Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases

[EN] In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody-antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.This work was funded by the EU-2020 program under grant agreement Nº 771649-SWINOSTICS project.Gómez-Gómez, MI.; Sánchez, C.; Peransi, S.; Zurita, D.; Bellieres, L.; Recuero, S.; Rodrigo, M.... (2022). Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases. Sensors. 22(3):1-14. https://doi.org/10.3390/s2203070811422

Integration of Microfluidics, Photonic Integrated Circuits and Data Acquisition and Analysis Methods in a Single Platform for the Detection of Swine Viral Diseases

[EN] Simple Summary: The control of several swine viral diseases relies mainly on evidence-based prevention protocols due to the lack of effective treatments or vaccines. To design these protocols, laboratory investigation of viral infections is critical to confirm their occurrence and determine their epizootiology. However, laboratory confirmation of certain swine viral diseases is a time-consuming and labor-intensive process, requiring scientific personnel with relevant expertise. Point-of-Care (POC) diagnostics are tests and devices that provide clinically relevant information on-site, facilitating decision-makers to swiftly take countermeasures for disease control. In the present study, novel photonic biosensors were integrated into a single, automated POC device that can record and analyze changes in the sensors' refractive index, allowing the detection of Porcine Parvovirus (PPV) and Porcine Circovirus 2 (PCV-2) in oral fluids within 75 min. The objective of this work was to validate this device using reference and field samples (oral fluids). The system was able to detect PPV and PCV-2 in oral fluid samples satisfactorily. The device can be directly deployed in farms for the fast diagnosis of these diseases, contributing to farm biosecurity.Viral diseases challenge the health and welfare of pigs and undermine the sustainability of swine farms. Their efficient control requires early and reliable diagnosis, highlighting the importance of Point of Care (POC) diagnostics in veterinary practice. The objective of this study was to validate a novel POC system that utilizes Photonic Integrated Circuits (PICs) and microfluidics to detect swine viral pathogens using oral fluids and Porcine Parvovirus (PPV) and Porcine Circovirus 2 (PCV-2) as proofs of concept. The sensitivity and specificity of the device were calculated for both viruses, and Receiver Operating Characteristic (ROC) curves were drawn. PPV had an Area Under Curve (AUC) value of 0.820 (95% CI: 0.760 to 0.880, p < 0.0001), and its optimal efficiency threshold of detection shifts was equal to 4.5 pm (68.6% sensitivity, 77.1% specificity and Limit of Detection (LOD) value 10(6) viral copies/mL). PCV-2 had an AUC value of 0.742 (95% CI: 0.670 to 0.815, p < 0.0001) and an optimal efficiency threshold of shifts equal to 6.5 pm (69.5% sensitivity, 70.3% specificity and LOD 3.3 x 10(5) copies/mL). In this work, it was proven that PICs can be exploited for the detection of swine viral diseases. The novel device can be directly deployed on farms as a POC diagnostics tool.This research was funded by E.U.'s H2020 SWINOSTICS project under the grant agreement ID 771649.Manessis, G.; Mourouzis, C.; Griol Barres, A.; Zurita-Herranz, D.; Peransi, S.; Sánchez, C.; Giusti, A.... (2021). Integration of Microfluidics, Photonic Integrated Circuits and Data Acquisition and Analysis Methods in a Single Platform for the Detection of Swine Viral Diseases. Animals. 11(11):1-18. https://doi.org/10.3390/ani11113193118111

Point-of-Care and Label-Free Detection of Porcine Reproductive and Respiratory Syndrome and Swine Influenza Viruses Using a Microfluidic Device with Photonic Integrated Circuits

[EN] Swine viral diseases challenge the sector's sustainability by affecting productivity and the health and welfare of the animals. The lack of antiviral drugs and/or effective vaccines renders early and reliable diagnosis the basis of viral disease management, underlining the importance of point-of-care (POC) diagnostics. A novel POC diagnostic device utilizing photonic integrated circuits (PICs), microfluidics, and information and communication technologies for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A (SIV) was validated using spiked and clinical oral fluid samples. Metrics including sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device. For PRRSV, the device achieved a sensitivity of 83.5%, specificity of 77.8%, and DOR values of 17.66, whereas the values for SIV were 81.8%, 82.2%, and 20.81, respectively. The POC device and PICs can be used for the detection of PRRSV and SIV in the field, paving the way for the introduction of novel technologies in the field of animal POC diagnostics to further optimize livestock biosecurity.This research was funded by the EU's H2020 SWINOSTICS project under the grant agreement ID 771649.Manessis, G.; Frant, M.; Wozniakowski, G.; Nannucci, L.; Bennedetti, M.; Denes, L.; Balka, G.... (2022). Point-of-Care and Label-Free Detection of Porcine Reproductive and Respiratory Syndrome and Swine Influenza Viruses Using a Microfluidic Device with Photonic Integrated Circuits. Viruses. 14(5):1-21. https://doi.org/10.3390/v1405098812114

How Nanophotonic Label-Free Biosensors Can Contribute to Rapid and Massive Diagnostics of Respiratory Virus Infections : COVID-19 Case

Altres ajuts: Generalitat de Catalunya /CERCAThis ACS article is provided to You under the terms of this Standard ACS AuthorChoice License. License: https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlThe global sanitary crisis caused by the emergence of the respiratory virus SARS-CoV-2 and the COVID-19 outbreak has revealed the urgent need for rapid, accurate, and affordable diagnostic tests to broadly and massively monitor the population in order to properly manage and control the spread of the pandemic. Current diagnostic techniques essentially rely on polymerase chain reaction (PCR) tests, which provide the required sensitivity and specificity. However, its relatively long time-to-result, including sample transport to a specialized laboratory, delays massive detection. Rapid lateral flow tests (both antigen and serological tests) are a remarkable alternative for rapid point-of-care diagnostics, but they exhibit critical limitations as they do not always achieve the required sensitivity for reliable diagnostics and surveillance. Next-generation diagnostic tools capable of overcoming all the above limitations are in demand, and optical biosensors are an excellent option to surpass such critical issues. Label-free nanophotonic biosensors offer high sensitivity and operational robustness with an enormous potential for integration in compact autonomous devices to be delivered out-of-the-lab at the point-of-care (POC). Taking the current COVID-19 pandemic as a critical case scenario, we provide an overview of the diagnostic techniques for respiratory viruses and analyze how nanophotonic biosensors can contribute to improving such diagnostics. We review the ongoing published work using this biosensor technology for intact virus detection, nucleic acid detection or serological tests, and the key factors for bringing nanophotonic POC biosensors to accurate and effective COVID-19 diagnosis on the short term

From Lab-on-chip to Lab-in-App: Challenges towards silicon photonic biosensors product developments

This work presents and evaluates different approaches of integrated optical sensors based on photonic integrated circuit (PIC) technologies for refractive index sensing. Bottlenecks in the fabrication flow towards an applicable system are discussed that hinder a cost-effective mass-production for disposable sensor chips. As sensor device, a waveguide coupled micro-ring based approach is chosen which is manufactured in an 8” wafer level process. We will show that the co-integration with a reproducible, scalable and low-cost microfluidic interface is the main challenge which needs to be overcome for future application of silicon technology based PIC sensor chips

Developments in Transduction, Connectivity and AI/Machine Learning for Point-of-Care Testing

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted

Surface Plasmon Resonance for Biosensing

The rise of photonics technologies has driven an extremely fast evolution in biosensing applications. Such rapid progress has created a gap of understanding and insight capability in the general public about advanced sensing systems that have been made progressively available by these new technologies. Thus, there is currently a clear need for moving the meaning of some keywords, such as plasmonic, into the daily vocabulary of a general audience with a reasonable degree of education. The selection of the scientific works reported in this book is carefully balanced between reviews and research papers and has the purpose of presenting a set of applications and case studies sufficiently broad enough to enlighten the reader attention toward the great potential of plasmonic biosensing and the great impact that can be expected in the near future for supporting disease screening and stratification

Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens: An Overview

With the advent of molecular technology, several isothermal techniques for rapid detection of zoonotic pathogens have been developed. Among them, recombinase polymerase amplification (RPA) is becoming an important technology for rapid, sensitive, and economical detection of zoonotic pathogens. RPA technology has the advantage of being able to be implemented in field settings, because the method requires minimal sample preparation and is performed at a constant low temperature (37–42°C). RPA is rapidly becoming a promising tool for the rapid detection, prevention, and control of zoonotic diseases. This article discusses the principles of RPA technology and its derivatives, including RPA coupled with lateral flow testing (RPA-LF), real-time fluorescence RPA, electrochemical RPA, and flocculation RPA, and their applications in the detection of zoonotic pathogens

Developing integrated optical structures, with special respect to applications in medical diagnostics

In my dissertation, I described two label-free optical biosensors based on integrated optical (IO) structures for the sensitive, rapid detection of pathogens - bacterial cells, viral proteins - from fluid samples, which can serve as a basis for rapid clinical tests. These types of devices provide a specific, cost-effective, user-friendly and portable way of detection with sufficient sensitivity by changing the optical signal. Thus, in practice, they could potentially be used as point-of-care (POC) or home rapid diagnostic tests, offering a promising alternative to traditional laboratory assays. Their realization is supported by their integration with microfluidic channels in a lab-on-a-chip (LOC) device, for handling small volumes of fluid. Based on these aspects, biosensors were designed as waveguides, integrated in a microfluidic channel on a glass substrate, performing evanescent-field sensing. The detection method is based on the fact that the light, propagating in the waveguide with total internal reflections, penetrates into the surrounding media at a limited extent, which is called the evanescent field. Material can enter this space and become bound to the surface, which can change the phase of the light, propagating in the structure, or even scatter it into the surrounding medium. These phenomena offer the possibility of specific detection of pathogens, adhering to the surface, pre-coated with a biological recognition element, such as an antibody. As a first application, an electro-optical biosensor was developed with an evanescent field-based detection concept, aiming at label-free, rapid, selective and sensitive detection of bacteria from body fluids. The usability of the measurement principle, based on the processing of light-scattering patterns, caused by evanescent waves, scattered on target cells, was demonstrated by quantitative detection of Escherichia coli bacterial cells from their suspensions. One of the keys to the applicability of biosensors is their sensitivity. To increase it in case of this device, I applied the phenomenon of dielectrophoresis using the polarizability of the target cells. It provides the possibility to selectively collect cells on the surface of electrodes placed close to the waveguide and then detect them based on the evanescent field. To test this, I wanted to sense bacteria in an artificial urine sample containing somatic cells, in this case endothelial cells, mimicking urine in an inflammatory state. By optimizing the parameters of the measurements, a rapid, sensitive bacterial detection of about 10 minutes was achieved. The detection limit of the biosensor was comparable to the characteristic pathogen concentration in body fluids. Furthermore, selective bacterial detection was also achieved from a fluid sample containing somatic cells, mimicking inflammatory urine. In my dissertation, a second application is also presented, in this case a miniature IO Mach-Zehnder interferometer-based biosensor was developed for the specific quantitative detection of viral proteins. Thanks to the interferometric measurement principle, a fast and accurate detection of target proteins can be achieved. With this device, the aim was to investigate the potential neuroinvasion of coronavirus (SARS-CoV-2) infection, from which point of view the pathological effects of viral surface spike proteins on the blood-brain barrier are of great importance in the case of severe symptoms. Furthermore, infection may also cause adverse effects in the intestinal tract. Thus, the specific aim of this application was to evaluate the ability of the S1 subunit of the coronavirus surface spike protein to cross the human in vitro blood-brain barrier and intestinal epithelial biological barrier system models using the biosensor. Experiments were designed to use the sensor for specific, quantitative detection of spike proteins, that may have been passed through permeability assays on biological barrier models prepared by our collaborators. To reach the specific sensing of the target protein, the waveguide surface of the interferometer’s measuring arm was functionalized with specific S1 protein antibody. To achieve optimal, stable measurement conditions, the operating point of the interferometer was adjusted thermo-optically. The results of the experiments with the biosensor were in agreement with the ones of the conventional immunological tests (ELISA) carried out in parallel. It was possible to determine that S1 protein could pass through the two types of barriers in different amounts. The findings of the experiments with the integrated optical Mach-Zehnder interferometer biosensor demonstrate that this detection approach can be used for similar medical diagnostic purposes, and thus can contribute to the investigation of the adverse effects of SARS-CoV-2 on the human body