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This article belongs to the Collection Diagnostic Sensors., Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria., This research was supported by CERCA program/Generalitat de Catalunya. ICN2 is supported by the Severo Ochoa Program from Spanish MINECO (Grant No. SEV-2017-0706). The VHIR-HUVH is supported by Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0003), co-financed by the European Development Regional Fund “A way to achieve Europe”. Authors acknowledge the support of project ASPIRE (funded by CIBER-BBN). J. Maldonado acknowledges the Mexican National Council for Science and Technology (CONACYT). This work has made use of the Biodeposition and Biodetection Unit from ICTS NANBIOSIS partially supported by MICINN/FEDER (FICTS- 1420- 27).

Integrated photonic biosensors have demonstrated low bulk detection limits down to 1e−7 refractive index units. Nevertheless, most rely on expensive optical sources, such as DFB lasers. Here, we experimentally demonstrate that with adequate sensor design comparable detection limits are achievable with a low-cost Fabry-Perot laser. European Union’s Horizon 2020, Marie Skłodowska-Curie grant agreement No 713721; Ministerio de Econom ́ıa y Competitividad (FEDER), Proyecto TEC2016-80718-R; Programa Operativo FEDER Andalucía 2014-2020 (UMA18-FEDERJA-219); Severo Ochoa program from Spanish MINECO, Grant No. SEV-2017-0706, Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Silicon photonics enables sensitive and label-free optical biosensors for the detection of chemical and biological substances. Different sensing architectures have been used to improve the limit of detection and increase the dynamic range response. Here, we show experimental limit of detection at state-of-the-art level using silicon nitride integrated Mach-Zehnder interferometers with coherent read-out. These preliminary results are concordant with theoretical results, showing that the proposed approach enables the use of simple read-out equipment using low-cost laser sources.

Nosocomial infections are a major concern at the worldwide level. Early and accurate identification of nosocomial pathogens is crucial to provide timely and adequate treatment. A prompt response also prevents the progression of the infection to life-threatening conditions, such as septicemia or generalized bloodstream infection. We have implemented two highly sensitive methodologies using an ultrasensitive photonic biosensor based on a bimodal waveguide interferometer (BiMW) for the fast detection of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), two of the most prevalent bacteria associated with nosocomial infections. For that, we have developed a biofunctionalization strategy based on the use of a PEGylated silane (silane-PEG-COOH) which provides a highly resistant and bacteria-repelling surface, which is crucial to specifically detect each bacterium. Two different biosensor assays have been set under standard buffer conditions: one based on a specific direct immunoassay employing polyclonal antibodies for the detection of P. aeruginosa and another one employing aptamers for the direct detection of MRSA. The biosensor immunoassay for P. aeruginosa is fast (it only takes 12 min) and specific and has experimentally detected concentrations down to 800 cfu mL-1 (cfu: colony forming unit). The second one relies on the use of an aptamer that specifically detects penicillin-binding protein 2a (PBP2a), a protein only expressed in the MRSA mutant, providing a photonic biosensor with the ability to identify the resistant pathogen MRSA and differentiate it from methicillin-susceptible S. aureus (MSSA). Direct, label-free, and selective detection of whole MRSA bacteria has been achieved, making possible the direct detection of also 800 cfu mL-1. According to the signal-to-noise (S/N) ratio of the device, a theoretical limit of detection (LOD) of around 49 and 29 cfu mL-1 was estimated for P. aeruginosa and MRSA, respectively. Both results obtained under standard conditions reveal the great potential this interferometric biosensor device has as a versatile and specific tool for bacterial detection and quantification, providing a rapid method for the identification of nosocomial pathogens within the clinical requirements of sensitivity for the diagnosis of infections.

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).

There is still the need for a compact and cost-effective solution for efficient light in-coupling in integrated waveguides employed in photonic biosensors, especially when these waveguides are of submicron dimensions and operate at visible wavelengths. The employment of a vertically stacked taper with a larger input area is proposed to meet this need. The design of the taper is divided into two stages: in the first stage, light is guided downwards by two vertically stacked tapers; in the second stage, an inverted taper directly confines the light inside the waveguide. The design parameters are optimized using commercial software, obtaining a total theoretical light coupling efficiency of 72.25%. The taper is manufactured using SU-8 polymer as the main material, employing standard photolithography techniques at wafer level. After characterization, the results show the practicality of the taper when coupling light from macrometric sources to nanometric waveguides, obtaining an experimental coupling efficiency of 55%. With this vertical taper, a compact, easy-to-couple and cost-effective solution is achieved for waveguide-based biosensors operating at visible wavelengths, opening the way for a truly portable point-of-care biosensor for low-cost and label-free diagnostics., This research was funded by Acción Estratégica Economía y Sociedad Digital (AEESD)-Ministerio de Industria, Energía y Turismo (MINETUR). The ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa programme of the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, grant SEV-2013-0295). This work has made use of the Spanish ICTS Network MICRONANOFABS partially supported by MEINCOM. D. Grajales acknowledges CONACYT (Mexican Council of Science and Technology) CVU 397275, contract SICOB 218103.

An interferometric nanobiosensor for the specific and label-free detection of the pollutant Irgarol 1051 directly in seawater has been settled. Due to the low molecular weight of Irgarol pollutant and its expected low concentration in seawater, the sensor is based on a competitive inhibition immunoassay. Parameters as surface biofunctionalization, concentration of the selective antibody and regeneration conditions have been carefully evaluated. The optimized immunosensor shows a limit of detection of only 3 ng/L, well below the 16 ng/L set by the EU as the maximum allowable concentration in seawater. It can properly operate during 30 assay-regeneration cycles using the same sensor biosurface and with a time-to-result of only 20 min for each cycle. Moreover, the interferometric nanosensor is able to directly detect low concentrations of Irgarol 1051 in seawater without requiring sample pre-treatments and without showing any background signal due to sea matrix effect.

A primary limitation preventing practical implementation of photonic biosensors within point-of-care platforms is their integration with fluidic automation subsystems. For most diagnostic applications, photonic biosensors require complex fluid handling protocols; this is especially prominent in the case of competitive immunoassays, commonly used for detection of low-concentration, low-molecular weight biomarkers. For this reason, complex automated microfluidic systems are needed to realise the full point-of-care potential of photonic biosensors. To fulfil this requirement, we propose an on-chip valve-based microfluidic automation module, capable of automating such complex fluid handling. This module is realised through application of a PDMS injection moulding fabrication technique, recently described in our previous work, which enables practical fabrication of normally closed pneumatically actuated elastomeric valves. In this work, these valves are configured to achieve multiplexed reagent addressing for an on-chip diaphragm pump, providing the sample and reagent processing capabilities required for automation of cyclic competitive immunoassays. Application of this technique simplifies fabrication and introduces the potential for mass production, bringing point-of-care integration of complex automated microfluidics into the realm of practicality. This module is integrated with a highly sensitive, label-free bimodal waveguide photonic biosensor, and is demonstrated in the context of a proof-of-concept biosensing assay, detecting the low-molecular weight antibiotic tetracycline.

With the aim to prevent the oceans ecosystems degradation, there is an urgent need to develop portable sensing tools able to operate directly in the environment, avoiding the transportation of the ocean samples to analytical laboratories. To achieve this long-term objective, we describe here the work carried out to develop and characterize a multiplexed photonic immunosensor for the direct analysis of toxic chemical targets in marine samples. We have employed immunosensors based on photonic Bimodal Waveguide (BiMW) interferometric devices fabricated in silicon technologies combined with specific receptors and antibodies for the targeted chemical targets. Several procedures for the functionalization of the S₃iN₄ sensor surfaces have been evaluated based on wet silanization methods and further covalent receptor immobilization. The developed immunosensors, based on competitive inhibition assays, show LODs at μg/L or ng/L levels, depending on the analyzed chemical target.

The field of environmental monitoring has experienced a substantial progress in the last years but still the on-site control of contaminants is an elusive problem. In addition, the growing number of pollutant sources is accompanied by an increasing need of having efficient early warning systems. Several years ago biosensor devices emerged as promising environmental monitoring tools, but their level of miniaturization and their fully operation outside the laboratory prevented their use on-site. In the last period, nanophotonic biosensors based on evanescent sensing have emerged as an outstanding choice for portable point-of-care diagnosis thanks to their capability, among others, of miniaturization, multiplexing, label-free detection and integration in lab-on-chip platforms. This review covers the most relevant nanophotonic biosensors which have been proposed (including interferometric waveguides, grating-couplers, microcavity resonators, photonic crystals and localized surface plasmon resonance sensors) and their recent application for environmental surveillance.

The bimodal waveguide (BiMW) sensor is a novel common path interferometric transducer based on the evanescent field detection principle, which in combination with a bio-recognition element allows the direct detection of biomolecular interactions in a label-free scheme. Due to its inherent high sensitivity it has great potential to become a powerful analytical tool for monitoring substances of interest in areas such as environmental control, medical diagnostics and food safety, among others. The BiMW sensor is fabricated using standard silicon-based technology allowing cost-effective production, and meeting the requirements of portability and disposability necessary for implementation in a point-of-care (POC) setting.In this chapter we describe the design and fabrication of the BiMW transducer, as well as its application for bio-sensing purposes. We show as an example the biosensor capabilities two different applications: (1) the immunodetection of Irgarol 1051 biocide useful in the environmental field, and (2) the detection of human growth hormone as used in clinical diagnostics. The detection is performed in real time by monitoring changes in the intensity pattern of light exiting the BiMW transducer resulting from antigen-antibody interactions on the surface of the sensor.

Carbon nanotubes (CNTs) have been widely used in many fields of chemical analysis to achieve more sensitive detection systems. In this work, we performed fundamental studies on grown or bottom-up fabricated MWCNTs (both non-oriented and oriented configurations), showing how variables like orientation, density, underlayer deposition, or synthesis time strongly determine their behavior (physical, electrochemical and analytical) as transducers. The electrochemical performance of these surfaces was demonstrated by cyclic voltammetry and chronoamperometry of dopamine (DA) solutions in 0.1 M H2SO4. The carbon nanotubes surfaces pre-treated with 1 M HNO3 lead to increased signals, sensitivity and enhanced limits of detection (LOD). The grown working electrodes (WE) were reproducible and stable over the time. The peak variations gave RSD values of 8%, 4% and 3% for high-density spaghetti-like and ITO or Al underlayered forest-like MWCNTs grown for 30 min, respectively. This study highlighted the importance of controlling the synthesis variables to achieve better analytical parameters.

We present recent results on the realisation and demonstration of an integrated optofluidic lab-on-a-chip measurement system. The system consists of an integrated on-chip automated microfluidic fluid handling subsystem, coupled with bimodal nano-interferometer waveguide technology, and is applied in the context of detection of antibiotics in seawater. The bimodal waveguide (BMWG) is a highly sensitive label-free biosensor. Integration of complex microfluidic systems with bimodal waveguide technology enables on-chip sample handling and fluid processing capabilities and allows for significant automation of experimental processes. The on-chip fluid-handling subsystem is realised through the integration of pneumatically actuated elastomer pumps and valves, enabling high temporal resolution sample and reagent delivery and facilitating multiplexed detection processes.

We review the most important achievements published in the last five years in the field of silicon-based optical biosensors. We focus specially on label-free optical biosensors and their implementation into lab-on-a-chip platforms, with an emphasis on developments demonstrating the capability of the devices for real bioanalytical applications. We report on novel transducers and materials, improvements of existing transducers, new and improved biofunctionalization procedures as well as the prospects for near future commercialization of these technologies., The nanoB2A is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (2014 SGR 624). ICN2 is the recipient of Grant SEV-2013-0295 from the “Severo Ochoa Centers of Excellence” Program of Spanish MINECO. Laura M. Lechuga acknowledges the Brazilian Program Science without Frontiers for International Cooperation—MEC/MCTI/CAPES/CNPq/FAPs, project No. 313690/2013-8. Daniel Grajales García acknowledges to the Mexican Council of Science and Technology (CONACYT) for the scholarship 218103-CVU 397275. Jhonattan C. Ramirez acknowledges to the National Counsel of Technological and Scientific Development (CNPq) Brazil., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

La investigación llevada a cabo durante la realización de esta tesis doctoral tiene su origen en la colaboración científica que se inició en 2004 entre los Laboratorio de Óptica Integrada (LOI) y de Inmunoelectroanalisis (LI).El LOI y el LI pertenecen a los departamentos de Física Aplicada y de Física y Química Analítica de la Universidad de Oviedo, respectivamente. Esta colaboración comenzó cuando el LI sugirió fabricación de microcanales grabados en sustratos vítreos, utilizando el sistema de litografía láser, para su aplicación en microchips de electroforesis capilar (MEC). La calidad de estos microcanales juega un papel esencial en la eficiencia de los biosensores basados en MEC. Esta colaboración se extendió posteriormente a otros aspectos del desarrollo y optimización de los MECs. Además, también permitió al LOI adquirir conocimientos y experiencia en el mundo de los biosensores ópticos, que requieren la combinación de tres disciplinas diferentes: óptica integrada, microfluídica y sistemas electroquímicos. El trabajo científico presentado en esta tesis ha perseguido el objetivo principal de establecer y desarrollar nuevos protocolos y procedimientos orientados al diseño, fabricación y optimización de técnicas de micrograbado para su aplicación en óptica integrada y en microfluídica analítica. A lo largo de la investigación, este objetivo principal a dado lugar a los siguientes objetivos específicos: a) Determinación del índice de refracción y el espesor de microcapas poliméricas. b)Fabricación de microcanales en el polímero PDMS. c)Diseño y fabricación de nuevas guías ópticas canal poliméricos embebidas en vidrio (GOCPEV). Propuesta de un biosensor óptico por interferometría MachW Zehnder (IMZ) fabricado con este tipo de guías ópticas y con la incorporación de microcanales poliméricos. d) Fabricación de microelectrodos de carbono para detección electroquímica para su aplicación en MEC.

Miniaturization is a well-established trend in the development of analytical devices, due to the important advantages it offers. Detection for this type of devices has to be in accordance and therefore easy integration and the employment of simple instrumentation are common requirements. These are fulfilled very well by the electrochemical detection (ED) and therefore, it is gaining relevance in many of the applications where miniaturized approaches are required. However, the use of very small sample volumes (i.e. in the case of microchip electrophoresis, ME) implies the use of very sensitive techniques. Even when this is the case of amperometry, sensitivity improvement is still needed. With this aim, nanotechnological approaches can be envisaged. Carbon nanotubes (CNTs) are one of the nanostructures that, from their discovery, remain generating relevant applications due to the possibilities and advantages they offer. Due to their conductive properties, they have been widely used as electrode modifiers. In this work, CNTs are directly grown on a glass surface and evaluated for electroanalytical purposes. However, simply by varying the procedure slightly, materials with very different properties can be obtained. For example, different configurations, vertically and non-vertically aligned CNTs, provide different analytical characteristics. Therefore, a careful choice of the nanomaterial has to be made in order to obtain the best performance of the devices.

Este trabajo ha sido realizado bajo la ayuda de los proyectos TEC2008-06844-CO2-01 del MEC, IB09-011C1 y una beca predoctoral “Severo Ochoa” de la FICYT.