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A plug and play PHOTOnics based bioSENSing platform for salmon pathogen detection

Periodic Reporting for period 6 - PHOTO-SENS (A plug and play PHOTOnics based bioSENSing platform for salmon pathogen detection)

Reporting period: 2023-06-01 to 2023-11-30

Zero hunger is the leading Sustainable Development Goal of the UN’s 2030 Agenda to eliminate hunger and malnutrition and ensure access to safe, healthful and sufficient food. While aquaculture is one of the most efficient food sources, it relies on controlled aquatic environments that must be kept in healthy conditions. As a consequence, the fish living environment must be monitored continuously with advanced biosensing.

PHOTO-SENS will connect pioneering biosensor technology and scaling-up procedures with aquaculture expertise to establish an aquaculture pathogen detection hub and a working prototype system for monitoring salmon pathogens and addressing key parameter selections such as sex discrimination in selected species.

This project builds upon the BIOCDx project (ID: 732309) which signifcantly advanced the state of the art and delivered a proof of principle prototype. However, due to lack of scalability, the overall costs of the biosensor remained very high. Thus, the principal aim of PHOTO–SENS is to reduce the costs by investigating the scalable production of this technology, and to validate the prototype with an end–user in the aquaculture market.
The PHOTO-SENS system will consist of a desktop reader, which can be used in combination with disposable microfluidic cartridges containing photonic biochips. To reduce the manufacturing costs of the cartridge, the price of the chip has to be reduced and the manufacturing and assembly process has to be improved.

Photonic chips are made out of silicon wafers by advanced cleanroom processes. The price of a photonic chip is mainly determined by the number of chips that fit on a wafer. Therefore, a biochip with a smaller footprint has been designed and fabricated. Optical components have been integrated on these chips using chip-level processes, and a read-out system for testing the new chips has been prepared. Currently, the PHOTO-SENS chip are being tested. A second batch of (dummy) chips has been fabricated, with which the validation of the wafer-level integration processes has been finalized.

After fabrication in the cleanroom, the chip has to undergo several processes to obtain a useful photonic biochip. These include integration of light source and detector on the chip, application of chemical and biological coatings, and cutting the wafer into single chips. Processes have been developed for wafer-level integration of components, application of chemical coatings, biofunctionalization, and singulation of the wafer into individual chips. Together, these processes constitute a scalable process flow for the manufacturing of integrated photonic biochips. Unfortunately, VCSEL failure was observed when testing the assembled cartridges with the hybrid PICs. Despite major efforts to identify the root cause and to find a solution, these issues are not yet satisfactorily solved. It is expected that a sufficient number of functional PICs will be available for validation tests of the cartridge and instrument prototypes within the project. However, the TRL of the hybrid PIC is not yet sufficiently advanced to be commercialized shortly after finalization of the project. The exploitation outlook has been adjusted accordingly.

The next step is the assembly of the photonic biochip in the microfluidic cartridge. Sample liquid will flow over the chip, so obviously a leak-tight interface is required. At the same time, electronic access to the chip is needed for actuation and read-out. These requirements, in combination with the reduced chip size to reduce cost, make chip-cartridge integration a challenging task. A basic cartridge has been designed and fabricated, with which the concept of chip-cartridge integration has been validated. Leak tightness, and optical and electrical functionality of the basic cartridge has been confirmed. Next, a microfluidic cartridge for DNA assays, including on-cartridge liquid storage, has been designed, with particular emphasis on the interfaces between the cartridge and the read-out instrument. The assay cartridge is currently being produced and assembled. Validation tests will be performed in Q1 2024.

The PHOTO-SENS prototype system will be used to monitor the presence of pathogenic microorganisms in salmon cultivation systems by detecting specific DNA biomarkers. Several relevant pathogens have been selected, and DNA biomarkers for their detection have been identified. qPCR assays have been developed to serve as a reference and benchmark for the results obtained with the PHOTO-SENS system. An assay to detect the biomarker for A. sal. has been succesfully developed on the photonic biochip. Direct detection of single stranded DNA at elevated temperature (required for high sensitivity) proved difficult. Therefore, an innovative alternative assay format has been developed based on the detection of double stranded DNA. As a second potential application for the PHOTO-SENS platform, a DNA assay for sex determination of sturgeon has been developed and optimized.

The requirements for the PHOTO-SENS prototype system, comprising the photonic biochip, the microfluidic cartridge, the desktop reader and software, have been finalized. The various interfaces between the microfluidic cartridge and the instrument have been defined, and technology concepts for cartridge handling, blister emptying, liquid actuation, and sample introduction have been developed. These have been translated into an integrated instrument design. The instrument has been realized, and will be tested in combination with the assay cartridge in Q1 2024.
Technically, PHOTO-SENS will go beyond proof-of-principle (shown in the previous project BioCDx) and demonstrate on a prototype level manufacturability of a device based on PIC based biosensors and microfluidics. Key requirements to achieve this are:
- reduction of the PIC footprint (expected result of the project)
- scalable manufacturing process of the bio-functional PIC (expected result of the project)
- design for manufacturing of the functional microfluidic cartridge including the integration of the PIC (expected result of the project)

From an application point of view, the PHOTO-SENS prototype aims to advance the state of the art by enabling on-site pathogen testing in aquaculture facilities. This is currently not standard procedure due to the lack of test equipment that meets the requirements in terms of costs, performance and time. PHOTO-SENS aims to deliver relevant results to users in aquaculture at an acceptable price and within an acceptable time (expected result of the project). This will result in improved fish health aquaculture sustainability and increased efficiency and yield of fish farms (expected result beyond the project).

In a broader socio-economic perspective, PHOTO-SENS contributes to the solutions required to feed the growing world population with sufficient nutritious and safe food. Owing to its high conversion efficiency from feed to protein, aquaculture is a key sector in the sustainable food production strategy for the future (expected result beyond the project).
PHOTO-SENS assay cartridge with hybrid PIC