), un component de la paret cel·lular bacteriana. En concret, la detecció d'aquest biomarcador va ser validada amb mostres clíniques de pacients amb TB i donants sans, mostrant la capacitat de la nostra plataforma POC per discriminar aquells pacients amb tuberculosi activa. A més, el disseny del sensor fotònic permet la detecció simultània de sis biomarcadors diferents. Tenint en compte això, hem dut a terme una prova de concepte de l'ús de la plataforma biosensora POC per a la detecció d'un panell de biomarcadors de TB utilitzant nanolitografía Dip-Pen per a la deposició de cada bioreceptor en cada sensor. Els nostres resultats, validats en estudis clínics més amplis, podrien tenir importants implicacions diagnòstiques. A més, el nostre biosensor POC ofereix una sèrie d'avantatges en comparació amb els mètodes recomanats per l'Organització Mundial de la Salut.Actualmente el diagnóstico de Tuberculosis (TB) se realiza en laboratorios centralizados, empleando equipos voluminosos, reactivos complejos y personal capacitado, aumentando los costes y el tiempo para obtener los resultados. Por esta razón, el objetivo de esta Tesis Doctoral es el desarrollo de una plataforma point-of-care (POC) capaz de ofrecer una respuesta rápida y fiable en el diagnóstico de TB. Para llevar a cabo este objetivo, la plataforma POC integra un novedoso sensor fotónico incorporado en un cartucho de micofluídica desechable. El sensor fotónico consiste en un conjunto de interferómetros Mach-Zehnder que ofrecen una alta sensibilidad. En primer lugar, se llevó a cabo una caracterización óptica para estudiar el rendimiento de la plataforma POC y su capacidad para ser empleada en aplicaciones biosensoras. Una vez caracterizada ópticamente, se evaluaron distintas estrategias de biofuncionalización para incorporar anticuerpos específicos como bioreceptores a la superficie del sensor. Después de un estudio en profundidad, se seleccionó y empleó la estrategia de biofuncionalización óptima para el análisis de los biomarcadores de TB. Los biomarcadores de TB se evaluaron tanto en solución tampón como en muestras biológicas, particularmente en orina humana. El biomarcador más prometedor y conocido de TB es el lipoarabinomanano (LAM), un componente de la pared celular bacteriana. En concreto, la detección de este biomarcador fue validada con muestras clínicas de pacientes con TB y donantes sanos, mostrando la capacidad de nuestra plataforma POC para discriminar a aquellos pacientes con Tuberculosis activa. Además, el diseño del sensor fotónico permite la detección simultánea de seis biomarcadores distintos. Teniendo esto en cuenta, hemos llevado a cabo una prueba de concepto del empleo de la plataforma biosensora POC para la detección de un panel de biomarcadores de TB utilizando nanolitografía Dip-Pen para la deposición de cada bioreceptor en cada sensor. Nuestros resultados, validados en estudios clínicos más amplios, podrían tener importantes implicaciones diagnósticas. Además, nuestro biosensor POC ofrece una serie de ventajas en comparación con los métodos recomendados por la Organización Mundial de la Salud.Nowadays, Tuberculosis (TB) diagnosis is carried out at centralised laboratories, employing bulky equipment, complex reagents, and trained staff, increasing costs and the time to obtain the results. For that reason, the aim of this Doctoral Thesis is to develop a point-of-care (POC) platform able to deliver a prompt and reliable response to TB diagnosis, taking advantage of a highly sensitive evanescent wave optical sensor. The POC platform integrates a novel photonic sensor consisting of a Mach-Zehnder Interferometer transducer array incorporated in a disposable microfluidic cartridge. Firstly, an optical characterisation was carried out to study the new POC performance and its ability to be employed for biosensing applications. Once the POC platform was optically characterised, diverse biofunctionalisation strategies were tested in order to incorporate specific antibodies as bioreceptors to the sensor surface. After an in-depth study, the optimal biofunctionalisation strategy was selected and employed for the analysis of the TB biomarkers. The TB biomarkers were evaluated in both buffer and biological samples, particularly human urine. The most promising and well-known TB biomarker was lipoarabinomannan (LAM), a bacterial cell wall component. In particular, this biomarker detection was validated with clinical samples from TB patients and healthy donors, showing the ability of our POC platform to discriminate those patients with active TB. Moreover, taking advantage of the photonic sensor design, which allows the simultaneous detection of six different biomarkers, we initiated the proof-of-concept of the POC platform for a TB biomarker panel detection using Dip-Pen Nanolithography for each corresponding bioreceptor deposition. Our results, if validated with larger clinical studies, could have important diagnostic implications taking into account the advantages added by our POC biosensor in comparison with the methods recommended by the World Health Organisation.Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologi

Development of a low-cost point-of-care test for Tuberculosis detection

Resumen del póster presentado al VIII International Congress on Analytical Nanoscience and Nanotechnology, celebrado en Barcelona (España) del 3 al 5 de julio de 2017.-- et al.Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a re-emerging disease impacting global health and affecting around 10.4 million people annually, with 1.8 million deaths. Registered incidence rates are low due to the lack of rapid and accurate diagnostic tools, especially in low-resource countries, posing serious hurdles for TB control. Since several years ago, a major effort is directed to develop a point-of-care (PoC) platform to facilitate the prompt and reliable TB diagnostics at low cost. In the frame of the European FP7 Pocket project, we have developed a novel PoC platform for the non-invasive technique detection of TB in human urine, accomplishing the above requirements. The new tuberculosis PoC is based on the combination of highly sensitive Mach-Zehnder integrated interferometers with an on-chip spectral filter, combined with a polymer microfluidic cartridge (disposable part), a SLED light source, a CCD camera for read-out and a graphical user interface for data processing. All the elements are incorporated in a PoC platform. The TB detection is achieved directly in the urine samples by an immunoassay employing high quality and selective antibodies against M. tuberculosis cell wall lipopolysaccharide lipoarabinomannan (LAM) and Ag85 complex. A preliminary Limit of Detection (LoD) of 956 pg/mL and 8.496 ng/mL for LAM and Ag85, respectively, have been achieved.European FP7 Pocket project.Peer reviewe

Low-cost point-of-care biosensor device for on-site tuberculosis diagnosis in developing countries

Resumen del póster presentado al 4th Scientific Meeting of BNC-b Students (JPhD), celebrado en Bellatera (España) del 6 al 7 de junio de 2019.Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, is considered the leading global cause of death from a single infectious agent. Registered incidence rates are scarce, especially in low-resource countries, due to the disadvantages of current diagnostic methods, which are slow, expensive, insufficiently accurate, time consuming, not portable or require trained technicians. For that reason, since several years ago, a major effort is directed to develop a low-cost point-of-care (PoC) diagnostics platform able to deliver a prompt response in order to reduce TB deaths. We have fully developed a novel PoC biosensor platform for fast TB detection in the frame of the European FP7 Pocket Project. The new PoC biosensor allows the detection of active TB directly in human urine, taking advantage of the high sensitivity offered by the evanescent wave optical sensors employed. The photonic sensor is based on a highly sensitive Mach-Zehnder Interferometer transducer with an on-chip spectral filter and is incorporated in a disposable microfluidic cartridge. The required elements for light coupling and optical readout are integrated in a prototype instrument, which allows real-time monitoring and data processing. To detect active M. tuberculosis we focus in several biomarkers present in the urine of TB patients, including lipoarabinomannan (LAM), early secretory antigenic target (ESAT-6), culture filtrate antigen (CFP-10) and immunogenic protein (MPT64), respectively. First results have been achieved for LAM biomarker, a lipopolysaccharide found in the mycobacterium cell wall. For the detection, the sensor chip surface was functionalized with high-quality and selective monoclonal antibodies against LAM. After the optimization of several parameters, a limit of detection of 475 pg/mL was achieved using a direct immunoassay. The analysis was performed in undiluted urine in less than 15 minutes. In addition, the results were validated by using 20 clinical samples from TB patients from Tanzania and healthy donors, showing an excellent correlation between the results from the PoC biosensor and those obtained with standard techniques. After these promising results for one biomarker and in order to take the advantage of the PoC design for the simultaneous detection of a panel of six different biomarkers in the same patient’s sample, all the optimizations for the evaluation of the other three biomarkers, mentioned above, is in progress.Peer reviewe

Label-free and real-time detection of tuberculosis in human urine samples using a nanophotonic point-of-care platform

Tuberculosis (TB) is the leading global cause of death from a single infectious agent. Registered incidence rates are low, especially in low-resource countries with weak health systems, due to the disadvantages of current diagnostic techniques. A major effort is directed to develop a point-of-care (POC) platform to reduce TB deaths with a prompt and reliable low-cost technique. In the frame of the European POCKET Project, a novel POC platform for the direct and noninvasive detection of TB in human urine was developed. The photonic sensor chip is integrated in a disposable cartridge and is based on a highly sensitive Mach-Zehnder Interferometer (MZI) transducer combined with an on-chip spectral filter. The required elements for the readout are integrated in an instrument prototype, which allows real-time monitoring and data processing. In this work, the novel POC platform has been employed for the direct detection of lipoarabinomannan (LAM), a lipopolysaccharide found in the mycobacterium cell wall. After the optimization of several parameters, a limit of detection of 475 pg/mL (27.14 pM) was achieved using a direct immunoassay in undiluted human urine in less than 15 min. A final validation of the technique was performed using 20 clinical samples from TB patients and healthy donors, allowing the detection of TB in people regardless of HIV coinfection. The results show excellent correlation to those obtained with standard techniques. These promising results demonstrate the high sensitivity, specificity and applicability of our novel POC platform, which could be used during routine check-ups in developing countries

Coherent silicon photonic interferometric biosensor with an inexpensive laser source for sensitive label-free immunoassays

Over the past two decades, integrated photonic sensors have been of major interest to the optical biosensor community due to their capability to detect low concentrations of molecules with label-free operation. Among these, interferometric sensors can be read-out with simple, fixed-wavelength laser sources and offer excellent detection limits but can suffer from sensitivity fading when not tuned to their quadrature point. Recently, coherently detected sensors were demonstrated as an attractive alternative to overcome this limitation. Here we show, for the first time, to the best of our knowledge, that this coherent scheme provides sub-nanogram per milliliter limits of detection in C-reactive protein immunoassays and that quasi-balanced optical arm lengths enable operation with inexpensive Fabry–Perot-type lasers sources at telecom wavelengths.Horizon 2020 Framework Programme (EuroNanoMed 3-H2020 DrNanoDAII); Ministerio de Economía y Competitividad (2019/PCI 2019-2, CTQ2017-86994-R, SEV-2017-0706); Junta de Andalucía, Proyectos Excelencia-Retos (P18-RT-1453, P18-RT-793); Fondos Europeos de Desarrollo Regional (CTQ2016-75870-P, PID2019-104293GB-I00, RD16/0006/0012, TEC2016-80718-R, UMA18-FEDERJA-007, UMA18-FEDERJA-219); H2020 Marie Skłodowska-Curie Actions (713721).Peer reviewe

Rapid and direct quantification of the SARS-CoV-2 virus with an ultrasensitive nanobody-based photonic nanosensor

Altres ajuts: this work has been funded by the European Commission - NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global - Iniciativa Estratégica de Diagnóstico). The ICN2 is funded by the CERCA Program/Generalitat de Catalunya. Work in CNB-CSIC was funded by the Spanish Ministry of Science and Innovation (MICIN) and the Spanish Research Council (CSIC) under grants PIE-RD-COVID 19 (No 202020E079) and PTI Salud Global REC_EU (No SGL 2103051, NextGenerationEU). Work at I2SysBio was funded by Ayudas de concesión directa a soluciones científico-innovadoras directamente relacionadas con la lucha contra la Covid-19 grant from the Generalitat Valenciana, Spanish National Research Council grant CSIC-COV19-082/104, Fondo Supera Covid-19 grant BlockAce, and European Commission - NextGenerationEU to Ron Geller.The coronavirus (CoV) disease 2019 (COVID-19) is expected to become endemic in the coming years, meaning that the worldwide society shall be prepared to routinely manage the highly contagious respiratory SARS-CoV-2. A rapid and early diagnosis of the SARS-CoV-2 infection is crucial for controlling the spread of the disease, interrupting the transmission chain, and providing timely medical attention to patients. We introduce an innovative nanophotonic biosensor for the quantitative detection of viral particles in less than 20 minutes total assay time. The nanosensor, based on the bimodal waveguide (BiMW) interferometric technology, has been functionalized with novel bioengineered nanobodies (Nb) targeting the SARS-CoV-2 receptor-binding domain (RBD). Our approach relies on the direct capture of the viral particles, and the optimized methodology allows the detection of the SARS-CoV-2 virus with outstanding sensitivity, below 200 TCID50 per mL, being able to provide accurate viral load determination within a broad dynamic range (102-106 TCID50 per mL). Both the nanobodies and the sensor nanotechnology can be produced at a large scale with highly-efficient cost-effective procedures, and they are being integrated into a user-friendly point-of-care device for multiplexed and decentralized operation. The implementation of this unique biosensor in primary care assistance, hospitals, pharmacies, or private laboratories could greatly aid in the relief and descongestion of the sanitary systems and the clinical and social management of COVID-19