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Sensing toxicants in Marine waters makes Sense using biosensors

Final Report Summary - SMS (Sensing toxicants in Marine waters makes Sense using biosensors)

Executive Summary:
SMS has delivered a novel automated networked system that enables in situ monitoring of marine water chemicals in coastal areas by the detection of a series of contaminants regulated by the Marine Strategy Framework Directive (MSFD). SMS designed a multi-modular apparatus that hosts in a single unit—the Main Box (MB)—a Sampling Module and an Analysis Module. The former contains sample collection and treatment components, whereas the latter includes three immunosensor sub-modules that enable the detection and measurement of algal toxins (i.e. Saxitoxin, Domoic Acid and Okadaic Acid) and a series of standard water quality parameters. The MB is equipped with a communication module for real-time data transfer to a control center, where data processing takes place, enabling alarm functionality to Health Warning Systems, whenever some critical value exceeds a pre-defined threshold. All work culminated in showcasing the project’s results in three demonstration sites: in La Spezia, Italy, in the Slovenian Adriatic Sea and in the Alonissos marine park in Greece. These three sites were chosen because they range from anthopegnic to chemical contamination to pristine conditions to offer a wide range of test conditions for monitoring. The consortium took advantage of different skills from industry and academia to address the objectives reported. The technology development has seen a multi-sectorial team of experts interacting with endusers and marine water stakeholders, demonstrating that ICT, biotechnology and nanotechnology can increase the potential of biosensors for marine applications.
Project Context and Objectives:
The main objective, among the others, of the SMS project was the real-time in situ monitoring of marine water chemicals in coastal areas by the detection of a series of contaminants regulated by the Marine Strategy Framework Directive (MSFD). SMS designs. For this purpose a multi-modular apparatus that hosts in a single unit—the Main Box (MB)—a Sampling Module and an Analysis Module was built . It contains sample collection and treatment components, whereas the latter includes four biosensor sub-modules that enable detection and measurement of algal toxins and their associated algal species, sulphonamides and a series of standard water quality parameters. The MB was equipped with a communication module for real-time data transfer to a control center, where data processing takes place, enabling alarm functionality to Health Warning Systems, whenever some critical value exceeds a pre-defined threshold. This instrument has been tested on a floating buoy positioned in loco at defined locations. All work culminates in showcasing the project’s results in three demonstration sites: in La Spezia, Italy, in the Slovenian Adriatic Sea and in the Allonisos marine park in Greece. These three sites were chosen because they range from anthropogenic to chemical contamination to pristine conditions to offer a wide range of test conditions for monitoring. The technology developed together within a multi-sectorial team of experts interacting with end-users and marine water stakeholders, demonstrated that ICT, biotechnology and nanotechnology can increase the potential of biosensors for marine applications.
The work was structured around three building blocks that interact with each other, whereas each block contains individual work packages: The first block deals with sensor development and adaptation for field use and includes WPs 1 through 4, with each WP developing independently a different type of biosensor, targeting a different pollutant group. Thus, WP1 focused on the development of optical aptasensors for four marine algal toxins (saxitoxin, palitoxin and okadaic and domoic acids), which are found in seawater in concentration levels of ng/l and accumulate in seafood, presenting serious threat to human health, because they can easily enter the food chain. WP2 focused on the development of a biocide detection microfluidic platform using nanotechnology principles to detect the relevant-to-marine-transport chemical tributyltin before it was definitively banned as a biocide compound in antifouling ship paints. Using the same techniques, it develops platforms for the herbicide diuron and the flame retardant pentabromodiphenyl ether (pentaBDE). WP3 focused on the development of an electrochemical sensor for the pharmaceutical product sulphonamide—one of the most frequently described antibiotics—found in human and animal excretion and ending up in treated municipal wastewater discharged to the sea. Finally, WP4 focused on the development of a new on line toxic algal biosensor and field-test the biosensor. Traditional water quality parameters, such as temperature, pH, conductivity, Dissolved Oxygen (D.O.) and nutrients were also measured because they are already available for use on the buoy systems in our project.
The second building block of this work plan was “system integration for field use” and includes 3 WPs each focusing on the development of units and prototypes that achieves the electronic engineering integration of all different modules developed in the first block together with a sampling device that automatically collects, filters and pre-concentrates samples preparing them for in situ analysis and allocation to the unit prototypes. Real-time data collection was realized through a wireless transmission system that includes a low-cost embedded device with processing and communication capabilities that enable communication with the Central Node over the Internet.
The third building block concentrated on validation in laboratory and field tests. In this important step, the modules built was validated for successful functioning in the laboratory and then mounted onto the buoy (at the three demonstration sites of SMS).

The objectives have been almost fulfilled thanks to a substantial amount of planned work, including collaborations among partners and across disciplines, which made possible to reach mostly of the objectives of the project.
UTV reached the objectives related to the development and the assemble of a multi-sensing platform for Saxitoxin (STX), Okadaic acid (OA) and Domoic acid (DA) detection (Multi-biosensor for the detection of marine algal toxins, D 1.1; month 24). Two main approaches have been developed during the full duration of the project. 1) Optical detection of algal toxins using nucleic acid aptamers. Different nucleic acid aptamers have been selected by Aptagen and fully characterized in terms of analytical performance by U2; Due to the low affinity and stability of DNA-based aptamers, none of them showed the required binding affinity, specificity and long-term stability to be further employed for the final application in the floating buoy. Thus we developed alternative assays to detect algal toxins according to the contingency plan. 2) Enzyme Linked Immuno-Magnetic Colorimetric assays for Okadaic Acid (OA), Saxitoxin (STX) and Domoic Acid (DA) have been developed and completely characterized in terms of analytical performance (Task 1.1 – 1.2). The colorimetric assays have been fully integrated into the automated flow prototype developed by Systea. A multi-toxins sensing device with high selectivity and sensitivity has been developed (Task 1.3). During this period U2 worked in close collaboration with Systea to optimize the analytical performance of the automated prototype. Moreover, relevant interactions have been also developed with NIB and ENEA partners, for in situ measurements of target analytes and for the validation of the analytical method respectively.
ICN2 was responsible for the development of sensing methods for the brominated flame retardants (PBDE), pesticides (Diuron) and anti-fouling compounds (TBT). Also ICN2 subcontracted Aptagen, a company responsible for the selection and synthesis of aptamers toward PBDE, Diuron, TBT. Unfortunately, Aptagen met a lot of difficulties during the process of aptamers selection. Therefore, we have received the first samples of aptamers between May and July 2017. Due to that, it was impossible to evaluate them, neither to develop any sensing approach. Being aware of these difficulties mentioned above, ICN2 in agreement with other partners, decided to run a contingency plan for PBDE and Glyphosate detection (new analyte). We performed all the necessary tests and proved that these approaches achieved the low limit of detection and are compatible with the seawater. Therefore, these methods were expected to be implemented in the prototype device. Despite the good performance of PBDE and Glyphosate tests, Systea (responsible for the prototype development) found the application of Glyphosate test too complicated due to the presence of organic solvents (derivatization step) which are not compatible with the tubing and other parts present in a prototype device. Therefore, the main effort was focused on the development of the prototype for PBDE however, the results obtained by Systea were not satisfactory. ICN2 were able to report several new detection approaches which benefit from the use of novel nanomaterials such as graphene or quantum dots. Thanks to this, the outcomes of ICN2 work were published in high-impact journals and therefore stimulated the academic community.
The objective of the WP3 was the development of electrochemical sensors based on nanostructured material for fast and sensitive detection of sulphonamide in marine environment. A various kinds of carbon nanomaterial were tested for sulphonamide detection. Furthermore, UH2MC and ICN had a strong collaboration by development of a nanocomposite material targeting sulphonamides via electrochemical impedance spectroscopy, which showed a high specifity and sensitivity for sulphonamide detection in marine water. Beside the electrochemical method, and as required by our SMS project partner W.P.7 (SYSTEA) UH2MC developed a sensitive spectrophotometric method for the determination of sulphonamide derivatives. A series of sulphonamide derivatives were analysed and tested using the developed automated µMac-Smart portable analyser developed by SYSTEA, which reached a ppb concentration levels.
The sample preparation of sulphonamide using the optimised pre-concentration procedure based on Oasis HLB columns and coupled to the spectrophotometric method was tested successfully for sulphonamides detection in real seawater samples.
To confirm the reproducibility of the method, the procedure was tested in the laboratory of our partner ENEA-Italy. This method shows satisfactory results leading to a good reproducibility of the procedure. Furthermore, sulphonamides were measured using the procedure of pre-concentration coupled with UPLC-MS. As an alternative,UH2MC developed a novel, easy and low-cost colorimetric method based on an android smartphone for sulphonamides determination. The developed technique was compared to the spectrophotometric method and the results obtained from both methods were well correlated.
MIC reached the objectives related to: a) Adaptation and improvement of a ELISA assay coupled to both colorimetric and electrochemical detection for the most important Mediterranean toxic microalgae species b) Calibration and validation at lab scale for 10 toxic species with environmental samples in batch mode c) Design of the fluidics analytical procedure for a new biosensor for single & multiple targets d) Proof of concept level for the autonomous toxic microalgae species biosensor in a disposable microfluidic cartridge format suitable to the adaptation in an autonomous analytical system e) Adaptation of MIC’s analytical procedure to SYS’s microMAC analyser system to build up the ToxAlg biosensor; f) Development and test of a new prototype for the pre-analytic part of the system comprising the automation of sampling, cell concentration and lysis procedures in collaboration with SYS (WP5). Perform the laboratory validation tests with the ToxAlg biosensor provided by SYS (WP8) and the system integration, buoy adaptation and in situ deployment (WP9) in collaboration with NIB and SYS.
SYSTEA partner developed the following module automated prototypes according with objectives of WP5: water sampling and SPE preconcentration unit was completed and then tested in laboratory by ENEA, coupled with the sulphonammide modular automated measurement prototype developed under WP6; water sampling cell preconcentration and lysis unit, coupled with specific toxic algae species modular automated measurement prototype developed under WP6, that was tested in laboratory and on the field; two 0.1 microns cut-off automatic filtration devices were improved and used in both field experiments under WP9: the first one coupled with WIZ in-situ nutrient probe in the NIB coastal buoy in Piran, Slovenia and the second one coupled with the algal toxins modular automated measurement prototype developed under WP6.
Furthermore the following modular automated prototypes based on proprietary µLFR fluidics were finally developed by SYSTEA. Sulfonamide module for sea water measurements was extensively tested in laboratory and finally coupled with the sampling and SPE preconcentration unit finalized under WP5. Algal toxins in sea water (domoic acid, saxitoxin and okadaic acid) measurement module was successfully tested in laboratory and in the field in the floating platform of La Spezia, Italy (under WP9). Toxic algae species in sea water measurement module was successfully tested in Microbia’s laboratory (WP4), coupled with the sampling, cells preconcentration and lysis automated module developed under WP5 and then the whole system was tested on field in the NIB coastal buoy in Piran, Slovenia (under WP9). PBDE and gluyphosate measurement module was developed and tested in laboratory. A multiparametric nutrient probe for the sequential analysis of ammonia, nitrite, nitrate and phosphate was built and installed and extensively tested on field in the same NIB coastal buoy in Piran. A probe suitable for the automated measurement of Sulfonamides in seawater was successfully designed, built and deployed in Volos, Greece.
Throughout the duration of the project UTH successfully managed and fulfilled the two main objectives that were required from WP7. UTH submitted two deliverables for the WP7 (M36 & M39) that described the process towards implementing the objectives of the work package. More specifically, UTH has developed a prototype communication device to be installed on SMS buoys and floating platforms in order to serve as the backbone plane between the remote sensing units and the experimenters/users. The communication device is connected with the in-field sensors in order to acquire their measurements and in turn to upload them to the SMS database for further processing and visualization. The prototype device features a vast number of communication technologies, able to establish network connection even in the most remote places. More specifically, the device features WiFi, LTE, LoRa, ZigBee and Iridium wireless interfaces. The idea was to exploit the best communication in each application scenario, towards saving as much as possible energy. Due to the fact that the device operates through batteries, it is of paramount importance to minimize the power consumption profile of the whole system as much as possible. To this end, UTH partner designed and implemented a power efficient scheme that orchestrates when each component should be active consuming energy and performing its task and when should be set in sleep state in order to save energy. Furthermore, UTH visited the NIB premises in Piran, Slovenia to install the prototype communication device on the NIB Buoy. Finally, in the context of the WP7 UTH designed and implemented an intuitive web GUI that enables the experimenters to monitor the measurements acquired and also control and manage the sensors deployed in each site through the SMS web GUI.
In accordance with WP9 objectives Partner NIB and ENEA accomplished the assembly, installation of developed biosensor prototypes in a floating platform (La Spezia, Italy) and on the buoy Vida (Bay of Piran, Slovenia) and their testing in natural environment. Then they also selected the sampling in National Marine Park of Allonisos (Greece; the largest marine protected area in Europe with limited pollution) to estimate the sensitivity of developed sensors; ENEA and NIB also monitored the ambient characteristics (state of the sea) monitored during in situ testing of the probes in their sites. Special attention was payed to monitor the biofouling effects on in situ installed biosensors. Various tests were performed in laboratory conditions to demonstrate the operational and functional performance of new biosensors. Identification of practical guides and useful information regarding the installation, maintenance, functioning and further improvement of developed biosensor prototypes have been provided.

Project Results:
Here below a bullet point list with the main S&T results from each WP.
WP1 – Biosensor for marine toxins.
UTV was responsible for the development of sensing methods for the algal toxins detection (i.e. saxitoxin, palytoxin, domoic acid and okadaic acid).
• A colorimetric assay based on the protein phosphatase-2A inhibition for Okadaic Acid detection has been developed and automated in a prototype produced by Systea.
• Enzyme Linked Immuno-Magnetic Colorimetric Assays, ELIMC for the detection of STX, OA and DA (Multi-biosensor for the detection of marine algal toxins, D1.1 - M24) have been developed and integrated in the system. A multi-toxins sensing platform with high selectivity and sensitivity has been developed. The prototype integrated with the multi-toxins sensing device was able to autonomously measure the concentration of OA, STX and DA in the range of low ppb dynamic range without any pre-concentration step in less than two hours.
• A multi-toxin sensing device with high selectivity and sensitivity has been developed (Method of analysis of algal toxins to be applied in laboratory in seawaters, D1.2 - M30) and it is actually operating in the buoy of La Spezia. Enzyme Linked Immuno-Magnetic Colorimetric assays for Okadaic Acid (OA), Saxitoxin (STX) and Domoic Acid (DA) have been developed and completely characterized in terms of analytical performance (affinity, specificity, detection limit, reproducibility, etc.).
WP2 - Biosensor for Biocidal and flame retardant Compounds.
ICN2 was responsible for the development of sensing methods for the brominated flame retardants (PBDE), pesticides (Diuron) and anti-fouling compounds (TBT). The main challenge of this work was to obtain such detection methods that allowed measurements in seawater and achieve the low limit of detection (LOD) required by the EU legislation. Although several novel approaches have been developed (PBDE, TBT) they couldn’t be applied in the prototype device. Moreover, due to difficulties linked to the chemical properties, no method for Diuron has been developed.
• ICN outcomes were published in high-impact journals (11 publications) and therefore stimulated the academic community.
• Several types of PDMS-based microfluidic chips have been developed mixing, electrochemical detection, optical detection, sample incubation;
• Novel composite PDMS-reduced graphene oxide (rGO-PDMS) has been synthesized and characterized. This material was found as an effective adsorbent of PBDE;
• A modular Lab-on-a-chip (LOC) platform for PBDE detection has been developed: LOC consists of incubation chip, detection chip (electrochemical) and removal chip (rGO-PDMS). The limit of detection was 0,018 ppb; this platform is suitable for the application of real samples, including seawater.
• MIP (molecularly imprinted polymers) which can specifically bind TBT and graphene quantum dots whose fluorescence depends on the amount of TBT bounded to composite (mSGP) have been developed. The limit of detection was 12,8 ppb (buffer) and 42,6 ppb (seawater).
WP 3 - Electrochemical sensor for sulphonamide.
The aim of this electrochemical method was the study of several types of electrodes based on carbon nanomaterial in order to choose the most sensitive for the determination of sulphonamides.
• The electrochemical determination of Sulfamethoxazole(SMX) on a variety of electrodes such as Carbon black (CB) nanoparticles N110, N220, N375, N772, Graphite, Carbon Nanopowder, Acetylene black (AB), Multiwall Carbon Nanotubes and Glassy carbon pastes were demonstrated. Cyclic Voltammetry (CV), Linear Sweep (LS), Differential Pulse (DPV) and Square Wave Voltammetry (SWV). Seven derivatives of sulphonamides (Sulfadiazine, Sulfacetamide, Sulafathiazole, Sulafamethiazole, Sulfamerazine, Sulfamethoxazole, Sulfadimethoxine) were fully characterized and determined by electrochemical method at a conventional carbon paste electrode based on graphite. The low limit of detection obtained by electrochemical method was 0.100mg/L 0.086mg/L 0.108mg/L for Sulfadiazine, Sulfacetamide and Sulfamethiazole respectively.
• The spectrophotometric method has been developed using the Griess test (nitrite assay). The protocol was reversed to measure sulfanilamide; This method permits the determination of sulfonamide derivatives in the range of μg/L.
• Pre-concentration and clean-up techniques mainly focus on the solid-phase extraction (SPE) based on different types of adsorbents, such as Oasis Hydrophilic–Lipophilic Balanced (HLB) have been developed.
• Test and validation of the developed sensors in the seawater and integration of them in the MB to be used on marine platforms and Buoys.
• A series of sulphonamide derivatives were analysed and tested using the developed automated µMac-Smart portable analyser, which gave an ng/mL concentration levels as a detection limit for the all tested sulphonamides.
• A novel, easy and low cost colorimetric method based on an android smartphone for determination of sulphonamides in the concentration range of 0.5 - 2.5 μg mL-1 has been published.
• A simple and more convenient device was created and developed as a tool for determination of sulphonamides, by using a colorimetric analyzer based on a camera integrated in a smartphone.
• The software program ‘’Sulphonamides Analysis’’ was developed, in the android operating system, to be used with the smartphone for collecting and analyzing the RGB color information of the picture and to carry out the colorimetric analysis of sulphonamides.
• A validation of the new method has been made by using the spectrophotometric method. The results obtained from the both methods correlated well, with detection limits of 0.11 and 0.12 μg mL-1 for the smartphone method and spectrophotometric method, respectively.WP.3 was participated in the tests performed in the selected marine sites.
WP4 - Biosensor for Toxic Algal Species.
MICROBIA ENVIRONNEMENT (MIC) conceived a brand-new prototype for toxic algae detection based on proprietary experimental assay. The original ALGADEC prototype showed relevant defaults that could not be corrected without a complete redesign. The new prototype was realized by SYSTEA and all components and microfluidic pathways were tested.
• Magnetic particles as support for the sandwich hybridization immune assay for toxic species identification (Biomagnetic assay) have been developed.
• MIC performed all calibration curves with RNA from toxic target species for the selected probes in lab-based assay. Colorimetric detection was identified as the more adapted for buoy integration. Relevant results were obtained in terms of sensitivity, the lowest detection limit obtained was of 1 ng/μl of RNA corresponding to a range of 20 to 500 of cells depending on the species tested.
• MIC conceived, designed and tested an extra pre-analytic module to concentrate algal cells in seawater and to perform cell lyses and SYSTEA built the automated device.
• MICROBIA ENVIRONNEMENT and SYSTEA finalized the prototypes’ testing and optimization experiments last July in Piran buoy Vida in a 15 day in situ deployment. This task was performed and detailed in the previous report and Deliverable D4.1.
• MIC achieved the goal of expansion of the repertoire of species detected on the ALGADEC through the design of new probes.
• Construction of a database of 172 probes for the identification of around 80 toxic species/groups.
• A brand-new prototype was conceived and designed by MIC and realized by SYSTEA (See also Deliverable 6.2).
• A protocol with magnetic beads bound to biotin labeled capture probes was developed (Biomagnetic assay). The protocol was developed and optimized to detect the genetic signatures of target toxic algae using both electrochemical and colorimetric detection systems.
• To validate the automation of the bench protocol developed by MIC to detect toxic micro algal species MIC used Lab-on-a-chip (LOC) cartridges (Deliverable 4.2).
• Development of an automatic pre-analytic module (See also WP9 Deliverable 9.1 and 9.2) to optimize cell pre-concentration and cell lysis tool (See also WP9 Deliverable 9.1 and 9.2).
• Adaptation and improvement of a sandwich hybridization immunoassay (SHIA) coupled to both colorimetric and amperometric detection for major Mediterranean toxic microalgae species (D4.3 - Orozco et al. Talanta 2016).
• Calibration and validation at lab scale for 10 toxic species with environmental samples in batch mode (D4.4).
• Design of the fluidics analytical procedure for single & multiple targets of toxic algae (D4.2) for the adaptation to the Systea’s MicroMac analyzer.
• Proof of concept level for the autonomous HAB biosensor in a disposable microfluidic cartridge format suitable to the adaptation in an autonomous analytical system as the micro loop flow analysis (uLFA) technology from SYSTEA (D4.2).
• Conceptualization and design of a pre-analytic system comprising: sampler, cell concentrator, cell lysis and reconditioning of the whole system for automation (M36-M45 Periodic Report and D9.2)
WP5 – Sampling and pre-concentration device development.
The prototype for sampling and preconcentration provided by Acromed did not function properly. In particular, the integration of all different parts has been a total failure. ACROMED refused to make amendments and, for this reason its activity was stopped by the coordinator in agreement with all the partners and substituted by SYSTEA .
• A sampling pre-concentration device for the analysis of toxic algae was designed, realized and tested by SYSTEA in collaboration with MICROBIA.
• SYSTEA designed and realized a water sampling and 0.1 micrometer cut-off filtration module with autocleaning by the same filtered water, that was used in Piran and La Spezia where it was connected with the modular prototype to measure algal toxins.
• A module for cell pre-concentration and lysis to be used in conjunction with the modular automated prototype for the detection of toxic algae has been developed.
• Device N. 1. For the SPE-based device, the functional stages included: sample injection, analyte separation, eluent evaporation, sample recovery.
• Device N. 2. A sampling device that collects seawater samples and performs algal cell pre-treatment processes, such as pre-concentration and lysis, and delivers the sample to the toxic algae sensor module was designed and the process was divided in the following stages: sampling, cell removal, sonication, washing.
• Device N. 3. Based on the previous expertise and commercial products developed before the beginning of the project, a sampling device to perform 0.1 μm cut-off filtration, enabling the automated modules to collect the filtered water sample and allowing the periodic autocleaning by backflush of the same filtered water stored in a bag was improved by SYSTEA.
• Development of the fluidic, optical, electrical and mechanical interfaces necessary for unattended operation and remote control of the different functions of sample treatment and analysis. The system was built around a Raspberry Pi 3 board, which was characterized by sufficient processing power capabilities (1.2 GHz 64-bit quad core ARMv8 CPU with 1GB RAM), low power consumption and several communication ports (4 USB ports, Ethernet port, Wireless LAN, Bluetooth 4.1 Bluetooth Low Energy, Full HDMI port and others) for interconnecting external hardware. A 7’’ touch screen was used for an easy and friendly user interface. The Peristaltic pump and Syringe pump were both connected via the two USB ports of the Raspberry Pi board. These pumps were controlled by RS232 commands.
• For the vacuum pump and Teflon chamber set-up, Arduino open-source electronics platforms were used for advanced control.
• A sampling device that collects seawater samples and performs pre-treatment processes, such as filtration and pre-concentration, and delivered the sample to the sensor modules was designed and constructed. Two versions of the system were built, in order to allow more simultaneous tests, validations and modifications.
• To concentrate and lyse the toxic algae cells eventually present in sampled seawater, a device based on micro hollow filter and sonication was developed.
• System validation and optimization (ACR then SYS, MIC, ENEA, ICN, UTH, UH2MC) System validation and optimization of the sampling system were carried out in the lab for each individual sensor system and with all sensors together and finally at several field tests for successive optimisation and validation of the complete system.
WP6- Development of modular automated measurement prototypes and Local Control Unit.
In the present project SYSTEA S.p.A. adapted an impressive variety of different procedures based on optical detection methods into a common configuration based on the μLFR technology and different modular automated measurement prototypes for the colorimetric detection of the analytical targets, suitable to be installed on field in a floating platform or a coastal buoy, for seawater measurements, were in the first place designed, built and tested.
• Measurement units providing automation of the antibody–based methods for the detection of PBDE, Glyphosate, Okadaic Acid, Domoic Acid and Saxitoxin were designed and realized to be calibrated and tested directly by academic partners ICN2 and University of Tor Vergata.
• Two multi–parameter probes for nutrient measurement and sulfonamide determination were constructed in SYSTEA and successively delivered to partners National Institute of Biology and University of Thessaly, respectively.
• Overall, seven on–line sensors (five portable instruments and two submersible probes), six of which were completely original models, have been designed, realized and tested within the frame of SMS project. As for the antibody-based modular prototypes, their architecture was identical to that used for the pioneer prototype realized for the identification of toxic algae species. The five portable on-line sensors integrated in the modular microfluidic system, respectively allowed: (i) SAA detection, (ii) enzyme–based detection of Okadaic Acid, (iii) identification of different toxic algal species, (iv) antibody–based measurement of marine toxins Domoic Acid, Okadaic Acid and Saxitoxin, and (v) antibody–based measurement of flame retardants PBDEs and herbicide Glyphosate.
• Design, realization, functioning and validation of the magnet system for toxix algae detection.
• µLFR modular modular microfluidic system.
• SYSTEA had the initiative to design and build a dedicated portable spectrophotometric/ fluorimetric analyzer which is fully controlled by an integrated PC with incorporated colour Touch Screen and USB ports, specifically addressing at site monitoring of seawater resources, to automate the measurement methods developed by the other partners in WP1-3, after the pre-concentration steps that were developed in WP5.
• The analyzer was integrated in a robust and easy-to-transport plastic case including room for reagents and all other potential accessories.
• In the end, five modular automated measurement prototypes (rather than three) were specifically designed to manage (i) a colorimetric method to measure sulphonamides developed in WP3, (ii) an enzyme-based fluorimetric method to measure Okadaic Acid developed in WP1, (iii) a colorimetric method to detect and quantify toxic algal species in the sea developed in WP5, (iv) immuno-magnetic colorimetric methods using antibodies developed in WP1 for algal toxins detection and (v) immuno-magnetic colorimetric methods using antibodies to measure biocidal and flame retardant pollutants developed in WP2.
• Multi–parameter probes for nutrient measurement and sulfonamide determination have been also developed.
WP7 - Integrated monitoring system and data management.
UTH has developed a prototype communication device to be used in buoys and floating platforms that are deployed for the purposes of the project. This prototype communication device features a number of different communication technologies that can be utilized under different scenarios and deployments. More specifically, the device features WiFi, LTE, LoRa, ZigBee and Iridium wireless interfaces.
• UTH has further developed the prototype communication device and its software in order to prepare and adapt it for a deployment in the NIB Vida coastal buoy.
• Several protocols and software has been developed to enable remote control mechanisms and management for the sensors developed in the context of the SMS project.
• The control and management of the sensors has been integrated in the SMS GUI which was extended to include the newly developed features.
• SMS GUI platform was configured to include and illustrate the measurements acquired from the SYSTEA commercial WIZlog data-logger, which was provided by SYSTEA to NIB during the previous year.
• Development of the data gathering/monitoring software and the corresponding graphical user interface. UTH developed a novel communication device to be used within the project for the monitoring and gathering of measurements acquired by the sensors developed in the SMS Project. This communication device features a number of devices in order to implement the required communication protocol.
• Development of the remote management protocols. For the purposes of the Task 2 UTH had to develop data gathering and monitoring software that enabled remote management of the sensors and automatic data collection.
• Implementation of remote control protocols and the integration of such mechanisms in the scope of the web GUI.
• UTH’s prototype communication device has extended to meet the requirements of the deployment in the NIB Vida Buoy.
• Deployment on the NIB Vida Coastal Buoy in Piran, Slovenia. Real-time measurement collection and illustration system. SMS’ remote sensor control and monitoring platform developed.
WP9 – Assembling the modules on selected sites and field tests.
• Assembly of analytical modules in a floating platform/buoy Vida (Bay of Piran).
• The toxic algae biosensor prototype developed by SYS and MIC was deployed and tested on NIB buoy Vida in the Bay of Piran.
• On the buoy Vida, also a new communication device (by UTH) was successfully integrated and tested. Testing biosensor prototypes in field conditions provided us with important information about sensor’s functionality and their maintenance.
• UTH team develop a new communication device, which was installed in the oceanographic buoy Vida and was tested on site with WIZ nutrient probe.
• The platform was equipped with solar panels, batteries, dataloggers, water level sensor, algal toxin prototype and sampling filter (0.1 µm).
• ToxAlg prototype developed by partners MIC and SYS was installed inside the buoy Vida. Two modules were installed, one for pre-concentration and cell lysis and the second unit for two toxic algae species detection (Alexandrium minutum and Pseudo-nitzschia spp.). The integrated ToxAlg biosensor is able to sample, concentrate, lyse and perform the detection of the target algal species through a combination of molecular methods that recognise the target’s unique genetic signature. The results are obtained in situ by a built-in colorimetric detection and the data is transmitted via SYS’s sea-side communication device installed on the buoy.
• UTH communication device was installed on the buoy Vida.
• Pre-concentration sampling device finalization achieved.
• Second deployment of WIZ probe on oceanographic buoy has been achieved.
• Field test in the Alonissos Marine Park has been performed.
• Control sampling and collection of phytoplankton samples has been performed.
• The following parameters: salinity (S), temperature (T), winds, dissolved oxygen concentrations (DO) and Photosynthetically Active Radiation (PAR) were measured during in situ testing of the WIZ probe and ToxAlg biosensors installed on the buoy Vida.
• Water samples were taken near the buoy Vida for the analyses of Chlorophyll a concentrations and phytoplankton community structure once per month.
• Additional samples for determination of toxic phytoplankton were taken at sampling stations located in three shellfish farms and were determined and counted under an inverted microscope.
WP10 - Exploitation, training demonstration and Dissemination, Intellectual Property
ALI developed communication tools and messages taking into account target groups, objectives to be reached (i.e. messages to be sent), also defining clearly also the activities and the expected outputs to be diffused. The targets groups involve people of different expertise and backgrounds related to the project topics. For communication matters, a coherent visual identity has been developed including a logo, and templates to be used by partners.
• Creating and keeping the project website. The SMS website ( was designed for communicating and disseminating key messages about the project and its results to policymakers, the industry, researchers, journalists and the public at large. It was ready by the beginning of month 4.
• A total of 46 original ‘News’ have been uploaded and the website has been visited by more than 8500 users
• The “Real time data” page on the public SMS website was made public in June 2017. This read only page shows all the results of the analysis made on field in Italy, Slovenia and Greece.
• In addition, ALI manages different social media channels for SMS in order to further disseminate the available material and the information included in the website to reach a larger number of people.
Potential Impact:
The potential impact that was attained by the completion of this project is high. The main reason was the development of a device located in marine areas where there is the need to close monitoring in real time the compounds selected in this proposal. The public organizations devoted to the control of coastal areas can rely now on a powerful tool able to give in real time information of the status of the waters obtaining information on a single analyte with the possibility to provide high quality of the data with a substantial reduction of the costs. All marine operators working in the coastal areas and operating on the buoys located in the places where there is high probability of pollution will receive useful information in real time and will be able to start the activities of remediation and alert the local governments.
The impact is extended to all the coastal areas Worldwide. In fact our SMEs working in the project were able to automate complex measurements that previously were possible only in the labs. the construction and assembling of a device able to measure the toxic algae, also the marine toxins contained in them was considered extremely useful not only by the people working in our marine areas but also by the organizations in US and in China where the problem of the toxic algae and their toxins in growing at exponential level.
The impact of the results obtained at the scientific level is excellent. the biosensor groups working in this project have developed innovative sensors for pesticide, flame retardant, marine toxins and sulphonamide. All this sensors have improved the detection of the selected compounds in terms of precision, accuracy and detection limits. The advanced Technologies developed for the flame retardants that are polluting our coasts have been published on the major scientific journal with high impact and several citations up to now. Also the new DNA technology has allowed the construction and assembling of a novel sensor for the detection of domoic acid that detects this toxin at ppb levels. A scientific impact that has led to the construction and assembling of a new instrument has been the new optical detection of sulphonamide, a pharmaceutical compound that is often present in the estuaries. This instrument has been used during our experimental activity in the sea areas selected in our proposal and the impact with the in loco operators has been quite positive because thanks to this early warning system the operators could alert the local authorities of the increasing pollution not easily detected earlier.
The impact of SMS project has been in line with the anticipating impact of the OCEAN of TOMORROW 2013. In fact the capabilities offered by the results obtained by SMS are undoubtedly of utmost importance towards improved monitoring coastal areas and thus will allow a higher quality of decision making.

The completion of the SMS project is having a major impact on marine water end users and relevant stakeholders. Through the use of novel Information and Communication Technologies, biotechnology and nanotechnology, SMS has made possible the real-time detection and monitoring of hazardous chemical concentrations in coastal waters. This is enhancing citizen awareness of the anthropogenic chemicals that threaten our marine ecosystems. Considering our dissemination activities during the project, information and coordinating activities with relevant environmental agencies, citizens can be made aware of the benefits of new technologies such as SMS technology for environmental monitoring and will acquire an increased social awareness to coastal problems.
SMS has successfully produced alerts if chemical concentrations exceed a pre-determined threshold; this way, citizens will feel more secure since they will know that through the use of biosensor technologies, a comprehensive seawater quality control is done. In fact, the improved detection and monitoring capabilities brought about by the developed sensors and relative instrumentation will contribute to a more controlled and better quality coastal environment. This can play a major role in restoring public confidence in the value of coastal waters and related maritime activities. . The use of our instrumentation in the selected areas of the marine coasts has an impressive impact on the local operators and will have an important societal impact in the years to come. In fact the SMS has put in place strategies that responded to all these demands.
Furthermore, the ability to detect emerging chemical pollutants, in seawater has been and will continue to be a major issue in environmental control. The subject has consistently been a matter of intense debate between governments and public and private industries. In particular areas, where pollution due to industrial activities and natural events continues to mount, the cost of water treatment and control becomes significant, with estimates putting the economic impact at a really high level. EU citizens must ultimately meet these multi-million euro costs. In this context, our smart biosensor technology, developed and applied to real-time, reliable and inexpensive is providing a solution.
SMS positively affects socioeconomic aspects related to maritime activities, such as the fish industry. Exploitation by them can be accomplished through focused dissemination actions of the project to the Fishery Ministries in the participating countries. Such actions, in combination with the increased confidence in marine water quality can raise citizen satisfaction and pride in their coastal areas and can lead to increasing economical revenues. This aspect is one of the main achievement obtained by our SMEs involved in SMS that were able to build three instruments that now are going to be commercialized.
SMS has responded positively to many of the issues addressed by Ocean 2013. Specifically, the proposed program has allowed SMS to develop a device to be located in marine areas where buoys can be assembled with a device that will give information on the status of the water in real time through the use of a wireless connection and remotely controlled processes.
An important benefit has been the substantial reduction of labor-intensive field sampling and laboratory analysis regimes. Moreover, the construction, assembling and testing of the sensors proved to be successful in terms of sensor sensitivity, detection limit and sensor robustness, then these sensors, linked to the sampling module, and optimized through the engineering integration and software contributed to a significant reduction of errors
Another useful impact is that the instruments are connected with a remote station so the operator not necessarily has to go to the place where the device is installed and this is quite important in terms of reducing labor and time. The combination of a real time data collection concerning the general status of the water (routine measurements) with the toxicity prototypes has lead to an early warning system that could elucidate some phenomena not observed earlier and thus could lead to a better understanding of phenomena and improved seawater management. The most relevant instrument of community water policy is the Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD), both important and ambitious legislations. As far as information management is concerned, these directives have made the new situation more demanding, as it involves all water bodies, all over Europe with ambitious goals. We believe that SMS has facilitated the information management process. The capabilities offered by biosensors, deployment system, and associated management system developed in SMS had a significant importance in an improved monitoring of coastal areas, and thus on better quality decision making.

The results achieved by SMS will promote and strengthen the EU’s scientific and technological excellence in the areas of biosensors, networking and data management technology for application on coastal water quality control and management. European research in biosensor technology as applied to seawater control will be enhanced and optimized, thus creating the conditions for joint and multi-sectorial exploitation and leading to a long-term commitment to integrated training and qualification. Identifying common strategic objectives for underpinning research is another important impact of SMS. Specific actions involving training and public awareness were undertaken with the numerous dissemination activities e.g. training PhD students and postdoc. training marine operators, giving demonstrations during the meetings and inviting experts in the evaluating the progress of our projects giving advices and suggestions.. Strong links between research and citizen education activities are now required in order to develop and insert the necessary resources within the European context so as to bring sensors at the forefront of smart devices, becoming critical elements to reinforce the seawater management. SMS has developed a strong exploitation plan to create a unified vision for future applied research and technology collaborations among Academics and SMEs and to provide both an exchange of personnel and an access to the facilities and ideas necessary to sustain both the fundamental research activity and the technological innovation necessary to deliver sensor-based products to the market. In fact the prototypes for sulphonamide, for the detection of toxic algae and for marine toxins is are in production and will be commercialized. the exploitation plan has been organized by partners Systea and Microbia in collaboration with Alienor. the demonstrations of the results achieved by SMS presented at many international congresses were successful. SMS will have in the future a profound and lasting impact on EU science and technology and will to contribute to the well being of all citizens in terms of improved environmental safety.
The economic impact of the results obtained in this project will be strong in particular areas, where pollution due to industrial activities and natural events continues to mount, the cost of water treatment and control becomes significant, with estimates putting the economic impact at levels reaching billions of € each year. The EU citizens must ultimately meet these costs. In this context, the instruments developed using optical sensor technology applied in seawater monitoring has provided an especially attractive solution. Our developed instruments were particularly amenable to exploitation by SMEs. As has been recognized under the Lisbon Agreement, SMEs are dependent on a climate of investment, innovation and entrepreneurship. Start-up costs are generally relatively low and niche markets can be addressed by the SMEs, which in many cases could then have immense potential for growth. In fact our instrument when presented at the international meeting and during the training and demonstration activities interested all the public and private organizations dealing with the marine activities. In fact it has been observed during this meeting that there is growing technology content in all marine sectors. Internationally, the Marine Technology sector, is characterized by many high-tech sub-sectors including software, underwater connectors, telemetry and communications and control systems. It is within these that the main opportunities for industry, including SMEs, exist. The market for marine technology products will continue to grow as development and management activity in the oceans increases. It will be characterised by increasingly sophisticated solutions involving the synthesis of a wide range of advanced Technologies and SMS results perfectly meet these requirements. The Greenwich Report (Marine Foresight Task Force (2009). The Greenwich Project - A Marine Information Strategy) estimated that the marine information sector would grow from €3bn to €7bn by 2020, with the largest growth areas expected to be in monitoring, forecasting and information systems. This will entail the development of new techniques in observation and data analysis and management.
One relevant scenario would be the well documented cases of start-up companies involving two or three people in the biosensor area that have subsequently grown into corporations with turnovers of billions of euro in the space of 10-15 years. An additional factor is that, unlike some “softer” markets, nanotech products (which can include sensors) have largely ridden out the down turn in financial markets and continue to be regarded as good investments by venture capitalists and other investors.
A vast range of product designs for the future incorporate elements of smart response to the user, but the constructive development of these strategies has been frustrated by the lack of biosensor technology transfer from universities to the private sector.
It was of great importance that two partners of the project is a SME had large experience in water pollution monitoring equipment. All activities in the project were done with the aim of future industrialization and implementation in real life and we think we reached this aim. Dissemination and promotion of the system have been done among potential end-users. This included as reported above, some demonstration activities in the project, finalized to study well specified and worldwide requested environmental problems.
The extensive demonstration and first use of the systems developed in this project has given the start to very precise and generally followed standards to detect threshold concentration limits in water pollution.
Having in the same Consortium two SME industrial companies, one of them already introduced in the specific market sector, producing in-situ nutrients multi-parametric probes and on-line analyzers for water quality analyses, has speed-up tremendously the process of industrialization and launch on the environmental market of the new products.
After a deep market evaluation the prototype that are going to be manufactured by the SME companies and marketed directly using their existing commercial network of distributors. However, it is clear to state that the market level of the new products is not simply Europe but the world market.
The positive economic effects of overcoming this barrier to technology transfer could be enormous. The commercialization of novel devices for real time water monitoring, while offering unique opportunities for business innovation, could also serve more established markets by stimulating the development of technology that complements the products of larger companies. Specifically, the SME Company SYSTEA, one of the SMS project partners, already started in organizing the commercialization of the and applications coming out from this project.
The general public naturally benefits from a wider choice of new and improved consumer products, but the benefits are more significant and direct when one considers the overall impact that application of advanced sensor technology could have on environmental quality, public health via the realization of new, cost effective, wireless monitoring devices.
The practical uptake of such activities on the part SMEs has resulted in the appearance of new products that will be put on the market and this means that SMS has successfully met the requirements asked by the EU for this call.
The main dissemination activities are carried out during the entire project by the research institutions and SMEs involved. one of the big dissemination activity was the demonstration of the instruments developed by partners Systea and Microbia that built a new ALGADEC prototype shown in many meeting allover the world including China and US. All the partners participated to congresses Symposiums and workshops (see list in the dissemination activities area) and the interest of the scientists as well as of the industries and public organizations was high.
The exploitation of the results were carried out using the strategy planned in the proposal: a close and intensive feedback between the SMEs and the research institution, also taking advantage of all the dissemination activities during the entire project. the coordinator managed the exploitation plans by the revision of the scientific activities during the first part of the project: changing the research activity based on aptamers with that of the contingency plan based on the antibodies that was more effective for the objectives of the proposal, deciding to build a new ALGADEC by the expertise of MICROBIA and SYSTEA . This decision resulted fundamental for the detection of the toxic algae., deciding to stop the activity of ACROMED and improving the prototype for sampling and preconcentration that was ready to work autonomously in the field, meanwhile partner Systea investigated all the relevant market segments to put the instruments developed by SMS on the market. Also Systea in collaboration with the other partners exploited the possibility to interact with a wider community to publicize the production of the new prototypes and with the partner MICROBIA made a join exploitation for the market interested in the marine environment control. All these exploitation plans were quite useful to interact with the government organizations and with the companies interested in the use of such instrumentation. One of the main goal of the industrial partners was an attentive examination of the technology transfer from the academies to them and this was done in accordance with the rules of the consortium Agreement. All the IPR management and knowledge management listed in the proposal has been fulfilled.

List of Websites: Contact details: Partner ALIENOR responsible of the dissemination activities Mrs Elise Regairaz []
Coordinator: Prof. Giuseppe Palleschi email address:

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