Final Report Summary - SEA-ON-A-CHIP (Real time monitoring of SEA contaminants by an autonomous Lab-on-a-chip biosensor)
Executive Summary:
Chemical contamination of estuarine and coastal areas is a highly complex issue with negative implications for the environment and human health (through the food chain) and related coastal industries such as fisheries. Early warning systems that can provide extreme sensitivity with exquisite selectivity are required.
Under this frame the main goal of the SEA-on-a CHIP project is to develop a compact, miniaturized and autonomous multianalyte immuno-sensor with electrochemical transduction. The electrochemical immunosensor will be integrated into an automated microfluidics system connected with a sample-pre-treatment chamber. Into the sample-pre-treatment chamber, a clean-up process and pre-concentration of the compounds to be measured will be done by immuno-recognition before the sensing step into the lab-on-a-chip electrochemical immunosensor. The signals will be communicated to a remote control centre. The platform will be able to perform the measure of eight representative compounds in duplicates simultaneously. The system will be build in order to work with one-month autonomy and measuring in real time at least once per hour. The units will be tested throughout the lifetime of the project and calibrated to state-of-the-art of chemical analytics.
SEA-on-a-CHIP achieved the project objectives and a miniaturized, autonomous, remote and flexible immuno-sensor platform based amperometric measurements using a fully integrated array of microelectrodes was developed and validated. The system is composed by a sampling module with an online extraction system to enrich the target substances. Then extracts are load to an immunosensors chamber where the target analytes are combined with specific antibodies. A microfluidic system loads this mixture to lab- on-a-chip unit where excess of antibodies are recognized by the modified microelectrodes giving an indirect proportional signal to the target substances. This permits the quantitative, selective and sensitive analysis of 8 analytes simultaneously for real time analysis of marine waters. The signals are communicated to the final user in land.
The system has been developed tested and validated in real aquaculture facilities achieving a technological maturity level TRL4. The validation procedure has been based on a step-by step approach with three levels of tests in each prototype: Laboratory, mesocosms and real aquaculture facilities test levels.
Along the project 3 prototypes has been constructed each time with advanced capabilities, able to sense a higher number of compounds with a reduced volume, weight and energetic consumption. The first prototype was based on impedimetric transduction and immunoextraction. This system operated autonomously for 3 days, detect two compounds. The representative compounds detected as prove of test were Irgarol and Sulfonamides. The main limitations of the first platform were in terms of robustness due to the high sensitivity of impedance in real environmental conditions. For that reason the second prototype changed the transduction element from Impedimetric measurements to Amperometry and the extraction and enrichment procedure were as well change in order to improve the sensitivity of the platform using an online solid-phase extraction module SPE-module. The second prototype was able to detect six contaminants in seawater and operate remotely during 7 days. In addition better performance was achieved in terms of robustness and sensitivity.
The final prototype incorporates the improvements of the second one with a more compact configuration presenting not only a better performance also reducing the energetic requirements and reducing the final weight and volume. The final prototype, as it was mentioned before permits the quantitative, selective and sensitive analysis of 8 analytes and one-month autonomy was proved.
The analytical performance of the platform along the development of the different prototypes was always tested in front of the state of the art of analytical chemistry techniques such as liquid and gas chromatography coupled to tandem mass spectrometry.
Another important activity during the project have been the diffusion and dissemination including: A Sea-on-a-Chip Website, leaflets, institutional poster, a yearly e-newsletter, a promotional video, several interview videos during the Sea-on-a-Chip events (Kick-off meeting, 3 annual meetings with 3 specific open workshops, a summer school and one Open Technical Meeting at the end of the project to show the platform in functioning to stakeholders and the scientific community. In addition during the project have been published in a total number of 36 peer-reviewed publications in SCI journals from the 1st quartile most of them, 3 book chapters and 2 books of abstracts. The work from the different WPs has been presented in different national and international meetings with a total number of 73 platform presentations and 52 posters. In addition, the project coordination team has received an editorial invitation from SPRINGER to create (with the participation of the consortium) a volume entitled “Biosensors for the Marine Environment: Present and Future Challenges” to be published either within “The Handbook of Environmental Chemistry” or within the “Springer Series on Chemical Sensors and Biosensors”. Finally, the dissemination of the project to the media has been done thorough 103 press releases and 62 media brief readings.
Finally, the potential impact expected from this project includes the improvement of the technology associated to immunosensors for marine pollution control (natural or anthropogenic) with a clear repercussion on aquaculture and related industries.
Project Context and Objectives:
European maritime regions account for over 40% of the EU gross national product (GNP). Between 3 and 5% of Europe’s GNP is estimated to be generated directly from marine based industries and services. Coastal waters generate 75% of the ecosystem service benefits for Europe’s coast and it is estimated to have an equivalent value of €18 billion/annum. In addition, the non-quantifiable value of the marine resource should not be underestimated as it has a direct impact on quality of life, health societal and business development in Europe.
Chemical contamination of estuarine and coastal areas is a highly complex issue with negative implications for the environment and human health (through the food chain) and related coastal industries such as fisheries. Early warning systems that can provide extreme sensitivity with exquisite selectivity are required.
SEA-on-a-CHIP aims to develop a miniaturized, autonomous, remote and flexible immuno-sensor platform based on a fully integrated array of micro/nano-electrodes and a microfluidic system in a lab- on-a-chip configuration combined with electrochemical detection (impedimetric measurements) for real time analysis of marine waters in multi-stressor conditions. The electrochemical immunosensor will be integrated into an automated microfluidics system connected with a sample-pre-treatment chamber. Into the sample-pre-treatment chamber, a clean-up process and pre-concentration of the compounds to be measured will be done by immuno-recognition before the sensing step into the lab-on-a-chip electrochemical immunosensor. The signals will be communicated to a remote-control center. The platform will be able to perform the measure of eight representative compounds in duplicates simultaneously. The system will be built to work with one-month autonomy and measuring in real time at least once per hour. The units will be tested throughout the lifetime of the project and calibrated to state-of-the-art of chemical analytics.
This system will be developed for application in aquaculture facilities, including the rapid assessment of contaminants affecting aquaculture production and those produced by this industry, but it is easy adaptable to other target compounds or other situations required by early warning systems for coastal waters contamination analysis.
The specific project objectives are thus the following:
• To develop and optimize specific immunoassays to detect eight representative contaminants (WP2)
• To Develop miniaturized immunosensors using integrated nano-electrodes specifically functionalized with the antigens of selected compounds to carry out indirect competitive immunoassays onto the electrodes surfaces. The interaction antigen antibody on microelectrodes surface will be studied by electrochemical measurements (impedimetric or amperometric) (WP3)
• To design and build an integrated microfluidic and electromechanical systems to manage: the motor controls, microfluidics, signal amplification and to control the wired signal transmission to the surface module, to provide a simple and robust underwater operating module (WP4)
• To design and develop a data processing and management. Data of each platform will be then transmitted to the central node sink (gateway) (WP5)
• To set up a data collector. This module will be designed to acquire the data from all the different surface devices and remotely send the information from the different modules to a central data base (WP6)
• The integration of the whole system including: Power management and the integration of the different modules (underwater unit, surface water unit and data-collector). In addition, the design of the watertight underwater module with easy accessibility to the battery and to the reservoirs for refilling (WP7)
• To develop the final used software for analytical results interpretation and system diagnostics (WP8)
• To carry out the verification and provide intercalibration and quality assessment of the System performance for real-time and in-situ measurement of marine water contaminants (WP9)
• To disseminate the main results of the project (WP10)
Project Results:
The main scientific and technological achievements have been reached using a step-by-step approach along the project. Each step allowed obtaining a more advanced prototype of autonomous biosensor platform according to the DoW.
The first SEA-on-a-CHIP prototype was able to operate autonomously for 3 days, detect two compounds (Irgarol and Sulfonamides) and to send their signals remotely. This prototype was evaluated at lab. scale, in mesocosms and, finally, in the real aquaculture facilities in Sagiada Greece.
The lessons learned from the first prototype showed that impedance measurements were very much sensitive but, in contrast, this type of measurement is very much affected by surrounding conditions influencing in a lack of robustness in real environmental situations. Therefore, to reach better repeatability, it was decided to change the transduction principle to amperometry. Nevertheless, amperometry is much more robust but less sensitive. On the other hand, the two-steps immunosensors for sample pre-concentration and clean up in the first prototype cannot concentrate the sample as much as is required to achieve enough sensitivity using amperometry. To overcome this lack, another important change was introduced and the pre-concentration/clean up system using general solid phase extraction (SPE) was incorporated in the system.
The second prototype showed a notorious volume reduction, thanks to the elimination of redundant system used to check the internal performance of the different modules conforming the first prototype. This second prototype was able to detect six contaminants in seawater and operate remotely during 7 days. In this case, the platform was also evaluated at laboratory scale, in mesocosms and in real aquaculture facilities in Bergen (Norway).
The final prototype has been characterized for another volume and weight reduction thanks to the improvement of building materials of some modules, a better space conformation and the compactness of different modules in a single unit. In addition, this system was able to detect 8 analytes in parallel and it was autonomous for one month. The final prototype was tested in the IPMA aquaculture facilities in Olhão (Portugal) with successful results.
In the next sections, the main scientific and technological results are described for each WP.
WP1: COORDINATION
The coordination team has been devoted to provide management of the consortium, to provide the necessary financial and administrative management of the project, to facilitate and ensure the exchange of information and transferring the knowledge among the partners and the correct evolution of the project providing the scientific and administrative coordination and, finally, to establish the relationships of the consortium with the end users and authorities through project meetings and workshops.
The coordination monitoring along the duration of the project has been based in the monitoring of progress of the different activities scheduled. This work was done by monthly video conferences, and extra video conferences when it was required. Different integration and validation meetings as well as the annual consortium meetings for promoting the knowledge interchange in the consortium were carried out. In addition, internal agreements and the adoption of contingency actions were done when required. This intensive follow-up has minimized delays and deviations and all the tasks, milestones and deliverables scheduled for each period have been fulfilled and submitted.
In order to facilitate the communication within the consortium, different internal communication tools have been set up, maintained and updated (timeline, calendar, document repository, contact details, guidelines, dissemination table, discussion forum, etc.). In addition, some meetings and the interchange of information among the different partners have been promoted by monthly video-conferences among interrelated work packages, working meetings between partners and work package clusters, consortium meetings, SEA-on-a-CHIP dropbox, SEA-on-a-CHIP Website (http://www.sea-on-a-chip.eu/). In the intranet, the partners can access to the meeting presentations, minutes of meetings and deliverables. In general, e-mail in combination with storage of files on the website has been used for communication, but phone and conference calls have been used regularly.
Finally, the dissemination of the project has been supervised by coordination team together with WP10 (devoted to the dissemination activities).
WP2: IMMUNOASSAYS AND IMMOBILIZATION
The main objective of the WP2 has been the development of the format of the immunoassay to be implemented in the sensor platform (WP3).
Within the 3 project periods, the main results have been based on different tasks and achievements. The main goals of the WP2 for the first period were i) the selection of the two compounds to be tested for the first prototype; ii) preparation of controlled batch of magnetic particles functionalized with antibodies; iii) to develop the two-step competitive immunoassays based on the magnetic particles for the analysis of 2 of the 8 selected compounds. The main results are summarized below.
• Different batches of magnetic nanoparticles were delivered for their functionalization, fully characterized and reproducible.
• The biofunctionalization was performed successfully and demonstrated their biofunctionality against the corresponding antigen.
• Different ELISAs for each target analyte were tested successfully in seawater, reaching good detectabilities allowing measures directly from sea water.
• The evaluation of the cross-reactivity between two pairs of immunoreagents was demonstrated (Irgarol and Sulfonamides), not showing any cross-reaction between them.
• The two-step immunoassay format proposed in the project was demonstrated. However, a high-background noise was observed and should be minimized.
During the second period, the decision to introduce a solid phase extraction column to preconcentrate the sample to reach the detectability requirements pointed by WP9 affected directly WP2. Within this period, the main objectives where i) the evaluation the effects of methanol (used for solid phase extraction elution) into the immunoassay to establish the maximum concentration without compromising the maximum signal and the detectability; ii) demonstrate the multiplexation of the different immunoreagents involved in the determination of the targeted pollutants by developing a multiplexed fluorescent microarray; iii) produce antibodies against pyrethroids and the development of an immunoassay; and iv) improve the analytical parameters of the domoic acid immunoassay. The main results showed that:
• The presence of a 10% of methanol in the final concentration of solid phase extraction eluates can be used without compromising biosensor functionality.
• The evaluation of the cross-reactivity between all immunoreagents showed that no cross-reaction between them exists, except for the case of the antibody specific for deltamethrin.
• The fluorescent microarray platform worked correctly for all immunoreagents tested, obtaining different calibration curves for each analyte in seawater, working with multiplexation analysis.
• New immunoreagents for the detection of deltamethrin were necessary to be produced. These allowed a new ELISA assay with improved features (including sensibility, detectability and limits of detection). The cross-reactivity between the new immunoreagents and the related ones of all other analytes showed that no cross-reaction exists.
• A new combination for the detection of domoic acid was implemented, with greater features including sensibility, detectability and limits of detection.
Finally, during the third period, WP2 aimed their contribution to characterize the immunoreagents for their implementation in the sensor platform (WP3) and the provision of the different immunoreagents for the calibration of the device and the final case study by means of: i) the evaluation of the effects of dimethyl sulfoxide (DMSO) in the immunoassay, for the analytical parameters of each assay; ii) set up the tribromophenol immunoassay as a contingency plan for BDE47 (see Deliverable 1.2 and 1.3); iii) accuracy studies with DMSO extracts and; iv) provision of immunoreagents for the evaluation of the 3rd prototype. The main results are summarized below:
• All the immunoreagents tested for the determination of the targeted pollutants were robust against the requested DMSO content.
• Tribromophenol was proposed as contingency plan for BDE47 detection. The ELISA developed was successfully tested in buffer and seawater, reaching good detectabilities.
• The ELISA developed for all the targeted pollutants resulted an accurate tool for the determination of DMSO spiked samples, being a helpful tool for the quantification of the pollutants.
• A set of different immunoreagents were provided to WP3 and WP7 for its implementation to the 3rd prototype.
WP3: Lab-on-a-Chip
The main objective of this WP has been the development of the functionalized Lab-on-a-chip unit. This unit contained devices with base on a polymeric substrate (polyimide (PI) and polyethylenenaphthalenedicarboxylate (PEN)), integrated with gold microelectrodes arrays, reference electrode based on silver/silver chloride, counter electrode based on platinum, and microfluidic structure, made on polydimethylsiloxane (PDMS). These devices allow the multiplexed detection and quantification of selected analytes by the interaction with specific antibodies immobilized on functionalized magnetic particles and impedimetric measurements after competitor-antibody binding events.
The main goal of this WP was the development and fabrication of miniaturized immunosensors using integrated nano-electrodes specifically functionalized with the antigens of selected compounds successfully. The microelectrodes were prepared to carry out indirect competitive immunoassays as it has been mentioned before. Finally, the interaction antigen antibody on microelectrodes surface was studied by impedance and amperomentric measurements.
The final Lab-on-a-chip has been based on the integration of eight microelectrodes. Each microelectrode based on gold working microelectrode (2.7 mm diameter) with integrated silver/silver chloride reference and carbon counter microelectrodes. External dimensions are L34 x W78 x H1 mm. Screen printing technology was used. These devices based on amperometric measurement have been used for the final trail.
Finally, a third prototype of the Lab-on-chip based on the amperometric transducing principle has demonstrated its feasibility and reproducibility during the last trial in Olhão for the determination of targeted pollutants (irgarol, sulfapyridine, chloramphenicol, estradiol, domoic acid and deltamethrin).
WP4: FLUIDICS MANAGEMENT AND ROBOTICS
The main objectives of this WP have been: i) to design, develop and test a water collecting device (or Sampling System, SS) and a microFluidic System (μFS) in order to manage the whole analytical procedure; ii) the SS has to be able to collect water samples autonomously from the sea at different depths and to deliver a controlled volume of solution to the μFS; iii) the SS and the μFS have to be provided with mini pumps and valves operated by a microcontrollers that cooperatively manages the whole fluidics; iv) the SS and the μFS couple has to be completely automated and particular care has to be dedicated to the initial cleaning phase and to the materials selection in order to avoid intra and cross contaminations; v) the μFS has to coordinate its activities with the bioSensor and to communicate with the Mote front-end device to send real time data (measurement values, telemetric information, warning and alarm) to receive end-user operating commands. In addition, during the last period the WP4 was devoted to test the refinements of the final fluidics management and robotics module. Regarding the main improvements of the final prototype in comparison with the 1st and the 2nd ones, these were the new design of the water collecting device (or Sampling System, SS) and the micro-Fluidic System (µFS), to rebuild those two systems and to test them and definitive analytical procedures in a long-term test field (at least 30 working days) at an aquaculture site. Furthermore, the µFS has to implement revised analytical procedures able to simultaneously reveal all the 8 analytes in the sea water sample.
The main results were achieved step-by-step according to the necessities of each prototype.
• During the development of the 1st prototype, an integrated microfluidic and electromechanical system to manage the electromechanical and microfluidics systems, signal amplification and to control the wired signal transmission to the surface module was developed and integrated into this first prototype. The system was autonomously working for 3 days.
• An integrated microfluidic and electromechanical system to manage the electromechanical and microfluidics systems, signal amplification and to control the wired signal transmission to the surface module was developed and integrated into the 2nd prototype, able to work autonomously for 7 days. Different improvements were carried out in the base of the lessons learned from the first prototype regarding pumps miniaturization and compactness.
• For the final and 3rd prototype, the fluidic management and robotic system was refined and tested showing excellent performance. The system integrates the new electromechanical system, a new electronics board (eBoard), a new firmware (FW) and a new operator/ end-user software (SW) running on PC. Other improvement in terms of compactness has been carried out as well. In addition, the prototype could work autonomously for 1 month detecting 8 compounds.
WP 5: DATA PROCESSING, MANAGEMENT AND TRANSMISSION TO SINK
The main objectives of WP5 has been to design, develop and test a sensor interrogation electronic module to acquire the sensor signal, and to be able to process the collected data and transmit it. To achieve this, the following goals must be performed: i) to design the sensor interrogation electronics interface; ii) supply the sensor with the appropriate voltage and frequency, collect the signal response of the sensor at this exciting voltage and frequency, amplify the signal response voltage and convert the analog value to digital information and, to make a pre-processing to digitally filtering the data and obtain the response of the sensor; iii) battery management for supplying the collecting data and the sensor electronic modules. Minimization of the power consumption of the acquisition system (including the sensor itself and all the electronics needed in the measurement system); iv) transmission of the collected data and some status information to the central surface module. However, during the last 3rd period, the objectives include the development of a backup system. It will allow to store the acquired data in a backup flash memory embedded in the microcontroller. It includes the different solutions to extract these data, deplete and initialize the backup for new measurements.
Within this WP, the Gateway module was designed, constructed and integrated in the 1st, 2nd and 3rd prototypes. The main results include:
• The development of the acquisition electronic system during the 1st period of the project. The electronic board was designed and fabricated to allow the measurement of the sensors and the whole system functioning. The first prototype was integrated in the Mote and tested during the 1st prototype trials. The electronic board was tested in laboratory and in offshore environments for some hours, proving their functionality and reliability. The developed system was integrated in the Mote structures with the other components (μFS, SS, battery, reservoir, OMHSC...) and was tested in laboratory environment during the 3rd trial meeting in Girona (March 2015) and in offshore tests in seawater during the 1st case study meeting in Sivota (April 2015). In both cases measurements were repeated automatically, proving the full functionality and robustness of the systems.
• An entirely new integrated unit was designed and fabricated for the 2nd prototype during the 2nd period to accomplish with the new measurement technique (solid phase extraction column and continuous flow through the bioSensors microchambers). The new unit, significantly smaller than in the 1st prototype system, was integrated into the new Mote and successfully tested in the Bergen/Norway test field.
• Finally, for the 3rd prototype, the BioLab on a Chip System electronic board was re-designed and applied to the new bioSensor µChs feeding during the 3rd period. These new µChambers feeding were built according the liquid multiplexing method producing a pulsing flow through µChs (European Pending Patent number EP17382269.3 14 claims). In addition, the backup system embedded in the flash memory of the bioLCS was successfully implemented. Furthermore, a new maximum mean value dynamic algorithm was developed in close collaboration with partners from WP4. Finally, the whole system was tested in CSIC (Barcelona), and at the last long-run test field in Olhão/Portugal.
WP6: ELECTRONIC SURFACE SINK SYSTEM (GATEWAY)
The overall objective of the WP6 has been to design and develop a reliable wireless communication and data transmission layers to wirelessly send Mote sensor collected data to the shore based Gateway and, then, to the cloud to be analyzed and finally presented to the end-users.
Definition of a Reliable, Flexible and expandable HW and SW Architecture.
The main results of this WP include:
• Development of sub-module Prototypes and Updated/Refined HW and SW for the 1st prototype.
• Improvement of the collector modules for the 2nd and 3rd prototypes.
• Integration of Submodules.
• Development of HW and SW tools to perform Testing in order to optimise functionality.
• Successful Stress Testing of HW and SW.
• Successful operation during different trials.
WP7: SYSTEM INTEGRATION
The work in WP7 has been dedicated to the comprehensive work of creating a technical framework for the integration of the different subunits that builds up the SEA-on-a-CHIP device for the 1st, 2nd and 3rd prototypes and the power management.
The focus in WP7 has hence been focused on harmonizing the extensive technological development work performed in WP2-6. Iteration of relevant question formulations related to the adaptation of the different subunits did result in an efficient workflow towards the integration of the first SEA-on-a-CHIP prototypes.
The main results during different project periods include:
• Integration of the first prototype system during the 1st period. It includes: power management, the integration of the different modules (underwater unit, surface water unit and data-collector) and the watertight underwater module with easy accessibility to the battery and to the reservoirs for refilling.
• During the 2nd period, the 2nd prototype system was refined including the same devices as the 1st one. However, in this case, a notable reduction of the volume was achieved thanks to the pumps and valves size reduction, compactness and the elimination of the redundant sensors included into the first prototype as system diagnostics.
• In the final phase of the SEA-on-a-CHIP project, the work regarding integration was focused on creating a refined technical framework of the further developed subunits that build up the SEA-on-a-CHIP device. The different subunits (i.e. microfluidics, immunosensor, liquid handling and storage, data communication/transmission and power management system) have all gone through extensive development and refinement throughout the project and were finally proved highly functional in the final long-term test period performed in Olhão, Portugal during March-May 2017.
WP 8: REMOTE APPLICATIONS SOFTWARE
The main goals of this WP have been: i) to get the data transmitted by the sensor network and maintain the records data-base ordered by time; ii) to display the data information to the final user in easy way and implement a friendly graphical user interface and; iii) Send commands to the remote sensor network to modify the performance of the sensor itself (frequency of analysis, set up clean-up procedures). Within the different periods of the project, the main achievements were summarized below.
• The Beta version of the users’ software for analytical results interpretation and diagnostics was developed during the 1st period to be used in the 1st prototype.
• An advanced version of the users’ software for analytical results interpretation and diagnostics was developed during the 2nd period.
• The final version of the users’ software for analytical results interpretation and diagnostics was developed and successfully tested during the last trial of the 3rd period with the 3rd prototype.
WP 9: VERIFICATION AND VALIDATION: CASE STUDIES
The main objective of WP 9 has been to test and validate the performance of the SEA-on-a-CHIP biosensor system through comparison with conventional analytical chemistry and to demonstrate its potential for in situ environmental and aquaculture related applications. With this aim, different trials to test some prepared blind samples with selected compounds during each period were carried out.
The main results include:
• A verification and evaluation of the whole system functioning as well as the different parts of the 1st prototype by means of a step-by-step approach that included: laboratory evaluations, mesocosms trials and a sea trial in aquaculture facilities for the two selected compounds.
• A verification and evaluation of the whole system functioning during the 2nd and the 3rd periods for the 2nd and 3rd prototypes, respectively. The second prototype was tested for 3 compounds samples along 7 days trial and comparing the results with the ones reported in laboratory facilities. The results were very promising, considering the incorporation of the new device of solid phase extraction in this prototype. Finally, during the last trial with the 3rd Sea on a Chip prototype for 1 month in aquaculture facilities for 8 compounds it was evaluated the good performance of the developed and miniaturized final device versus the laboratory results.
WP 10: DISSEMINATION AND EXPLOITATION PLAN
The main objectives of WP10 have been i) to ensure exchange of information and transferring the knowledge and the correct evolution of the project to the partners provided by the scientific and administrative coordination; i) to coordinate the dissemination and exploitation of the results and to establish the relationships of the consortium with the end users and authorities through open technical meetings, publications, and newsletters; iii) to interact directly with legislation authorities in order to evaluate the implementation into national and European legislations and; iv) to involve stakeholders to the project.
The main results within the project are as follows:
• A dissemination plan was performed during the 1st period of the project in order to assure the fluency of information between the consortium and to inform outside the consortium about the main achievements and results of SEA-on-a-CHIP.
• Different dissemination activities were conducted according to the dissemination plan during the 2nd and 3rd periods. Also, monthly teleconferences with the WP leaders, consortium, and working/integration meetings were as well carried out to assure the fluency of information between the consortium. Besides, the main results of the project were presented in different international conferences related to the field of the research in the SEA-on-a-CHIP.
Potential Impact:
Potential impact:
The development of a miniaturized unattended immunosensor system for real-time testing and applications in aquaculture with commercialization potential is expected.
SEA-ON-A-CHIP project has improved technology associated to immunosensors for marine pollution control (natural or anthropogenic) with a clear repercussion on related industries such as fisheries and aquaculture facilities. Recent technological developments in the miniaturization of electronics and wireless communication technology have led to the emergence of Environmental Sensor Networks (ESN). These will greatly enhance monitoring of the natural environment and in some cases, open new techniques for taking measurements or allow previously impossible remote deployments of sensors.
Societal implications:
At least 16% of the EU population lives in coastal areas, many more depend on the sea for work, leisure, food or other products and services. Between 3 and 5% of Europe’s gross national product is estimated to be generated directly from marine based industries and services. The chemical contaminations of marine environments influence negatively related coastal industries such as fisheries and aquaculture facilities, but also human health and the general ecosystem. Identification of types of hazards and their temporal and spatial scale are crucial for an analysis of the associated risks. These issues are regulated by law under global, regional or national statutes, such as the EU Water Framework Directive (WFD) (2000/60/EC) and the EU Marine Strategy Framework Directive (MSFD) (2008/56/EC).
During the last decade, a range of global and regional monitoring programs have been developed to protect human and environmental health and prevent economic losses caused by marine hazardous substances in an integrated manner. Among the different groups of pollutants representing a relevant danger to human health, to the environment and to economic resources like fishering and aquaculture facilities, endocrine disrupting compounds are related the decrease of aquaculture production due to their interference with the endocrine system; persistent organic pollutants and marine biotoxins present accumulation and toxicity; antibiotics and pesticides used in the aquaculture production may pose a risk in the environment and the human health. All these elements could have an additive impact on marine habitats and marine resources. To combat this problem early warning systems are required that can provide extreme sensitivity with exquisite selectivity. SEA-ON-A-CHIP project will improve technology associated to immuno-sensors for marine pollution control (natural or anthropogenic) with a clear repercussion on related industries such as fisheries and aquaculture facilities. Recent technological developments in the miniaturisation of electronics and wireless communication technology have led to the emergence of Environmental Sensor Networks (ESN). These will greatly enhance monitoring of the natural environment and in some cases open up new techniques for taking measurements or allow previously impossible remote deployments of sensors.
Dissemination activities:
Different levels of dissemination and communication activities have been carried out:
Scientific publications:
• Peer-reviewed publications: 36 in SCI journals from the 1st quartile most of them,
• Book chapters: 3
• Book of abstracts: 2
Participation in congresses and conferences (National and International):
• Platform presentations: 73
• Poster presentations: 52
Participation in mass media:
• Press releases: 103
• Media brief readings: 62
• Promotional videos: 11
Book:
Editorial invitation from SPRINGER to create (with the participation of the consortium) a volume entitled “Biosensors for the Marine Environment: Present and Future Challenges” to be published either within “The Handbook of Environmental Chemistry” or within the “Springer Series on Chemical Sensors and Biosensors”.
More detailed information can be seen in Annex A.
Exploitation activities
a) Patents:
During the last period, a patent has been generated too: “Devices and methods for multiplexing liquid in biosensor micro-chambers” by Giacomo Saviozzi, Juan Pablo Salvador, Manuel Lopez, Francisco Palacio, Raquel Pruna, M.-Pilar Marco, Cecilia Laschi. European patent application: EP17382269.3. Date: 12/05/2017.
b) Exploitable foreground:
It is expected to generate exploitable foreground with the SEA-ON-A-CHIP final autonomous system to monitor water contaminants on-site sending remotely the results or alarms to the end-users.
List of Websites:
http://www.sea-on-a-chip.eu/V1/SOC50V4_Main.php
Deputy coordinator: Marinella Farré (mfuqam@cid.csic.es); IDAEA-CSIC
Chemical contamination of estuarine and coastal areas is a highly complex issue with negative implications for the environment and human health (through the food chain) and related coastal industries such as fisheries. Early warning systems that can provide extreme sensitivity with exquisite selectivity are required.
Under this frame the main goal of the SEA-on-a CHIP project is to develop a compact, miniaturized and autonomous multianalyte immuno-sensor with electrochemical transduction. The electrochemical immunosensor will be integrated into an automated microfluidics system connected with a sample-pre-treatment chamber. Into the sample-pre-treatment chamber, a clean-up process and pre-concentration of the compounds to be measured will be done by immuno-recognition before the sensing step into the lab-on-a-chip electrochemical immunosensor. The signals will be communicated to a remote control centre. The platform will be able to perform the measure of eight representative compounds in duplicates simultaneously. The system will be build in order to work with one-month autonomy and measuring in real time at least once per hour. The units will be tested throughout the lifetime of the project and calibrated to state-of-the-art of chemical analytics.
SEA-on-a-CHIP achieved the project objectives and a miniaturized, autonomous, remote and flexible immuno-sensor platform based amperometric measurements using a fully integrated array of microelectrodes was developed and validated. The system is composed by a sampling module with an online extraction system to enrich the target substances. Then extracts are load to an immunosensors chamber where the target analytes are combined with specific antibodies. A microfluidic system loads this mixture to lab- on-a-chip unit where excess of antibodies are recognized by the modified microelectrodes giving an indirect proportional signal to the target substances. This permits the quantitative, selective and sensitive analysis of 8 analytes simultaneously for real time analysis of marine waters. The signals are communicated to the final user in land.
The system has been developed tested and validated in real aquaculture facilities achieving a technological maturity level TRL4. The validation procedure has been based on a step-by step approach with three levels of tests in each prototype: Laboratory, mesocosms and real aquaculture facilities test levels.
Along the project 3 prototypes has been constructed each time with advanced capabilities, able to sense a higher number of compounds with a reduced volume, weight and energetic consumption. The first prototype was based on impedimetric transduction and immunoextraction. This system operated autonomously for 3 days, detect two compounds. The representative compounds detected as prove of test were Irgarol and Sulfonamides. The main limitations of the first platform were in terms of robustness due to the high sensitivity of impedance in real environmental conditions. For that reason the second prototype changed the transduction element from Impedimetric measurements to Amperometry and the extraction and enrichment procedure were as well change in order to improve the sensitivity of the platform using an online solid-phase extraction module SPE-module. The second prototype was able to detect six contaminants in seawater and operate remotely during 7 days. In addition better performance was achieved in terms of robustness and sensitivity.
The final prototype incorporates the improvements of the second one with a more compact configuration presenting not only a better performance also reducing the energetic requirements and reducing the final weight and volume. The final prototype, as it was mentioned before permits the quantitative, selective and sensitive analysis of 8 analytes and one-month autonomy was proved.
The analytical performance of the platform along the development of the different prototypes was always tested in front of the state of the art of analytical chemistry techniques such as liquid and gas chromatography coupled to tandem mass spectrometry.
Another important activity during the project have been the diffusion and dissemination including: A Sea-on-a-Chip Website, leaflets, institutional poster, a yearly e-newsletter, a promotional video, several interview videos during the Sea-on-a-Chip events (Kick-off meeting, 3 annual meetings with 3 specific open workshops, a summer school and one Open Technical Meeting at the end of the project to show the platform in functioning to stakeholders and the scientific community. In addition during the project have been published in a total number of 36 peer-reviewed publications in SCI journals from the 1st quartile most of them, 3 book chapters and 2 books of abstracts. The work from the different WPs has been presented in different national and international meetings with a total number of 73 platform presentations and 52 posters. In addition, the project coordination team has received an editorial invitation from SPRINGER to create (with the participation of the consortium) a volume entitled “Biosensors for the Marine Environment: Present and Future Challenges” to be published either within “The Handbook of Environmental Chemistry” or within the “Springer Series on Chemical Sensors and Biosensors”. Finally, the dissemination of the project to the media has been done thorough 103 press releases and 62 media brief readings.
Finally, the potential impact expected from this project includes the improvement of the technology associated to immunosensors for marine pollution control (natural or anthropogenic) with a clear repercussion on aquaculture and related industries.
Project Context and Objectives:
European maritime regions account for over 40% of the EU gross national product (GNP). Between 3 and 5% of Europe’s GNP is estimated to be generated directly from marine based industries and services. Coastal waters generate 75% of the ecosystem service benefits for Europe’s coast and it is estimated to have an equivalent value of €18 billion/annum. In addition, the non-quantifiable value of the marine resource should not be underestimated as it has a direct impact on quality of life, health societal and business development in Europe.
Chemical contamination of estuarine and coastal areas is a highly complex issue with negative implications for the environment and human health (through the food chain) and related coastal industries such as fisheries. Early warning systems that can provide extreme sensitivity with exquisite selectivity are required.
SEA-on-a-CHIP aims to develop a miniaturized, autonomous, remote and flexible immuno-sensor platform based on a fully integrated array of micro/nano-electrodes and a microfluidic system in a lab- on-a-chip configuration combined with electrochemical detection (impedimetric measurements) for real time analysis of marine waters in multi-stressor conditions. The electrochemical immunosensor will be integrated into an automated microfluidics system connected with a sample-pre-treatment chamber. Into the sample-pre-treatment chamber, a clean-up process and pre-concentration of the compounds to be measured will be done by immuno-recognition before the sensing step into the lab-on-a-chip electrochemical immunosensor. The signals will be communicated to a remote-control center. The platform will be able to perform the measure of eight representative compounds in duplicates simultaneously. The system will be built to work with one-month autonomy and measuring in real time at least once per hour. The units will be tested throughout the lifetime of the project and calibrated to state-of-the-art of chemical analytics.
This system will be developed for application in aquaculture facilities, including the rapid assessment of contaminants affecting aquaculture production and those produced by this industry, but it is easy adaptable to other target compounds or other situations required by early warning systems for coastal waters contamination analysis.
The specific project objectives are thus the following:
• To develop and optimize specific immunoassays to detect eight representative contaminants (WP2)
• To Develop miniaturized immunosensors using integrated nano-electrodes specifically functionalized with the antigens of selected compounds to carry out indirect competitive immunoassays onto the electrodes surfaces. The interaction antigen antibody on microelectrodes surface will be studied by electrochemical measurements (impedimetric or amperometric) (WP3)
• To design and build an integrated microfluidic and electromechanical systems to manage: the motor controls, microfluidics, signal amplification and to control the wired signal transmission to the surface module, to provide a simple and robust underwater operating module (WP4)
• To design and develop a data processing and management. Data of each platform will be then transmitted to the central node sink (gateway) (WP5)
• To set up a data collector. This module will be designed to acquire the data from all the different surface devices and remotely send the information from the different modules to a central data base (WP6)
• The integration of the whole system including: Power management and the integration of the different modules (underwater unit, surface water unit and data-collector). In addition, the design of the watertight underwater module with easy accessibility to the battery and to the reservoirs for refilling (WP7)
• To develop the final used software for analytical results interpretation and system diagnostics (WP8)
• To carry out the verification and provide intercalibration and quality assessment of the System performance for real-time and in-situ measurement of marine water contaminants (WP9)
• To disseminate the main results of the project (WP10)
Project Results:
The main scientific and technological achievements have been reached using a step-by-step approach along the project. Each step allowed obtaining a more advanced prototype of autonomous biosensor platform according to the DoW.
The first SEA-on-a-CHIP prototype was able to operate autonomously for 3 days, detect two compounds (Irgarol and Sulfonamides) and to send their signals remotely. This prototype was evaluated at lab. scale, in mesocosms and, finally, in the real aquaculture facilities in Sagiada Greece.
The lessons learned from the first prototype showed that impedance measurements were very much sensitive but, in contrast, this type of measurement is very much affected by surrounding conditions influencing in a lack of robustness in real environmental situations. Therefore, to reach better repeatability, it was decided to change the transduction principle to amperometry. Nevertheless, amperometry is much more robust but less sensitive. On the other hand, the two-steps immunosensors for sample pre-concentration and clean up in the first prototype cannot concentrate the sample as much as is required to achieve enough sensitivity using amperometry. To overcome this lack, another important change was introduced and the pre-concentration/clean up system using general solid phase extraction (SPE) was incorporated in the system.
The second prototype showed a notorious volume reduction, thanks to the elimination of redundant system used to check the internal performance of the different modules conforming the first prototype. This second prototype was able to detect six contaminants in seawater and operate remotely during 7 days. In this case, the platform was also evaluated at laboratory scale, in mesocosms and in real aquaculture facilities in Bergen (Norway).
The final prototype has been characterized for another volume and weight reduction thanks to the improvement of building materials of some modules, a better space conformation and the compactness of different modules in a single unit. In addition, this system was able to detect 8 analytes in parallel and it was autonomous for one month. The final prototype was tested in the IPMA aquaculture facilities in Olhão (Portugal) with successful results.
In the next sections, the main scientific and technological results are described for each WP.
WP1: COORDINATION
The coordination team has been devoted to provide management of the consortium, to provide the necessary financial and administrative management of the project, to facilitate and ensure the exchange of information and transferring the knowledge among the partners and the correct evolution of the project providing the scientific and administrative coordination and, finally, to establish the relationships of the consortium with the end users and authorities through project meetings and workshops.
The coordination monitoring along the duration of the project has been based in the monitoring of progress of the different activities scheduled. This work was done by monthly video conferences, and extra video conferences when it was required. Different integration and validation meetings as well as the annual consortium meetings for promoting the knowledge interchange in the consortium were carried out. In addition, internal agreements and the adoption of contingency actions were done when required. This intensive follow-up has minimized delays and deviations and all the tasks, milestones and deliverables scheduled for each period have been fulfilled and submitted.
In order to facilitate the communication within the consortium, different internal communication tools have been set up, maintained and updated (timeline, calendar, document repository, contact details, guidelines, dissemination table, discussion forum, etc.). In addition, some meetings and the interchange of information among the different partners have been promoted by monthly video-conferences among interrelated work packages, working meetings between partners and work package clusters, consortium meetings, SEA-on-a-CHIP dropbox, SEA-on-a-CHIP Website (http://www.sea-on-a-chip.eu/). In the intranet, the partners can access to the meeting presentations, minutes of meetings and deliverables. In general, e-mail in combination with storage of files on the website has been used for communication, but phone and conference calls have been used regularly.
Finally, the dissemination of the project has been supervised by coordination team together with WP10 (devoted to the dissemination activities).
WP2: IMMUNOASSAYS AND IMMOBILIZATION
The main objective of the WP2 has been the development of the format of the immunoassay to be implemented in the sensor platform (WP3).
Within the 3 project periods, the main results have been based on different tasks and achievements. The main goals of the WP2 for the first period were i) the selection of the two compounds to be tested for the first prototype; ii) preparation of controlled batch of magnetic particles functionalized with antibodies; iii) to develop the two-step competitive immunoassays based on the magnetic particles for the analysis of 2 of the 8 selected compounds. The main results are summarized below.
• Different batches of magnetic nanoparticles were delivered for their functionalization, fully characterized and reproducible.
• The biofunctionalization was performed successfully and demonstrated their biofunctionality against the corresponding antigen.
• Different ELISAs for each target analyte were tested successfully in seawater, reaching good detectabilities allowing measures directly from sea water.
• The evaluation of the cross-reactivity between two pairs of immunoreagents was demonstrated (Irgarol and Sulfonamides), not showing any cross-reaction between them.
• The two-step immunoassay format proposed in the project was demonstrated. However, a high-background noise was observed and should be minimized.
During the second period, the decision to introduce a solid phase extraction column to preconcentrate the sample to reach the detectability requirements pointed by WP9 affected directly WP2. Within this period, the main objectives where i) the evaluation the effects of methanol (used for solid phase extraction elution) into the immunoassay to establish the maximum concentration without compromising the maximum signal and the detectability; ii) demonstrate the multiplexation of the different immunoreagents involved in the determination of the targeted pollutants by developing a multiplexed fluorescent microarray; iii) produce antibodies against pyrethroids and the development of an immunoassay; and iv) improve the analytical parameters of the domoic acid immunoassay. The main results showed that:
• The presence of a 10% of methanol in the final concentration of solid phase extraction eluates can be used without compromising biosensor functionality.
• The evaluation of the cross-reactivity between all immunoreagents showed that no cross-reaction between them exists, except for the case of the antibody specific for deltamethrin.
• The fluorescent microarray platform worked correctly for all immunoreagents tested, obtaining different calibration curves for each analyte in seawater, working with multiplexation analysis.
• New immunoreagents for the detection of deltamethrin were necessary to be produced. These allowed a new ELISA assay with improved features (including sensibility, detectability and limits of detection). The cross-reactivity between the new immunoreagents and the related ones of all other analytes showed that no cross-reaction exists.
• A new combination for the detection of domoic acid was implemented, with greater features including sensibility, detectability and limits of detection.
Finally, during the third period, WP2 aimed their contribution to characterize the immunoreagents for their implementation in the sensor platform (WP3) and the provision of the different immunoreagents for the calibration of the device and the final case study by means of: i) the evaluation of the effects of dimethyl sulfoxide (DMSO) in the immunoassay, for the analytical parameters of each assay; ii) set up the tribromophenol immunoassay as a contingency plan for BDE47 (see Deliverable 1.2 and 1.3); iii) accuracy studies with DMSO extracts and; iv) provision of immunoreagents for the evaluation of the 3rd prototype. The main results are summarized below:
• All the immunoreagents tested for the determination of the targeted pollutants were robust against the requested DMSO content.
• Tribromophenol was proposed as contingency plan for BDE47 detection. The ELISA developed was successfully tested in buffer and seawater, reaching good detectabilities.
• The ELISA developed for all the targeted pollutants resulted an accurate tool for the determination of DMSO spiked samples, being a helpful tool for the quantification of the pollutants.
• A set of different immunoreagents were provided to WP3 and WP7 for its implementation to the 3rd prototype.
WP3: Lab-on-a-Chip
The main objective of this WP has been the development of the functionalized Lab-on-a-chip unit. This unit contained devices with base on a polymeric substrate (polyimide (PI) and polyethylenenaphthalenedicarboxylate (PEN)), integrated with gold microelectrodes arrays, reference electrode based on silver/silver chloride, counter electrode based on platinum, and microfluidic structure, made on polydimethylsiloxane (PDMS). These devices allow the multiplexed detection and quantification of selected analytes by the interaction with specific antibodies immobilized on functionalized magnetic particles and impedimetric measurements after competitor-antibody binding events.
The main goal of this WP was the development and fabrication of miniaturized immunosensors using integrated nano-electrodes specifically functionalized with the antigens of selected compounds successfully. The microelectrodes were prepared to carry out indirect competitive immunoassays as it has been mentioned before. Finally, the interaction antigen antibody on microelectrodes surface was studied by impedance and amperomentric measurements.
The final Lab-on-a-chip has been based on the integration of eight microelectrodes. Each microelectrode based on gold working microelectrode (2.7 mm diameter) with integrated silver/silver chloride reference and carbon counter microelectrodes. External dimensions are L34 x W78 x H1 mm. Screen printing technology was used. These devices based on amperometric measurement have been used for the final trail.
Finally, a third prototype of the Lab-on-chip based on the amperometric transducing principle has demonstrated its feasibility and reproducibility during the last trial in Olhão for the determination of targeted pollutants (irgarol, sulfapyridine, chloramphenicol, estradiol, domoic acid and deltamethrin).
WP4: FLUIDICS MANAGEMENT AND ROBOTICS
The main objectives of this WP have been: i) to design, develop and test a water collecting device (or Sampling System, SS) and a microFluidic System (μFS) in order to manage the whole analytical procedure; ii) the SS has to be able to collect water samples autonomously from the sea at different depths and to deliver a controlled volume of solution to the μFS; iii) the SS and the μFS have to be provided with mini pumps and valves operated by a microcontrollers that cooperatively manages the whole fluidics; iv) the SS and the μFS couple has to be completely automated and particular care has to be dedicated to the initial cleaning phase and to the materials selection in order to avoid intra and cross contaminations; v) the μFS has to coordinate its activities with the bioSensor and to communicate with the Mote front-end device to send real time data (measurement values, telemetric information, warning and alarm) to receive end-user operating commands. In addition, during the last period the WP4 was devoted to test the refinements of the final fluidics management and robotics module. Regarding the main improvements of the final prototype in comparison with the 1st and the 2nd ones, these were the new design of the water collecting device (or Sampling System, SS) and the micro-Fluidic System (µFS), to rebuild those two systems and to test them and definitive analytical procedures in a long-term test field (at least 30 working days) at an aquaculture site. Furthermore, the µFS has to implement revised analytical procedures able to simultaneously reveal all the 8 analytes in the sea water sample.
The main results were achieved step-by-step according to the necessities of each prototype.
• During the development of the 1st prototype, an integrated microfluidic and electromechanical system to manage the electromechanical and microfluidics systems, signal amplification and to control the wired signal transmission to the surface module was developed and integrated into this first prototype. The system was autonomously working for 3 days.
• An integrated microfluidic and electromechanical system to manage the electromechanical and microfluidics systems, signal amplification and to control the wired signal transmission to the surface module was developed and integrated into the 2nd prototype, able to work autonomously for 7 days. Different improvements were carried out in the base of the lessons learned from the first prototype regarding pumps miniaturization and compactness.
• For the final and 3rd prototype, the fluidic management and robotic system was refined and tested showing excellent performance. The system integrates the new electromechanical system, a new electronics board (eBoard), a new firmware (FW) and a new operator/ end-user software (SW) running on PC. Other improvement in terms of compactness has been carried out as well. In addition, the prototype could work autonomously for 1 month detecting 8 compounds.
WP 5: DATA PROCESSING, MANAGEMENT AND TRANSMISSION TO SINK
The main objectives of WP5 has been to design, develop and test a sensor interrogation electronic module to acquire the sensor signal, and to be able to process the collected data and transmit it. To achieve this, the following goals must be performed: i) to design the sensor interrogation electronics interface; ii) supply the sensor with the appropriate voltage and frequency, collect the signal response of the sensor at this exciting voltage and frequency, amplify the signal response voltage and convert the analog value to digital information and, to make a pre-processing to digitally filtering the data and obtain the response of the sensor; iii) battery management for supplying the collecting data and the sensor electronic modules. Minimization of the power consumption of the acquisition system (including the sensor itself and all the electronics needed in the measurement system); iv) transmission of the collected data and some status information to the central surface module. However, during the last 3rd period, the objectives include the development of a backup system. It will allow to store the acquired data in a backup flash memory embedded in the microcontroller. It includes the different solutions to extract these data, deplete and initialize the backup for new measurements.
Within this WP, the Gateway module was designed, constructed and integrated in the 1st, 2nd and 3rd prototypes. The main results include:
• The development of the acquisition electronic system during the 1st period of the project. The electronic board was designed and fabricated to allow the measurement of the sensors and the whole system functioning. The first prototype was integrated in the Mote and tested during the 1st prototype trials. The electronic board was tested in laboratory and in offshore environments for some hours, proving their functionality and reliability. The developed system was integrated in the Mote structures with the other components (μFS, SS, battery, reservoir, OMHSC...) and was tested in laboratory environment during the 3rd trial meeting in Girona (March 2015) and in offshore tests in seawater during the 1st case study meeting in Sivota (April 2015). In both cases measurements were repeated automatically, proving the full functionality and robustness of the systems.
• An entirely new integrated unit was designed and fabricated for the 2nd prototype during the 2nd period to accomplish with the new measurement technique (solid phase extraction column and continuous flow through the bioSensors microchambers). The new unit, significantly smaller than in the 1st prototype system, was integrated into the new Mote and successfully tested in the Bergen/Norway test field.
• Finally, for the 3rd prototype, the BioLab on a Chip System electronic board was re-designed and applied to the new bioSensor µChs feeding during the 3rd period. These new µChambers feeding were built according the liquid multiplexing method producing a pulsing flow through µChs (European Pending Patent number EP17382269.3 14 claims). In addition, the backup system embedded in the flash memory of the bioLCS was successfully implemented. Furthermore, a new maximum mean value dynamic algorithm was developed in close collaboration with partners from WP4. Finally, the whole system was tested in CSIC (Barcelona), and at the last long-run test field in Olhão/Portugal.
WP6: ELECTRONIC SURFACE SINK SYSTEM (GATEWAY)
The overall objective of the WP6 has been to design and develop a reliable wireless communication and data transmission layers to wirelessly send Mote sensor collected data to the shore based Gateway and, then, to the cloud to be analyzed and finally presented to the end-users.
Definition of a Reliable, Flexible and expandable HW and SW Architecture.
The main results of this WP include:
• Development of sub-module Prototypes and Updated/Refined HW and SW for the 1st prototype.
• Improvement of the collector modules for the 2nd and 3rd prototypes.
• Integration of Submodules.
• Development of HW and SW tools to perform Testing in order to optimise functionality.
• Successful Stress Testing of HW and SW.
• Successful operation during different trials.
WP7: SYSTEM INTEGRATION
The work in WP7 has been dedicated to the comprehensive work of creating a technical framework for the integration of the different subunits that builds up the SEA-on-a-CHIP device for the 1st, 2nd and 3rd prototypes and the power management.
The focus in WP7 has hence been focused on harmonizing the extensive technological development work performed in WP2-6. Iteration of relevant question formulations related to the adaptation of the different subunits did result in an efficient workflow towards the integration of the first SEA-on-a-CHIP prototypes.
The main results during different project periods include:
• Integration of the first prototype system during the 1st period. It includes: power management, the integration of the different modules (underwater unit, surface water unit and data-collector) and the watertight underwater module with easy accessibility to the battery and to the reservoirs for refilling.
• During the 2nd period, the 2nd prototype system was refined including the same devices as the 1st one. However, in this case, a notable reduction of the volume was achieved thanks to the pumps and valves size reduction, compactness and the elimination of the redundant sensors included into the first prototype as system diagnostics.
• In the final phase of the SEA-on-a-CHIP project, the work regarding integration was focused on creating a refined technical framework of the further developed subunits that build up the SEA-on-a-CHIP device. The different subunits (i.e. microfluidics, immunosensor, liquid handling and storage, data communication/transmission and power management system) have all gone through extensive development and refinement throughout the project and were finally proved highly functional in the final long-term test period performed in Olhão, Portugal during March-May 2017.
WP 8: REMOTE APPLICATIONS SOFTWARE
The main goals of this WP have been: i) to get the data transmitted by the sensor network and maintain the records data-base ordered by time; ii) to display the data information to the final user in easy way and implement a friendly graphical user interface and; iii) Send commands to the remote sensor network to modify the performance of the sensor itself (frequency of analysis, set up clean-up procedures). Within the different periods of the project, the main achievements were summarized below.
• The Beta version of the users’ software for analytical results interpretation and diagnostics was developed during the 1st period to be used in the 1st prototype.
• An advanced version of the users’ software for analytical results interpretation and diagnostics was developed during the 2nd period.
• The final version of the users’ software for analytical results interpretation and diagnostics was developed and successfully tested during the last trial of the 3rd period with the 3rd prototype.
WP 9: VERIFICATION AND VALIDATION: CASE STUDIES
The main objective of WP 9 has been to test and validate the performance of the SEA-on-a-CHIP biosensor system through comparison with conventional analytical chemistry and to demonstrate its potential for in situ environmental and aquaculture related applications. With this aim, different trials to test some prepared blind samples with selected compounds during each period were carried out.
The main results include:
• A verification and evaluation of the whole system functioning as well as the different parts of the 1st prototype by means of a step-by-step approach that included: laboratory evaluations, mesocosms trials and a sea trial in aquaculture facilities for the two selected compounds.
• A verification and evaluation of the whole system functioning during the 2nd and the 3rd periods for the 2nd and 3rd prototypes, respectively. The second prototype was tested for 3 compounds samples along 7 days trial and comparing the results with the ones reported in laboratory facilities. The results were very promising, considering the incorporation of the new device of solid phase extraction in this prototype. Finally, during the last trial with the 3rd Sea on a Chip prototype for 1 month in aquaculture facilities for 8 compounds it was evaluated the good performance of the developed and miniaturized final device versus the laboratory results.
WP 10: DISSEMINATION AND EXPLOITATION PLAN
The main objectives of WP10 have been i) to ensure exchange of information and transferring the knowledge and the correct evolution of the project to the partners provided by the scientific and administrative coordination; i) to coordinate the dissemination and exploitation of the results and to establish the relationships of the consortium with the end users and authorities through open technical meetings, publications, and newsletters; iii) to interact directly with legislation authorities in order to evaluate the implementation into national and European legislations and; iv) to involve stakeholders to the project.
The main results within the project are as follows:
• A dissemination plan was performed during the 1st period of the project in order to assure the fluency of information between the consortium and to inform outside the consortium about the main achievements and results of SEA-on-a-CHIP.
• Different dissemination activities were conducted according to the dissemination plan during the 2nd and 3rd periods. Also, monthly teleconferences with the WP leaders, consortium, and working/integration meetings were as well carried out to assure the fluency of information between the consortium. Besides, the main results of the project were presented in different international conferences related to the field of the research in the SEA-on-a-CHIP.
Potential Impact:
Potential impact:
The development of a miniaturized unattended immunosensor system for real-time testing and applications in aquaculture with commercialization potential is expected.
SEA-ON-A-CHIP project has improved technology associated to immunosensors for marine pollution control (natural or anthropogenic) with a clear repercussion on related industries such as fisheries and aquaculture facilities. Recent technological developments in the miniaturization of electronics and wireless communication technology have led to the emergence of Environmental Sensor Networks (ESN). These will greatly enhance monitoring of the natural environment and in some cases, open new techniques for taking measurements or allow previously impossible remote deployments of sensors.
Societal implications:
At least 16% of the EU population lives in coastal areas, many more depend on the sea for work, leisure, food or other products and services. Between 3 and 5% of Europe’s gross national product is estimated to be generated directly from marine based industries and services. The chemical contaminations of marine environments influence negatively related coastal industries such as fisheries and aquaculture facilities, but also human health and the general ecosystem. Identification of types of hazards and their temporal and spatial scale are crucial for an analysis of the associated risks. These issues are regulated by law under global, regional or national statutes, such as the EU Water Framework Directive (WFD) (2000/60/EC) and the EU Marine Strategy Framework Directive (MSFD) (2008/56/EC).
During the last decade, a range of global and regional monitoring programs have been developed to protect human and environmental health and prevent economic losses caused by marine hazardous substances in an integrated manner. Among the different groups of pollutants representing a relevant danger to human health, to the environment and to economic resources like fishering and aquaculture facilities, endocrine disrupting compounds are related the decrease of aquaculture production due to their interference with the endocrine system; persistent organic pollutants and marine biotoxins present accumulation and toxicity; antibiotics and pesticides used in the aquaculture production may pose a risk in the environment and the human health. All these elements could have an additive impact on marine habitats and marine resources. To combat this problem early warning systems are required that can provide extreme sensitivity with exquisite selectivity. SEA-ON-A-CHIP project will improve technology associated to immuno-sensors for marine pollution control (natural or anthropogenic) with a clear repercussion on related industries such as fisheries and aquaculture facilities. Recent technological developments in the miniaturisation of electronics and wireless communication technology have led to the emergence of Environmental Sensor Networks (ESN). These will greatly enhance monitoring of the natural environment and in some cases open up new techniques for taking measurements or allow previously impossible remote deployments of sensors.
Dissemination activities:
Different levels of dissemination and communication activities have been carried out:
Scientific publications:
• Peer-reviewed publications: 36 in SCI journals from the 1st quartile most of them,
• Book chapters: 3
• Book of abstracts: 2
Participation in congresses and conferences (National and International):
• Platform presentations: 73
• Poster presentations: 52
Participation in mass media:
• Press releases: 103
• Media brief readings: 62
• Promotional videos: 11
Book:
Editorial invitation from SPRINGER to create (with the participation of the consortium) a volume entitled “Biosensors for the Marine Environment: Present and Future Challenges” to be published either within “The Handbook of Environmental Chemistry” or within the “Springer Series on Chemical Sensors and Biosensors”.
More detailed information can be seen in Annex A.
Exploitation activities
a) Patents:
During the last period, a patent has been generated too: “Devices and methods for multiplexing liquid in biosensor micro-chambers” by Giacomo Saviozzi, Juan Pablo Salvador, Manuel Lopez, Francisco Palacio, Raquel Pruna, M.-Pilar Marco, Cecilia Laschi. European patent application: EP17382269.3. Date: 12/05/2017.
b) Exploitable foreground:
It is expected to generate exploitable foreground with the SEA-ON-A-CHIP final autonomous system to monitor water contaminants on-site sending remotely the results or alarms to the end-users.
List of Websites:
http://www.sea-on-a-chip.eu/V1/SOC50V4_Main.php
Deputy coordinator: Marinella Farré (mfuqam@cid.csic.es); IDAEA-CSIC
