Project Results M51

MOLOKO activities were conducted within 10 Work Packages (WPs). The strategy which guided the development of the project was the minimization of the number of feedback and loop actions among the WPs necessary to (i) realize the monolithic integration approach (ii) deploy the functional prototype in the milk production chain and iii) validate the functional prototype in real-setting environment and in the safety frameworks.

In brief the results of the project have been:

• Innovative miniaturised and monolithic-integrated optical biosensor utilising patented surface plasmonic resonance nanostructure.
• Plasmonic-based quantitative, self-calibrating and multiple measurements.
• Multiplexing detection of quality and safety parameters simultaneously on the same chip with 14 minutes long protocol of use.
• Microfluidic control, packaging and automation of analyser module suitable for milking parlours

WP1 started at month 1 and ended at Month 10, with the leadership of CNR.

The specifications of the single-device components were defined by SMEs and end-users according to a system-engineering viewpoint, comprising the final bio-optical sensor in order to meet the requests of the end-users in terms of sensitivity, limits of detection, measure time, usability. Thus, specifications for each component or module of MOLOKO sensor (optoelectronic devices, plasmonic grating, microfluidics) were provided by the technological partners in order to guarantee the best trade-off between the highest integrated architecture (miniaturization/portability) and the best implementation (reusability, stability and user-friendliness)

Also, the list of the analytes to be detected in relation to real applications scenarios was defined: quality vs safety detection, position along the value chain of milk (milking, collecting and processing), measure protocol.

WP2 started at month 4 and ended at Month 42, with the leadership of CNR.

WP2 was dedicated to the fabrication and characterization of the organic photonic module to be implemented in the MOLOKO biosensor. WP2 and WP3: were strongly correlated because they included the development of the optoplasmonic module, including the organic photonic module (array of highly integrated organic optoelectronic devices, OPDs onto OLETs), and the plasmonic grating as label-free transducer.

The first activities of WP2 were focused to the realization and optimization of the single components, the definition of the photonic chip layout and finalization of the integrated optoplasmonic chip. The final photonic module is composed of a series of monolithically integrated organic devices (OLEDs as the light sources, and OPDs collecting the modulated light) and is fabricated at FhG FEP. The photonic module was completed with the biofunctionalized nanoplasmonic grating on top of the encapsulation glass and constitutes the optoplasmonic module and characterized.

​Operation stability of the photonic module and the dependence of its characteristics on temperature​ has been evaluated. The organic photonic module is compatible with the functionalization block and the electronics​ and the operational characteristics are in line with the requirements.

WP3 started at month 3 and ended at month 36, with the leadership of Plasmore Srl.

The main objective of WP3 has been the development and the sequential functionalization of the nanoplasmonic grating, intended as sensitive and reproducible surface for multiplexing detection of milk specific target analytes. In this WP we completed the layout of fabrication and optical characterization of the nanoplasmonic grating, and we fabricated the optoplasmonic module. Subsequently, the integration of the components from WP2, WP4, WP5 with the nanoplasmonic grating allowed the realization of a MOLOKO manually controlled sensor prototype. The development of self-testing functionality in MOLOKO sensor had a delay due to the stop of lab activity at JRC (where Plasmore operates) between M27 and M30, as a consequence of the Covid19 emergency. Nevertheless, from M31 the lab activity restarted and thanks to a close collaboration among all the partners involved in WP3, all the planned deliverables and expected results could be completed and submitted. The milestones MS3 (“Determination of Dose-Response curves in controlled medium”) was achieved accordingly.

WP4 started at month 9 and ended at month 30, with the leadership of Fraunhofer.

The main objective of WP4 has been to develop the microfluidic module for the automated fluid handling. Final aim is to reduce manual handling by end-users through an automatic sampling and regeneration procedure. Interfacing technology for optoplasmonic module (OPM – OLED + OPD + nanoplasmonic grating) and microfluidic module has been developed. Within this task, Milkline re-planned the in-field activities foreseen in Task 4.5 due to the COVID-19 outbreak that reduced the availability of person-power and access to farm sites necessary for performing the in-field activity at that time. FhG (ENAS) (in close cooperation with CSEM for instrument integration and testing) worked to the design, realization, and testing of the actuator block, that includes all mechanical actuators necessary for the execution of the assay in the automated MOLOKO sensor.

WP5 started at month 11 and ended at month 51, with the leadership of CSEM. This WP has been focused on the development of the Electronics (driving and readout electronics) and the Software (component drivers, signal analytics, and graphical user-friendly interface (GUI)) in order to obtain a final automatic-controlled MOLOKO sensor that could be used in an industrially relevant environment such as the milking parlour in farm.

A demonstrator was developed and delivered at month 32, consisting of two PCB assembled in a dedicated housing in which OPM can easily connected. The stability of the OPM + sensor PCB was tested, with very positive results.

Further work was dedicated to the development of the user interface necessary to configure and control the prototypes of the MOLOKO sensor. The most crucial points were (i) the interface between the MOLOKO device and the microfluidic setup embedding the OPM; (ii) the characteristics of the optoelectronic sensors (SNR, dispersion, drift with temperature and time, etc.). Finally, the Graphical User Interface (GUI) and the Web Database were developed and the assembly of the automatic version of the sensor was completed which included all the necessary modules and components.

WP6 started at month 4 and ended at month 42, with the leadership of WFSR. The main objectives were to develop the best immunoreagents for the sensitive and selective detection and quantification of the target compounds in milk, and to develop a biocartridge composed of multiplex biosensor immunoassays, to be integrated into MOLOKO sensor, and optimization of conditions.

Single assays can be successfully combined to obtain multiplexing for both low-molecular weight and high weight analytes of interest in milk. In the case of high-molecular weight assays the non-specific interaction issue was identified for a couple of parameters.

The innovative technology of recombinant antibody assays some analyties have been developed and tested for SEA/SEB and cephalosporins, the binding site of the three most promising recombinant antibody clones were mapped and two antibodies binding to different epitopes were further studied in different diagnostic platforms.

The prototype 4-plex assay was developed and tested for providing the kit cartridge for the use in real-setting conditions. Reusability of the sensing surface was demonstrated by using iNPx system.

WP7 started at month 23 and ended at month 51, with the leadership of NEBIH.

In WP7 we assessed and confirmed the validation criteria pre-set in WP1 (the definition of the method and device validation criteria) for the internal and external validation process of the MOLOKO sensor for on-line detection of various target parameters in milk. A pre-validation testing of the MOLOKO sensor was conducted in order to enhance the validation process and the composition of the Standard Operating Procedure.

Collaboration with an external company was started in order to develop a platform employing an automatic data ingestion processes and artificial intelligence procedure to create predictive model capable of providing relevant key-performance indicators (KPIs) for the sensor, such Limit of Detetection.

Moreover, actions were implemented towards (i) the comparison of the limit of detection of MOLOKO sensor with respect to golden benchtop method for SPR-based detection with calibration standards, and (ii) the measurement campaign performed in collaboration with Parmalat end-user in order to analyse relevant raw milk samples and discuss the positioning of the MOLOKO technology with respect to screening methods implemented in the milk supply chain.

WP8 started at month 1 and ended at month 51, with the leadership of ISS.

In this WP the MOLOKO sensor was transferred to enduser partners and integrated into a diagnostic platform already working in farms. Use-validation in real-setting allowed us to characterize and transfer MOLOKO parameters in sentinel animal populations as One Health indicators during food production.

The WP8 put scientific efforts for translational research and, in particular, for boosting collaboration between the different actors (food business operators, official control, and technological industry) and make their mutual understanding closer. Starting from the comparison of advantages and weakness of what is already on the market (D8.1), MOLOKO provides ad hoc tools for consolidated technology assessment once the technology will reach the necessary technological level (D8.5). From the technological viewpoint, the automatic version of the MOLOKO sensor prototype was designed to better fit the end-user’s needs in the farm environment (D8.6) and to exploit different sensor devices already working in the dairy chain (D8.7). Different level of data treatment, from statistical comparison to multivariate analysis to neural networks have been applied including parameters already in use in the dairy chain to improve the understanding of MOLOKO/BEST (D8.1, D8.4, D8.7). Among others, MOLOKO/BEST parameters are able to detect early udder inflammation and antibiotics residues, thus supporting the prevention or early correction of important food safety, food security and issues. WP8 proved well in exercising MOLOKO/BEST system (HACCP; ClassyFarm; remote data sharing and auditing; artificial intelligence) in the official control system (current, emerging and expected, at national/Italian and European level) (D8.1, D8.3, D8.4, D8.5), also incorporating lesson learned from the COVID-19 pandemics. WP8 demonstrates the benefits of the adoption of a sensor system in the whole chain, and the feasibility of the “whole chain” approach starting from primary food production (including application for traceability and anti-fraud systems, D8.8).

WP9 started at month 1 and ended at month 51, with the leadership of QCL.

Dissemination and exploitation activities aimed to establish engagement and commitment from different stakeholders.

The IPR strategy has been investigated and externally examined for due-diligence and commercialisation activities. Individual results for partners have been identified and examined for commercial potential. The MOLOKO platform business plan has been written and used with external organisations. There are currently 2 commercialisation actions in progress with external organisations looking at ways to bring the MOLOKO platform technology to market, examining both manufacturing and IP licencing aspects.

.

WP10 started at month 1 and ended at month 51, with the leadership of CNR

The project management and coordination activities ensured that the planned work would take place smoothly and without major issues. Unfortunately, the Covid-19 hit the world during progression of MOLOKO project, and this led to a stop of technical activities for several months. For this reason, the project registered a delay of a few months, but it finally could reach all objectives.

Moreover, the exploitation paths and strategies for each main exploitable result/innovation identified by the MOLOKO consortium, the related contribution-benefit matrixes and IP ground definition and the preliminary assessment of IP rights were defined in order to maximize the impact for each MOLOKO partner. The Innovation Table was expanded in order to identify possible paths to increase the TRL and to support further commercialization and industrialization of products and services by project partners or external stakeholders.