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FP7

AQUAVIR Report Summary

Project ID: 604069
Funded under: FP7-NMP
Country: Denmark

Final Report Summary - AQUAVIR (Portable Automated Water Analyser for Viruses)

Executive Summary:
Water-borne viral diseases pose high risks for public health worldwide. The urban wastewater contains a large number of pathogen viruses, and even the most advanced wastewater treatment cannot remove all virus particles.
The conventional biological water quality indicators do not provide adequate information about the presence of pathogenic viruses. The currently available reliable virus test - based on molecular biology - is expensive, time consuming and labor intensive, thus limited to few laboratories with sophisticated facilities and well-trained personnel, even though the protection of water networks against pathogenic viruses is crucial.
In the AquaVir project we aimed to develop a novel, cost effective, portable, on-site detection system, which is capable for monitoring human enteric viruses in different freshwater bodies.
The Portable Automated Water Analyser for Viruses (AquaVir) is a three-year FP7-funded project designed to develop a monitoring system for detecting pathogenic viruses in different water sources. In the project 14 partners from universities, research institutes, manufacturers, laboratories, end-users and standardization bodies worked together from Denmark Germany, Portugal, Romania, Sweden, Belgium, Hungary, and Italy.
The Aquavir Virus Monitoring system is intended to detect virus particles in a concentrated water sample on basis of electrical readout. The measurement data can be sent to a monitoring station.
We have developed a computer controlled automated system, which consists of a water sampling and filtering unit and a virus concentration and detection unit based on the input from the end-user partners and on the required virus detection limits.
The system comprises two plastic chips as core components fabricated with state-of- the-art mass producible micro- and nanotechnologies. In these chips, the viruses are concentrated and detected, respectively. The data from the measurements can be sent to a monitoring station, which is used for early warning and risk assessment for infections by the pathogenic viruses. The obtained data ensures the prevention of further infections and outbreak of the pathogenic viruses.
We have also developed a standardization document, a CEN Workshop Agreement (CWA) for detection viruses in water. The standardization strategy provides a map on the standardization landscape on national, European and international level as well as the differentiation to industry standards.

The main expected impact of the project is that the system will radically improve the water quality monitoring and thereby will give safe water for the society.

Project Context and Objectives:
The Portable Automated Water Analyser for Viruses (AquaVir) is a three-year FP7-funded project designed to develop a monitoring system for detecting pathogenic viruses in different water sources.
Enteric viruses exist frequently in various types of environmental water samples, such as wastewater, bathing water, ground or surface water and drinking water, constituting a primary source of gastroenteritis or hepatitis outbreaks.
The infection by enteric viruses is associated with considerable costs resulting from hospitalization and loss of time at work.
Pathogenic viruses cannot replicate outside of the host, but - based on their structures and composition - are generally much more resistant than bacteria or other microorganisms, they can survive extreme conditions for long time in the environment. The primary source of pathogens waterborne viruses is the urban wastewater. Human faecal materials of infected persons discharged to the urban wastewater, contain large number of pathogen viruses (107-1011 virus/gram faeces).
The small sizes and the extreme resistance of the enteric viruses causes that even the most advanced wastewater treatment technologies - such as membrane bioreactors with tertiary treatment including filtration, heat and UV light inactivation and chlorination - cannot fully eliminate viral particles.
Microbial water quality often varies rapidly in time and over a wide range. Short-term peaks in pathogen concentration may increase the disease risks considerably and may trigger outbreaks of waterborne disease. Water-borne viral diseases are important risks in the climate change, which causes floods and extreme climate events.
The problem of microbiological/virological measurements in water samples is the time requirement of the laboratory analyses, which is typically 24-72 hours after sampling. By the time the microbial contamination is detected, many people have already been exposed to the infection. Therefore an on-site continuous virus-monitoring probe is crucial for the human health.
The conventional biological water quality indicators do not provide adequate information about the presence of pathogenic viruses . The currently available reliable virus test - based on molecular biology - is expensive, time consuming and labour intensive, thus limited to few laboratories with sophisticated facilities and well-trained personnel, and not used routinely.
The AquaVir project aimed to deploy nanotechnology in affordable, mass-produced sensor for continuous monitoring of pathogen viruses in water. The applications include monitoring of raw water, process water for the food industry, wastewater discharge, irrigation water, bathing water, surface water and aquacultures.
The project included also i) laboratory and field tests ii) development an early warning system, iii) exploitation possibilities at the end-users, iv) economical assessment for positive production capacity and v) preparation for standardisation.
The results of the project will radically improve the water quality monitoring and contribute to creating new standards. Standardisation is an important tool for bringing the results beyond the research community and to the market.
The product design is based on the input of the end-users including the user-friendliness, adaptable to other water quality indicators and maintenance and further development strategies.
The core components of the virus-monitoring probe in AquaVir project are two microfluidic plastic chips for concentration and selective detection of viruses, respectively. The detection chip is a consumable product that has to be replaced regularly (every time if viruses are detected or in normal cases after several weeks) depending on the contamination in water to be monitored. The project includes the development of a method for manufacturing of the two chips in cost effective way.

The system consists of a Water Sampling and Filtering Unit (WSFU) and a Concentration and measuring system (CMS), which includes a Virus Concentration Unit (VCU) and a Virus Detection Unit (VDU). The different units are described in the following sections.
The overview of the sensor system can be seen on Figure 1.

Water sampling and filtering unit
The Water sampling and filtering has the function to collect the raw water from the original source. In the next step the water will be filtered through a rough filter to remove the large objects and particles. After this, a pre-concentration step is included, in which the water is slowly filtered through a glass wool filter. An elution buffer is used for recovering the viruses from the glass wool. The collected sample is stored in a sample reservoir for further processing.
Concentration and measuring system
The concentration unit
The concentration unit is utilized in an effort to further increase virus concentration in the liquid sample to improve the limit of detection (LOD) of the system. The principle is to push the virus particles towards the centre of a microchannel, where a fraction of the liquid containing all the virus particles is collected. The remaining liquid is discarded and thereby increasing the virus concentration in the sample liquid and decreasing the total sample volume, while retaining the virus particles. With careful electrode design, the virus particles are pushed towards to the middle of the channel and divided into three, they are then collected in the central channel. The concentration factor can be optimized by adjusting the flow rate in the three channels. By dividing the flow into 45 %, 45 % and 10 % fractions, one can achieve a 10-fold increase in virus concentration. In the AquaVir chip 128 parallel channels are used to increase the throughput. These chips can be stacked up for futher improvement of turnout.

The detection unit
The detection unit is a single-use inexpensive plastic chip, which has the function of selectively and accurately detecting the targeted viruses. In the sensor chip several sets of electrodes are exposed to a liquid sample. The electrodes are functionalized with the specific recognition molecules (aptamers) that have high affinity to the pathogens to be detected. The recognition molecule, the aptamer is a short oligonucleotide sequence (single stranded DNA) with a function similar to antibodies. The detection of the target pathogens is based on electrical measurement. There is a possibility to detect different analytes in the same time on the different electrodes. After detection of any pathogen or after a pre-defined running time, the detection chip should be replaced with a new one.
1.3. The achieved results
- In the project both the detection chip and the concentration chip have been developed based on micro- and nanotechnological methods.
- The description of the process and tests protocols for mass production of these chips has also been achieved.
- The two chips have been produced with in advanced industrial processes for the pilot testing.
- A computer controlled prototype sensor systems have been developed for controlling the concentration chips and for measuring the water samples with the detection chip.
- The project has included laboratory tests of the different components of the sensor system.
- A RT-qPCR to detect norovirus and rotavirus was developed in the project as a reference method.
- In the project an early warning model and epidemic risk assessment have been developed, providing with exploitable possibilities at the end-users.
- The feasibility of the AquaVir system and economical assessment for positive production capacity was also investigated.
- A CEN Workshop Agreement (CWA, a standardization document) has been developed regarding virus detection in water
- Over 300 water samples have been collected and pre-concentrated in different countries from different water sources. These samples were divided into two parts. One part was analyzed with classical laboratory methods by one of the chosen partner´s lab, and the other part came for testing in laboratory with the AquaVir system.

Project Results:
The project aimed to develop and test an inexpensive nanotechnology-based virus-monitoring unit, capable of detecting waterborne viruses in different water bodies.
The virus monitoring system is able to semi-continuously analyse the pathogenic virus content of the water automatically for longer periods giving alarm signal to a remote observer if the virus concentration is increasing. Inexpensive, disposable and mass-producible chips are used, which have to be exchanged when needed (either regularly or in case when viruses have been detected).
The product design is based on the input of the end-users including the user-friendliness, adaptable to other water quality indicators and maintenance and further development strategies.
The main achievements in the reported periods are described below.
In WP2, WP3, WP5 and WP6 the project was focusing on the development and fabrication of the monitoring system. The achieved results in the period are:
- Both the detection chip and the concentration chip have been developed based on micro- and nanotechnological methods.
- The nanotechnological method used in the concentration chip is filed in a patent application on. 1 March 2016.
- Description of the process and tests protocols for mass production of the microfluidic chips has also been achieved.
- The two chips have been produced with advanced industrial processes in the required numbers for the pilot testing.
- The High Level Product and Architecture Specifications for the AquaVir Sensor System have been described.
- A computer controlled prototype sensor systems have been developed for controlling the concentration chips and for measuring the water samples with the detection chip based on the Architecture Specifications.
In WP4, WP7 and WP8, the plan was the validation of the prototype monitoring system in laboratory and test on field, including building up a conceptual sensor-models system for early warning of risk of infection. The achieved results are:
- Collection, pre-concentration and laboratory analysis of more than 300 water samples from different sources and form different countries.
- Laboratory tests of the different components of the sensor system.
- Development of an early warning model and epidemic risk assessment, providing with exploitable possibilities at the end-users.
- Preliminary tests of few water samples for noroviruses with the developed detection unit. Although the number of the analysed samples did not allow making statistical conclusions, the results are promising. Further analysis is going on beyond the AquaVir project.
In WP9 a market analysis have been planed. The achieved result is:
- The feasibility of the AquaVir system and economical assessment for positive production capacity has been investigated.
WP10 includes the activities in dissemination, exploitation and standardization. The achieved results are:
- The results from the project have been published on conferences, on exhibitions and in Fact sheets for wider audience (end-users and authorities).
- The work on the standardization strategy for the AquaVir project. A CEN Workshop Agreement (CWA) has been developed regarding virus detection in water. A CEN Workshop Agreement (CWA) is a standardization document published by the European Committee for Standardization (CEN). The workshop is open for everyone and a contribution by direct participation of interested parties is explicitly desired.
The results from the WPs are described in the followings:
WP2: System engineering and scientific coordination
Objectives:
‒ Scientific coordination and meetings
‒ Specification of end-user requirements, agreement on overall system specifications and
Results:
- Target Product Profile including end user requirements have been developed for the AquaVir monitoring system
- High Level Product and Architecture Specifications have been defined for the AquaVir system.
- System verification test have been defined for the AquaVir system.
- The system verification test has been conducted with good results.
WP3: Design of virus sensor
Objectives:
- To design a microfluidic chip for concentration of the viruses from the water
- To develop a the virus capturing microchip
- To develop a technique for mass-production of conductive polymer microelectrodes
- To develop a fast method for the functionalization of conductive polymer electrodes with aptamers for selective detection of virus particles
- To integrate the virus concentration unit with the virus capturing unit
- To develop and injection molding processes for mass production of the virus sensor chip
Results:
- The virus detection unit (VDU) is developed and tested in laboratory
- The virus concentration unit (VCU) is developed and tested in laboratory
- The electrode fabrication and functionalization methods for mass production are developed and tested.
- The design of the virus concentration unit is optimized for mass production by injection molding
- Simplified glass wool filtration method was developed and tested for the pre-concentration of the viruses, which has also been integrated into the AquaVir monitoring system.
WP4: Early warning of risk of virus infection
Objectives:
- To develop an integrated sensor-model concept for early warning of water related risks of virus infections
- To setup a prototype for an early warning system based on sensor measurements and dynamic hydraulic/hydrodynamic models
- To optimize and evaluate the early warning system prototypes
- To develop Graphical User Interface for the Monitoring Station
Results:
- Conceptual models for surface drinking water supply (Case: KOV) and urban flooding (Case: Copenhagen) including estimation of the analytical requirements for the different application scenarios
- The scientific background for describing the decay of NoV, RoV and HaV has been been established.
- A hydraulic model of the sewage system for a selected area of Copenhagen (Nørrebro) has been established and connected to a surface 2D model.
- Health risk assessment was successfully integrated in the hydraulic models.
- A graphical interface was successfully developed in cooperation with Philips and DELTA
WP5: Virus Sensor prototyping
Objectives:
- To create mold concepts for mass production of plastic parts
- To develop an injection molding process for the sensor and the virus concentration unit
- To develop a concept for electrode fabrication
- To define an assembly process suitable for mass production
To define an electrode functionalization method suitable for mass production
- To manufacture functional prototypes
Results:
- The injection molding concept is defined and the tools are produced.
- The parts of the detection chip and the concentration chips are manufactured.
- The concept of electrode printing is defined and the electrodes are fabricated on both chips
- The electrode functionalization methods is developed and used in the detection chips
- The detection and the concentration chips have been assembled with gluing and with thermal bonding, respectively.
WP6: System integration and prototype manufacturing
Objectives:
- To develop a Control and Measuring Unit (CMU)
- To develop a Water Sampling and Filtering Unit (WSFU)
- To test an verify the measuring system
- To integrate the total measuring system
- Manufacturing of prototype Water Monitoring Probe (WMP)
- Integration of data transmission with monitoring station
Results:
- The Control and Measuring Unit (CMU) has been developed, manufactured and verified
- The Water Sampling and Filtering Unit (WSFU) has been developed, manufactured and verified
- The total monitoring system has been integrated
- Four prototypes of Water Monitoring Probe (WMP) have manufactured
- The data transmission has been integrated with the monitoring station
WP7: Laboratory test
Objectives:
- Lab test of the prototype virus sensor
- Lab test of the final prototype virus sensor
Results:
- Development of standardized methods for validation of the virus sensor.
- URV and BME in collaboration with DTU and PBC developed a pre-concentration unit in order to find solution for the challenge that the virus particles tend to attach to larger particles.
- Laboratory test of the detection chip have been performed for the salinity and pH of the water and the stability of the sensor for longer time period in flowing water.
- Samples have been analysed with the detection unit with promising results.

WP8: Field test and validation
Objectives:
- Field test of the Water Monitoring System
- Seasonal dependence of virus concentration
Results:
- Systematic collection of 365 water samples from different sources in different countries
- Concentration of viruses in smaller volume of water for analysis with the simplified filtration method
- Laboratory analysis of the water samples and freezing one part of the samples for further analysis with the AquaVir system
- Evaluation of the laboratory analysis data for an “European” map of viruses
WP9: Feasibility study
Objectives:
- To assess the production capacity for the sensor (consumable) and for the control unit (equipment)
- To assess the economic feasibility of the developed Water Monitoring System.
Results:
- A market survey have been performed: It shows three potential markets: detection in raw water intake to produce drinking water, detection in treated water after waste water treatment and detection in the laboratories from different types of water, for example recreational/bathing water, raw water or waste water.
WP10: Dissemination and exploitation
Objectives:
- To disseminate the results of the project to academia, governmental end-users and industry
- To prepare for exploitation of the results
- To disseminate the results to academia and industry
- To contribute to standardisation
Results:
- The project website have been updated with Fact Sheets about the results of the project: http://www.aquavir.eu/fact-sheets
- Research articles, conference papers have been published
- The road map for exploitation has been evaluated
- Exploitation workshops have been organized
- CEN Workshop Agreement have been drafted and published

Potential Impact:
The key trends in the water quality sensor market demonstrate the need for reliable, portable sensors for on-site monitoring of water quality and transferring the data to a remote observer in real time.
The developed virus monitoring system fulfils the requirements for a reliable, portable sensor and therefore could supply the lack on the market for water monitoring.
The materials in the sensor are inexpensive plastics, and the sensor is reagent-free with an electrical readout, hence the system is cost-effective. Since the proposed nanosensor can be adapted for detection of any other contaminants in the water, a development of a portable multi-sensor using inexpensive chips could reduce the costs of the monitoring requirement by the Water Framework Directive.
The availability of regular, routine, on site water test for enteric viruses as the result of AquaVir could reduce the spread of the waterborne infections.
The regular water quality test can reduce the pathogen contaminations in the drinking water, agriculture, fishery and food production.
In the European Union, pertinent legislation is manifested as a series of acts principally relating to environmental protection and water and wastewater managements. Whilst these pieces of legislation (typically in form of Directives) serve to provide Europe-wide standards, individual countries are able to interpret the Directives nationally and determine their implementation plans within the framework provided.
The European countries are responsible for employing the relevant water and wastewater treatment technologies to comply with the treatment standards of various EU Water Directives. Driven by strict regulations, it is required to develop reliable sensor technologies that can effectively monitor different water quality parameters, including pathogens.
Unfortunately, in these directives (and neither in the WHO guidelines) the determination of the pathogen virus contamination levels is not standardized and therefore not required. Because of the high resistance of the viruses to temperature, chemicals and disinfection, the E. coli or, alternatively, thermo-tolerant coliforms are not reliable indicators for the presence/absence of pathogen viruses in water bodies.
The results of this project will greatly contribute to improve monitoring and early warning of health risks related to water source, and minimize the viral infections in Europe. In addition, the sensor would be valuable in quality assessment of the many small water supplies.
The AquaVir sensor system will help people to have access to clean water, which is a major problem worldwide. The low cost virus sensor could be used in African and Asian countries where the quality of water, intended for drinking, is poor. Also Albania and the Slavic countries could encourage virological monitoring of heavily polluted surface waters.
The developed early warning system and “European” map of virus contamination of surface/bathing waters can also be used by the water works for prediction/prevention of virus contamination of different water bodies.
One of the objectives of the project was to develop a standardization document, called CWA, is to disseminate and exploit project results. The CW (CEN Workshop Agreement) is a standardization document published by the European Committee for Standardization (CEN). It is an agreement that is developed and approved in a CEN workshop. The workshop is open to everyone and a contribution by direct participation of interested parties is explicitly desired.
Defining the detection process in a CWA enhances sustainable exploitation of the project results and makes them accessible for any stakeholder in the field of water analysis.
The CWA addresses a wide range of stakeholders, by defining a system that describes how viruses can be detected in water.
The CWA defines a sensor system that monitors unacceptable levels of rotavirus, norovirus and hepatitis A virus in various types of water intended for human use. The system is characterised by the attributes: rapid, simple and economic.
Even though the application of the CWA is voluntary the project consortium of AquaVir highly recommends its use as a guideline on how to ensure high-quality results when detecting viruses in water. The following stakeholders have been considered as main target groups for the use and application of the defined sensor system for monitoring viruses in water:
‒ Developers / operators / producers of sensor chips
‒ Water supply companies
‒ Environmental authorities
‒ Health care companies and the public health sector
‒ Authorities and companies from the disaster management and civil protection
‒ Companies from the food industry
‒ R&D community
‒ Peace keeping military force
Posing a high risk of viruses in different kinds of water is not just a national or regional problem. This topic is relevant on European and even international level. Publishing a standardization document enhances the importance of a sustainable virus detection to minimise the risk of infection of serious diseases dramatically.

List of Websites:
www.aquavir.eu

Contact: Anders Bjerrum, DTU Nanotech, email anders.bjerrum@fp7consult.dk

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DANMARKS TEKNISKE UNIVERSITET
Denmark
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