Final Report Summary - AQUALITY (On-line industrial water quality analysis system for rapid and accurate detection of pathogens)
Executive Summary:
Diseases caused by bacteria, viruses, fungi and other parasites are major causes of death, disability, and social and economic disruption for millions of people. Water is an important environmental source and agent for transmission of many of these pathogens. The AQUALITY solution is an online water monitoring device for microbiological contamination analysis, that allows industries and environmental protection agencies to replace the routine activities of sampling and laboratory testing of pathogens. The new system is able to real time monitor the quality of industrial process water and effluents basing on an opto-ultrasonic device and on a lipid-based diagnostic kit. To realize the device, the focus of the first project period was on the conceptual design and development of the single components. During the last phase of the second project period, the AQUALITY consortium dedicated its efforts to the system integration and verification.
Starting from the analysis of the User and System requirements, the technical specification of all the components have been defined on the base of the requirements and specification set up by the beneficiaries and end users. The final architecture of the AQUALITY system has been drawn. The main components of the system are: the mechanical layout; the Ultrasound Unit; the Optical Unit and the Control system. Thanks to the effort dedicated to this delicate phase, the following steps of design, development and integration preceded with less risks and uncertainties, permitting to recover the extra time invested in this phase and, above all, permitting to have an integrated device able to measure and detect bacteria.
The part of the AQUALITY project concerning liposomes was driven by the CNR and involved efforts in three main activities: synthesis of lipid components (positively charged lipids) to engineer liposomes and make them more sensitive to the interaction with bacteria; formulation and characterization of liposomes and evaluation of the liposome/bacterium interaction. Two liposomes formulations have been developed, composed of the natural lipid DOPC, a cationic surfactant and the fluorescent probe umbelliferone, suitable to be efficient nanosensors to be integrated in the AQUALITY device.
The system integration was led by LABOR, with the support of CNR and USOUTH. The main output of the activities performed was the integrated AQUALITY device. The system can be considered made up by two main subsystems: the hydraulic and the electronic (control) with their external interfaces which permit the integration of the two subsystems in the complete prototype. After the system integration, the system has been tested at lab scale. The purpose of the initial AQUALITY prototype system tests is to verify the overall system functionality and to perform tests with calibration beads. As first step, each single component was tested separately. Then, new tests with the entire system were performed. These tests were needed to verify that every single operation ran with the others in the correct way. Therefore, these were a verification that the hydraulic system worked properly. These were necessary also to verify that the phases of loading and discharging of all liquids in tanks ran properly. In conclusion, the hydraulic system worked properly and the results reported were coherent with the input.
Finally, the prototype was finally ready to be installed on site (Norway) for the final tests and validation. The pilot project will be set up for demonstration purposes in Norway at ROROS water treatment plant. In this task TI will take care of the installation of the AQUALITY online monitoring system at ROROS plant. TI will be the responsible, in this task, of realizing the report on the installation and will take care of validating the system and of granting the possibility, for ROROS, to remotely monitor its facilities through AQUALITY technology. The activity includes test run and training session as well as preparation of instruction manual for the operator of the AQUALITY prototype. The results of the testing and operation of the AQUALITY system for a 3 weeks period has been evaluated and the results from of the microbiological parameters is compared to water samples analyzed at an accredited laboratory. In addition, the system validation includes identification of key provisions according to Norwegian legislation (“Drikkevannforskriften”) with regards to water quality and monitoring program. Existing practices and costs for water quality monitoring at Rørosmeieriet and Røros Water Plant have been also identified and described.
Project Context and Objectives:
Diseases caused by bacteria, viruses, fungi and other parasites are major causes of death, disability, and social and economic disruption for millions of people . Despite the existence of safe and effective interventions, many people lack access to needed prevention methods and treatment. The lost productivity, missed educational opportunities and high health care costs caused by infectious diseases directly impact families and communities. Infections are prevalent in developing countries, where co-infection is common.
Water is an important environmental source and agent for transmission of many of these pathogens. Water-related pathogens that have emerged or re-emerged recently include Cryptosporidium, Legionella, Escherichia Coli 0157, Rotavirus, Hepatitis E virus, and Norovirus (formally Norwalk virus) (WHO, 2003).
It is well known that many industries require huge quantities of water for their processes and need to be supplied by water with specific characteristics, since water quality may seriously affect production units and quality of finished products. Currently, the identification and quantification of pollutants in water are mostly carried out manually through sampling and subsequent laboratory analysis (off-line analysis. These methodologies of work involve some significant costs in terms of displacement to sampling points, reagents and specialized personnel dedicated to the operation, leading to time consuming and economically challenging approaches, causing the number of analyses performed to be kept at the bare minimum.
The industry therefore is calling for novel, cost-effective solutions to meet these new challenges: there is an urgent need for rapid methods for detecting the major waterborne pathogens, which can be implemented both online and at laboratory scale; this achievement would represent a huge competitive advantage for the enterprise proposing it and would open up a significant international market.
We propose to develop an online water monitoring device for microbiological contamination analysis, that allows industries and environmental protection agencies to replace the routine activities of sampling and laboratory testing of pathogens. The new system will be able to real time monitor the quality of industrial process water and effluents basing on an opto-ultrasonic device and on a lipid-based diagnostic kit. The novelty of our approach is the use of engineered liposomes for detecting bacteria in water: these are nanoparticles formed by a lipid bilayer enclosing an aqueous compartment displaying features that can be different (pH, ionic strength, composition) with respect to the bulk. We will load liposomes with a chromophore and will engineer them in order to make them specifically react with target bacteria; this is the simple operating system of the AQUALITY system, which is completed by an ultrasonic unit to concentrate bacteria and an optical unit for detecting the sample colour change following to the interaction between liposomes and bacteria.
Therefore, the tangible outcomes of AQUALITY project will be:
o A new method for rapidly detecting the presence of bacteria strains in water and wastewater, that relies on the interaction of liposomes with bacteria without specific antigen- antibody interactions;
o The implementation of an opto-ultrasonic unit to concentrate the bacteria and detect the sample color changes;
o The implementation of this method in a new device, which will be integrated in a real industrial production line (online mode) at an end-user facility.
THE LIPOSOMES
Liposomes are nanoparticles formed by a lipid bilayer enclosing an aqueous compartment displaying features that can be different (pH, ionic strength, composition) with respect to the bulk. Because of their morphology, since their discovery in 1964 [J. Mol Biol 1964, 8, 660-668] liposomes have been exploited as models of biological membranes.
The use of liposomes as sensor elements for the detection of different species has been widely described and is inspired to some functions of biological membranes [J. Am. Chem. Soc. 2011, 133, 9720; J. Am. Chem. Soc. 2008, 130, 5010; Trends Anal. Chem. 2005, 24, 9; Sens. and Actuators B 2005, 107, 82; Anal. Chim. Acta 2006, 556, 127; Biotechnol. Prog. 2006, 22, 32; Biosensors and Bioelectronics 2007, 22, 2848]; actually the basic concept is mimicking the role of biomembranes in signal transduction and information processing, based on the reorganization of the lipid components upon the interaction with defined entities (bio molecules, ions, bacteria, viruses….). The interaction of bacteria with the lipid membrane of defined liposomes might trigger processes such as lipid reorganization or membrane disruption, or induce changes in the electrical properties of the liposome surface, the kind of perturbation depending both on lipid composition and on the interacting entity. The changes induced on the lipid bilayer by the interaction with bacteria might in turn affect the optical features of a proper dye included in the liposomes and thus be revealed as an optical response.
The initial goal of the project was developing engineered liposomes capable of interacting specifically with target bacteria, namely E. Coli, S. Aureus, E. Faecalis, and loading their internal aqueous compartment with a proper dye (pH indicator, chromophore), so that its released into the bulk upon disruption of liposomes due to their specific interaction with bacteria, would have produced an optical signal (changes in the absorbance or emission) (Figure below).
Figure 1 - Interaction of liposomes with bacteria.
However, as the project developed it became evident that the lipid bilayer stability, necessary for storage, was in contrast with the attitude to disrupt upon interaction with bacteria, and that specificity toward target bacteria could detract efforts from other features such as high sensitivity and fast response. Therefore we changed means of detection of the perturbation caused by the interaction of liposomes with bacteria, and chose a chromophore sensitive to changes in the electrical features of liposome surface, exploiting the fact that the interaction of positively charged liposomes with bacteria (that are negatively charged) changes dramatically the electrical features of liposome surface. Further we set as a goal the development of aspecific liposomes, i.e liposomes capable of giving an optical signal (due to the change of surface potential) upon interaction with various bacteria.
THE ULTRASOUND UNIT
A major obstacle in the implementation of rapid pathogen detection methods in drinking water is the low concentration of microorganisms in samples, requiring laborious methodological steps to increase the microbial concentration to a level sufficient for analysis1.
Concentration of bacteria in microfluidic systems has been achieved by different means, including electrical forces2-5, hydrodynamic effects6, evaporation7,8, and ultrasound-enhanced sedimentation9.
The use of ultrasound (US) in microfluidic environments has recently emerged as a non-invasive way of manipulating cells and particles for a range of applications10, such as sample enrichment11,12, intra-cellular drug delivery13-16, sensing and bio-detection17-19, sample filtration20-22, cell and particle sorting23-25 and trapping26,27. Continuous-flow concentration of particles by acoustophoresis is usually performed by generating an ultrasonic standing wave (USW) within a fluid chamber, causing suspended particles to focus in a confined liquid volume under the action of primary acoustic radiation force28. Either one-dimensional (1-D) or two-dimensional (2-D) focusing can be achieved, depending on the properties of the acoustic field. In order to obtain a significant increase in particle concentration, the fluid flow must be split into the particle-rich and the particle-depleted fractions28,29 at finely controlled flow rate ratios. Based on this functioning principle, concentration of a range of particles including bacterial spores, biological cells and polystyrene spherical micro-beads 11,24,30-35 has been demonstrated.
Manipulating bodies as small as bacteria by means of acoustic radiation forces is however a considerable physical challenge27. This is mainly due (i) to the fact that the acoustic primary radiation force scales with the cube of particle radius29, and to the increased particle susceptibility with respect to hydrodynamic drag force resulting from acoustic streaming or thermal convection36,37. It has been found that the critical particle size at which a transition to drag-dominated behaviour occurs is ~1 μm, at operating frequencies of approximately 2 MHz36,38. For this reasons, only few studies have demonstrated concentration of flowing particles with diameter <2μm32,33 and, to the best of our knowledge, continuous-flow concentration of bacteria by acoustophoresis has not been previously demonstrated. Recently, Hammarström et al. reported on highly efficient trapping of E. coli by half-wave resonance in glass microcapillaries27; however they employed 12 μm diameter seed particles to increase the trapping efficiency through the use of secondary, inter-particle radiation forces.
In this project we initially set ourselves the goal of producing a 4x bacterial concentration with a half-wave device, however as the project developed it became evident that higher concentration factors would be required to meet system performance requirements. In order to meet these higher concentration factors we developed a unit based around a thin-reflector acoustic mode.
Project Results:
System Requirements
The main aim of the work package “System Requirements” was to perform a study of the industry addressed in the AQUALITY project. The intention of the study was that of comprehending the actual characteristics of the target industrial scenario and of identifying the relevant aspects to be reached during the project.
To do this, the consortium identified and reported in a document the main information related to water monitoring technologies actually in use across Europe together with the main EU regulations in the fields of water control and quality assessment.
Three possible scenarios have been taken into account and described accurately in our reports:
• Scenario 1: Food & Beverage Industry
• Scenario 2: Private and Public Utilities
• Scenario 3: Bathing Waters
The AQUALITY online monitoring device is mostly intended for industrial processing waters. Thus, the choice of the application scenario for the project has been made consistently with the objective of the project of reaching the water suppliers, and the industrial processors making great use of water of high quality.
On the basis of the application scenarios, the user requirements have been collected and organized. However, stated that several applications could be possible for the AQUALITY technology, the Consortium focussed the priority on the second identified application scenario, that of Public & Private Utilities. This scenario, in fact, best represents the target industrial sector for the project, and best fits with the AQUALITY water analysis method, which, according to the technical discussions following the realization of the application scenario description, is intended for industrial processing more than for the drinking water market.
The project partners were involved in the definition of all the user requirements of the project. This activity, carried out as an iterative process, led to the identification of slightly different bacteria to be detected by the system, respect to what was originally planned. Furthermore, the functionalities, the alarms and interface requirements, the size and properties of the device have been indicated in this data collection.
After the user requirements collection, the system requirements have been identified, collected and organized. The goal was obviously that of assessing a complete, consistent and clear set of requirements through which the different components of the system could be outlined.
The techniques used for the identification of the AQUALITY device requirements have been the following:
- Plenary meeting (kick-off meeting in Rome) for performing a brainstorming session,
- Interviews in the target industry performed at a Consortium level and at a restricted external group of stakeholders/potential end-user in the sector, in Italy and Norway,
- Use of a supporting model for requirements collection and sharing of the chosen (IEEE) shell structure with the explanation of how these should be filled in,
- Exchange of further contributions via email/ phone with the Consortium members,
- Iterative revision of the document for updates and improvements.
At the end of these activities a complete set of system requirements was available for the further technical specifications of all the system component.
Hardware development
Starting from the analysis of the User and System requirements, the technical specification of all the components have been defined on the base of the requirements and specification set up by the beneficiaries and end users.
The final architecture of the AQUALITY system has been drawn, and the responsibilities of each partner in the design and implementation of the specific units have been highlighted; the final version of the architecture can be considered the results of a close collaboration among the partners (RTDPs in particular).
The main components of the system are:
1. The mechanical layout;
2. The Ultrasound Unit;
3. The Optical Unit;
4. The Control system.
The technical specification of the single components has been a fundamental phase. Thanks to the effort dedicated to this delicate phase, the following steps of design, development and integration preceded with less risks and uncertainties, permitting to recover the extra time invested in this phase and, above all, permitting to have an integrated device able to measure and detect bacteria.
The Mechanical Layout
The design activity of the mechanical layout has been performed using 3D CAD technology by LABOR, and the images of the digital mock-up are very well representative of the prototype.
The results of the design activities are of three different types:
• manufacturing drawings of the parts to be prototyped in mechanical workshop,
• assembly drawings, which serves as a guide for assembling and installing the system,
• BOM (bill of material), to source parts and off the shelf components.
The device consists of a parallelepiped, 410mm width X 330mm depth X 252mm height, with the inlet filter mounted on the right side, for a total width of 550mm, and handles on top cover, for a total height of 301mm. The main section and components are outlined. The structure of the AQUALITY is supported by four identical plastic legs (represented in black), which are connected and fixed in position by the internal main plate and the six covers.
The top cover is equipped with two robust handles, for easy handling and transportation of the device, and a window, which can be removed, without any additional tools, by means of four knurled head screws, to replace the consumables inside the device (i.e. filling the liposome storage tank), to make fine regulations (i.e. adjusting the pressure of the CO2 regulator), or to allow monitoring the internal of the device during testing.
The Ultrasound Unit
The picture 3 of the attachment shows electrical impedance plots for Half-wave (HW) (a) and Thin-reflector (TR) (b) devices. Impedance minima were identified at fR = 1.43 MHz (HW) and fR = 831 kHz (TR), for water-filled devices under static fluidic condition.
Final verification of the resonant frequency was carried out using a digital oscilloscope, and adjusted resonant frequencies of 1.46 MHz and 826 kHz were determined for HW and TR device, respectively.
Assessment of devices functioning was also performed qualitatively, by in situ microscope observation of bacteria behaviour in the absence and in the presence of US. In a series of experiments, the total flow rate was conveyed into OBR and bacteria flow behaviour in the outlet slot was visualized. In figure 4, (a) shows images of bacteria flowing into the slot (i.e. in a direction perpendicular to the image plane) of a HW device, both in the presence and in the absence of US (fR = 1.46 MHz; 2.5Vpp; QIN = 20 ml/h). Acoustic focusing of E. coli in a narrow fluid layer located approximately at the centre of the slot can be appreciated, whilst bacteria distributed uniformly when US was deactivated. This was confirmed quantitatively by performing a z-scan analysis in the fluid chamber. Results show that the large majority of bacteria (~90%) are focused within a ~30 μm thick fluid layer centred on the chamber axis (b).
The picture 5 (a) shows microscope images of bacteria flowing on the reflector surface of the TR device (QIN = 20ml/h and QBR/QIN = 0.1). It can be observed that the acoustic field was capable of effectively constraining the bacteria in close proximity or in contact with the glass reflector. (b) shows microscope images of bacteria flowing into QBD slot, in the presence (left) and in the absence (right) of US. Microscope focus was set at the very bottom (in z-direction) of the slot, and a nearly bacteria-free slot was observed when US was activated signifying that most of E. coli were forced to flow above the slot towards OBR (c). (d) shows a microscope image of bacteria flowing into OBR, where the presence of lateral modes can be appreciated.
Ultrasonic bacterial manipulation: E. coli vs S. epidermidis
1. Half-wave device
The figure 6 (a) shows the % number of bacteria along the z-coordinate, determined from z-scan analysis (taking successive images at varying focal positions) at a fixed x- and y-position (QIN = 20 ml/h; f = 1.46 MHz; 3.1Vpp). Representative results for both E. coli and S. epidermidis are reported, demonstrating that the HW device is capable of effectively manipulating both bacterial species. The average performance is illustrated in (b), where the average % number of bacteria located in a 40 μm thick fluid layer (centred on the chamber mid-plane) is reported. Device performance slightly reduced when the inlet flow rate was increased from 10 ml/h to 20ml/h, with no statistical difference between E. coli and S. epidermidis. Figures c-e and f-h show representative microscope images of E. coli and S. epidermidis, respectively; taken at z = 100, 190 and 300 μm (where z = 0 μm corresponds to the carrier surface and z = 390 μm corresponds to the reflector surface ).
2. Thin-reflector device
The figure 7 (a) shows the % number of bacteria along the z-coordinate, determined from z-scan analysis at a fixed x- and y-position (QIN = 20 ml/h; f = 826 kHz; 22Vpp). Representative results for both E. coli and S. epidermidis are reported, demonstrating that the TR device is capable of effectively manipulating both bacterial species. The average performance is illustrated in (b), where the average % number of bacteria located in a 10 μm thick fluid layer (from the reflector surface) is reported. As for HW device, no statistically significant difference between the two bacterial species was detected, at both inlet volumetric flow rates applied. The figures c-e and f-h show representative microscope images of E. coli and S. epidermidis, respectively; taken at z = 0, 50 and 100 μm (where z = 0 μm corresponds to the macor surface and z = 100 μm to the reflector surface).
Minimization of bacteria attachment on the reflector surface in TR device
Bacterial loss due to adhesion to the glass reflector surface was a major cause of low performance in initial devices. A number of coatings were evaluated to solve this problem. For each surface experiments were carried out with the aim of quantifying the total % loss of bacteria due to attachment on the glass surface. The table 1 shows the % loss of bacteria, for both silane- and PMOX-coated glass reflectors. In the preparation of silane coatings, two different strategies were investigated, namely (i) silane was dissolved in anhydrous dimethylformamide (DMF) (as described in Section 5.5.6) and (ii) silane was dissolved in hexane. The latter approach was associated with reduced formation of undesired patterns on the glass surface, due to the higher solubility of silane in hexane compared to DMF.
Results show that silane dissolved in DMF provided the best performance (i.e. lowest % number of bacterial loss), for both bacterial species. This is coherent with in situ results illustrated in Section 6.5.1. PMOX demonstrated to be an effective repellent for E. coli, but was less effective in preventing attachment of S. epidermidis (~9 % loss in bacteria). Silane dissolved in hexane performed less effectively compared to the other surface treatments, with % bacterial loss of ~22 % (E. coli) and ~15 % (S. epidermidis). Results from these studies confirm the suitability of hydrophobic silane (dissolved in DMF) as a bacterial repellent coating in TR devices. However, it is worth mentioning that there appears to be a time evolution of the attachment process which could lead to different results depending on the duration of the experiment. This, and other aspects of bacterial adhesion in the presence of US, is currently under investigation in our laboratories.
Performance assessment of TR device
Figure 8 shows the concentration increase achieved with TR device, at QBR/QIN ranging from 0.0025 to 0.2. Experimental values are compared with theoretical values calculated assuming that all bacteria flow into OBR. The percentage difference between experimental and theoretical values is reported.
Results show that the TR device is capable of achieving significant increase in bacterial concentration and thus it may represent a powerful technological platform in pathogens analysis systems for water quality examinations. A maximum 60-fold concentration increase was achieved at QBR/QIN = 0.005. However, by comparing experimental with theoretical results, it can be observed that there is still a large margin for performance improvement. Notably, device performance was observed to degrade with reducing QBR/QIN, as it can be observed in Figure 8 where theoretical and experimental results are plotted together. This is likely due to a significant number of bacteria escaping the bacteria-rich layer, caused by insufficient radiation force to constrain the bacteria in a fluid layer as thick as the characteristic size of the bacteria itself.
While the device was observed to perform very effectively in the central region of the chamber, a loss of performance was observed in close proximity to the channel side walls, which may be the cause of the observed degradation of device performance with reducing QBR/QIN. Other sources of performance degradation may include bacteria sedimentation in the syringe or attachment on the tubing inner surfaces, particularly in the long-term. Given that E. coli and S. epidermidis displayed a similar behaviour when exposed to US in both HW and TR resonators, we expect that analogous concentration increase will be achieved with S. epidermidis.
Effect of initial bacterial concentration on TR device performance
Figure 9 shows the increase in bacterial concentration (θ) achieved using the TR device (at a fixed QBR/QIN = 0.05) at varying initial concentrations of bacteria in the inlet suspension (CIN), corresponding to 10, 102, 103 and 104 CFU/ml.
Results show that device performance slightly decreases with reducing the initial bacterial concentration from 104 CFU/ml (θ = 14.31) down to 103 CFU/ml (θ = 12.07). Notably, TR performance remains substantially invariant with further decreasing bacterial concentration (i.e. θ = 11.23 at CIN = 10 CFU/ml), and likely no statistical difference in performance subsists at these lower inlet concentrations. Increased performance at the higher CIN may be attributed to the effect of acoustic secondary radiation forces, which becomes stronger when the distance between bacteria reduces (conforming to higher bacterial concentration). However, it should be noted that approximations in the calculation of device performance at the lower CIN may have caused marginal underestimation of TR performance. Overall, TR device is capable of achieving significantly high concentration increase under a wide range of bacterial concentrations in the inlet suspension.
Comparison between HW and TR devices
Table 2 shows the concentration increase achieved with using TR and HW resonators at QIN = 20 ml/h and QBR/QIN = 0.2 0.1 and 0.05. Results show that at the higher QBR/QIN, the two resonators performed similarly (percentage difference in concentration increase: 17.4%). However, with reducing QBR/QIN, the TR configuration displayed a significantly improved performance compared to the HW configuration. The concentration factor for the HW device reached a maximum of 3.39 at QBR/QIN = 0.1 and reduced to 2.98 at QBR/QIN = 0.05; likely due to an increased number of bacteria escaping the top and bottom outlets of the device.
Results show that the TR device is capable of achieving significantly higher concentration increase (~1 order of magnitude) compared with traditional HW configurations. The latter performed similarly to previously reported half-wave or quarter-wave devices32,33.
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The Optical Unit
Taking into consideration the Technical specification for all the components the Optical Unit has been developed by LABOR. The Optical unit is connected and driven by and electronic Control Board: a microcontroller based board which controls the light source and the detection unit has been implemented. The picture is the CAD design of the positioning of the Optical Unit inside the system.
The unit is made up by 5 main sections:
1. Light source: an emitting diode; this is to obtain the peak of emission of umbelliferone, the marker used by CNR as fluorescent dye;
2. Two optical fiber (source side and detection side): the first one placed on the source side is for transmitting the irradiation from the light source to the optical chamber and the second one placed on the detection side is for the signal detection device;
3. Optical chamber: this is made up by a steel cuvette square-based in order to permit an angle of 90° between light source and detection, except for the optical windows which will be made of optical glass to perform a fluorescence analysis;
4. Two optical filters: these are necessary in order to focus the signal on the selected wavelengths;
5. Light detector: Photomultiplier module which takes in input the output signal from the Optical chamber and communicates the measurement to the electronic control system.
The Control system
The main sections of the AQUALITY control system are the electronics, the firmware and the software.
The control system developed for the AQUALITY process is a particular hardware and software configuration controlling the entire functioning of the AQUALITY system. More in detail, the control system is made up by:
An Electronic main board (which can execute commands given by a running software on the central unit): microcontroller based circuit, that runs a dedicated firmware;
A central unit (industrial panel PC) which runs automation software which manages all the operations needed in the process, with an human machine interface (HMI) which shows the process status and the results to the user.
The firmware of the AQUALITY system is the part of the program embedded in electronics card that allows the software to use it as passive command executor. The control system has pumps valves, an optical unit, temperature sensors and heater unit actuator. Since the card simply actuates commands, it must have all the operative links for all single input or output connection. The serial interface has the role of centralize all the peripherals which are cross-present through all the pinout layout.
The control software of the AQUALITY automation system handles all the logic operations by controlling the operations of the electronic subsystem to the microcontroller via a serial protocol. LABOR has defined a communication protocol with a set of intuitive controls. The software also decodes the string communicated by the microcontroller to obtain information about the status and operation of the system, and in particular of all the valves and the pumps in the hydraulic subsystem and the temperature sensor. The latter is the only transparent value for the end user and displayed in a box in order to allow the monitoring of the internal temperature of the system. All other values of the string are used automatically by the software, in order to ensure the correct sequence of operations and reduce the human error. The home screen of the interface appears as shown in the picture 11.
Nanosensors Development
The part of the AQUALITY project concerning liposomes involved efforts in three main activities:
- Synthesis of lipid components (positively charged lipids) to engineer liposomes and make them more sensitive to the interaction with bacteria.
- Formulation and characterization of liposomes.
- Evaluation of the liposome/bacterium interaction
Synthesis of lipid components.
The ten cationic lipid components reported in Figure 2 were synthesized. Compounds 1 and 2 were chosen based on the results of a study where they were included in formulations to deliver a drug to MRSA [C. Bombelli et al. Mol. Pharm. 5, 672-679, (2008)], compounds 3 were chosen because structurally related to compounds 1 and 2, whereas compounds 4 and 5 were chosen because unsaturated lipids are reported to interact with bacterium wall.
Formulation and characterization of liposomes.
A number of liposomes formulations composed of different ratios of one of the cationic components and a phospholipid both in the presence and in the absence of cholesterol were prepared and evaluated for their capability of retaining in the internal aqueous compartment a water soluble dye (calcein or phenol red). This evaluation allowed selecting a number of stable formulations that were further investigated for the interaction with bacteria.
The idea was that interaction of the lipid nanoparticles with bacteria could disrupt the lipid bilayer thus allowing the release of the dye from liposomes. Calcein is a fluorescent molecule generally used to evaluate the extent of release of liposome content into the bulk medium; its fluorescence is quenched when it is confined at high concentration in the aqueous compartment of liposomes, but quenching ceases in dilute conditions. Therefore its release from liposomes and its dilution in the bulk is accompanied by the appearance of a fluorescent signal. In the case of phenol red the appearance of an optical signal is triggered by the change of pH when moving from the aqueous compartment of liposomes to the bulk medium.
We evaluated the optical response of calcein or phenol red loaded liposomes to the interaction with bacteria. We tested three different bacterial strains: Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 28212 and Stafilococcus aureus ATCC 29213. Unfortunately none of the selected formulations gave the expected optical signal. The expected optical signal was not observed or was very slow to appear.
It was then necessary to understand whether the lack of an optical response upon the addition of different bacteria to the liposome suspensions had to be ascribed to the lack of interaction, to the nature of formulations or to the selected chromophore. The occurrence of interaction could be investigated by Z potential measurements.
Z potential ( is the difference of potential between the dispersion medium and the stationary layer of fluid attached to a nano- or microparticle in solution (picture below). Bacteria and liposomes are characterized by a certain determined by the surface charge and by the ions associated to the surface. The interactions of particles characterized by different values of will give a new value of as shown in the picture below.
measurements were carried out on liposomes selected on the basis of the previous experiments, i.e those formulated with cationic lipids 1b, 4 and 5. In themeasurements the concentrations of bacteria and liposomes were chosen to obtain an acceptable ratio between the surface area of liposomes and bacteria. The values obtained in the case of the liposome suspensions in the presence of bacteria demonstrated that the interaction actually occured. Therefore the absence of an optical response in liposomes loaded with a dye indicated that the selected formulations are too stable to disrupt in the presence of bacteria.
It was therefore decided to include in the lipid bilayer of liposomes a chromophore whose optical features depend on the electrical features of liposome surface, i.e. 4-heptadecyl-umbelliferone (from now on umbelliferone) and is generally used to measure the surface potential. [N.J. Zuidam, Y.Barenholz Biochim. Biophys. Acta 1997, 1329, 211-222].
The formulations that according to all previous investigations were evaluated as the most promising, i.e. 7/3 DOPC/4c and 7/3 DOPC/1b (DOPC=1,2-dioleoyl-sn-glycero-3-phosphocholine), were formulated with 3-7% of umbelliferone in the presence and in the absence of cholesterol (the presence of cholesterol could further stabilize the formulation). The following investigations were carried out on the engineered liposomes:
- a preliminary evaluation of the occurrence of a fluorescent signal upon the interaction with bacteria, to verify if it was worthwhile to follow the ‘surface potential strategy’. Actually the addition of bacteria to umbelliferone loaded liposomes changed its fluorescence.
- evaluation of their stability as fluorescent devices (leakage of the fluorescent probe upon storage), to check the stability of the liposome sensors to storage. This allowed to set the best experimental conditions (lipid formulations, lipid/umbelliferone ratio, concentration, pH).
- assessment of stability/morphology in the presence of umbelliferone by dynamic laser light scattering (DLS) experiments. In the preparation of liposomes it is possible to impose a defined size to the lipid nanoparticles; after preparation the size is measured by DLS to check if liposomes feature the expected size and are mono dispersed, this being also an evaluation of their stability.
- interference by ions commonly present in water with the probe fluorescence. In fact, the actual water to be analyzed might contain ions that could change the fluorescence features of umbelliferone included in the liposome formulations. Therefore, in order to identify any possible interference with our detection system, we investigated the fluorescence of umbelliferone included in the liposome lipid bilayers in the presence of a number of the ions present in all Berlin water wells for drinking water (a 2011document given by ARGUS, one of the partners of the project). We considered concentration ranges between the maximum value actually found in water and the maximum value admitted by the law. It was observed that: i) the fluorescence of liposome included umbelliferone decreases of~ 20% in the presence of 150 mg/L calcium; ii) the fluorescence of liposome included umbelliferone increases of ~300% in the presence of 140 mg/L carbonate.
- evaluation of the stability to contamination. We had to find the best preparation conditions to guarantee sterile conditions for the different liposomes formulations, thus avoiding contamination of liposome suspension by environmental bacteria during preparation. We found that the extrusion of liposomes directly in sterile plastic tubes used as furnished by the producer (Falcon) is the best procedure, and no differences between 4°C and room temperature storage were observed.
Evaluation of the liposome/bacterium interaction
And finally we were ready for a detailed investigation of the interaction of the two selected formulations with three different bacteria namely E. Coli, S. Aureus, E. Faecalis. The investigation was aimed at finding the best conditions to have a fluorescent signal from the umbelliferone included in the liposome formulations upon the interaction of liposomes with low concentrations of bacteria. We set up the conditions of the fluorescence experiments to meet engineering requirements and explored different concentrations of 7/3 DOPC/4c and DOPC/1b liposomes, containing different amounts of umbelliferone (3%, 6%, 7%), in the presence and in the absence of cholesterol (3% and 5%), in the absence and in the presence of added salts.
Fluorescence experiments in Phosphate Buffer Saline (PBS). First experiments were carried out in the buffer used in all previous experiments (PBS). The best results obtained are reported in Figure 14 that shows the decrease of fluorescence observed when different bacteria were added to the 7/3 DOPC/4c (Figure 14a) and 7/3 DOPC/1b (Figure 14b) formulations. Both formulations gave a change of fluorescence signal upon the interaction with 102 CFU/mL bacteria solutions.
Fluorescence experiments in Rome pipeline water. Analogous experiments were carried out in a medium more similar to the actual media to be analysed by the AQUALITY device, i.e. a pipeline water (from one of Rome pipelines). As shown in Figure 15, in pipeline water, due to presence of different salts, an increase of the fluorescence of umbelliferone was observed. This result, confirmed by analogous experiments in samples of water enriched with mixtures of salts indicate that the response of the lipid sensors to the presence of bacteria depends also on the medium and that a calibration has to be done on each specific implant.
In order to simulate a real situation, we carried out experiments in pipeline water cointaining at the same time the three bacteria, e. coli, s. aureus and e. faecalis. The total concentration of bacteria was set to 102 CFU/mL, as in previous experiments, in order to maintain constant the ratio between liposomes and bacteria. The results are reported in Figure 6. As expected, also in the presence of the mixture, liposomes formulated with the surfactant 4c display a higher sensitivity.
In conclusion our liposomes sensors are able to detect three different bacteria, i.e. e. coli, s. aureus and e. faecalis, at a concentration of 102 CFU/mL. This means that by combining our liposomes with the opto-ultrasonic device the AQUALITY device will be able to detect these bacteria at 10 CFU/mL.
It is important to note that though during the development of the project it was decided to skip over the specificity of liposome sensors toward defined bacteria in order to focus our efforts on important features such as high sensitivity and fast response, the different response obtained from the two selected formulations suggests that specificity might not be a too far goal to accomplish.
System Integration
The system integration was led by LABOR, with the support of CNR and USOUTH. The main output of the activities performed is the integrated AQUALITY device.
At a glance, the system can be considered made up by two main subsystems: the hydraulic and the electronic (control) with their external interfaces which permit the integration of the two subsystems in the complete prototype.
PHASE 1 - THE HYDRAULIC SYSTEM
The design activities regarding mechanical layout have been included in the design of the thermostated box for containing the hydraulic system.
All the single components have been placed in the hydraulic system.
The hydraulic subsystem has the following electronic interfaces towards the control unit:
sma connector;
BNC connector;
40 plugs connector: this is an industrial connector and is used in AQUALITY for connecting the hydraulic system with the control one. It includes cables for connecting the sensors, the pumps and the valves.
PHASE 2 - THE CONTROL UNIT
The electronic board, was mounted on a specific plate. On the reverse side of the plate were installed the power supply modules for the electronics. This plate was housed into the box, previously prepared.
The panel PC was housed in the case cover and connected to the electronics.
PHASE 3 - INTERGRATION OF THE TWO SUBSYSTEMS
The integration of the two subsystem (hydraulic and control) has been designed to be easy: 3 connectors interface the 2 cases.
In the picture 18, the complete and final prototype is showed.
Generally speaking, systems integration is potentially the most problematic phase in a research project, since all the sections (previously tested stand-alone) have to “talk” together. In the particular case of the AQUALITY project, the system integration went smoothly.
After the system integration, the system has been tested at lab scale. The purpose of the initial AQUALITY prototype system tests is to verify the overall system functionality and to perform tests with calibration beads.
These tests were performed at lab scale, just before the installation on the target site. From the technical point of view, these tests were the most important because, in a lab, we were able to detect the device functioning in a corrupted environment (which means water with bacteria). Due to the dangerously of the bacteria involved, obviously, it was not possible to check the functioning of the device in a water municipality (where the probability to detect bacteria is unlikely and it is not possible to flow bacteria in the main pipeline). For the reason above, particular attention has been paid in the management of these tests and the necessary timeframe has been dedicated to them in order to check the real detection power of the system, in case of bacteria.
The tests has been performed in two different phases:
• Phase 1: tests were performed on the single components before testing the entire system functioning; in particular the following components were tested: Piezoelectric pumps, the Ultrasonic unit and the Optical unit.
• Phase 2: Description of the tests performed on the entire system, as a black box.
Here below a summary of the two phases is reported.
PHASE 1
1 Piezoelectric Micro Pumps
The pumps were tested with a range of driving signals in order to find the right control signals to produce the needed flow rate.
The experimental setup for the pumps calibration is represented in the picture 19. It consisted of a very simple arrangement. The backer in the foreground contained the water to be injected in the pumps. The calibrated column in the foreground contained the water in output from the pumps.
From the analysis of the values obtained, we observed the most suitable value in line with the flow rate specification established and calibrated the pumps on that value.
Other tests have been carried on in order to optimize the amplitude and frequency rate to drive the pump with the higher possible frequency value.
2 Us Unit
The ultrasonic unit was tested with the piezoelectric pumps characterized in the previous section, in order to evaluate the resonance frequency of the transducer and the concentration performance in the real working conditions.
Resonance frequency
The resonance frequency of the transducer is a critical parameter for the good concentration performance. During the AQUALITY preliminary tests, LABOR and USOUTH agreed to use an oscilloscope.
The value of the resonance frequency can be different depending on the flow rate of the liquid which flows into the US unit and some parameters of fabrication (such as the thickness of the silane glass).
In the case shown in the figure above (representing one of the preliminary tests), the resonance frequency was 785 KHz.
Bacteria count
In order to evaluate the performance of the system, a solution of bacteria at known value was prepared and was measured by bacteria count on plates. Then, this sample was flowed into the unit. The volume of the water collected after concentration was counted again.
This test was repeated 3 times. In the first two repetitions, a concentration increasing approximately of 2x was observed.
In the last repetition, no concentration was observed, but a check of the unit revealed a crack on the silane glass: so this case was not considered as valid.
A 2x concentration is smaller than the values obtained by studies in Southampton (8x), but this can depend on the different operating conditions of the integrated system in respect the lab test for the single component (such as flow rate and frequency).
3 Optical Unit
Validation of the optical response of fluorescently labelled liposomes with other pathogens.
Before performing the final tests on the prototype, a detailed investigation was carried out to explore the behaviour of the previously selected liposome formulations in the presence of two other bacterial strains, Clostridium perfringens (ATCC 13124) and Salmonella thyphimurium (ATCC 14028).
Liposomes containing a fixed amount of umbelliferone (7%) were used at fixed total lipid concentration.
The fluorescence experiments were carried out in the emission mode, collecting the fluorescence of umbelliferone. Experiments were carried out in a medium similar to the actual media to be analysed by the AQUALITY device, i.e. a pipeline water (from one of Rome pipelines). These tests were carried on by the CNR. The results indicate that variation of the fluorescence of umbelliferone can be observed upon the addition of bacteria to liposomes.
In order to simulate a potential real situation, CNR carried out experiments in pipeline water containing at the same time S. thyphimurium , E. coli, S. aureus and E. faecalis. The total concentration of bacteria was set as in previous experiments, in order to maintain constant the ratio between liposomes and bacteria. As expected, also in the presence of the pathogen mixture, liposomes formulated with the surfactant display a higher sensitivity.
Tests on the AQUALITY optical unit
The experiments described below concern the set-up of the optical unit of the AQUALITY device and were carried out both off line and on line. The tests in the presence of bacteria were carried out in the presence of the following pathogen strains: E. faecalis, S. aureus and E. coli. These tests were carried on by LABOR and the CNR.
Liposome preparation
To guarantee sterile conditions for the different liposomes formulations, we performed liposome extrusions in 15 ml sterile plastic tubes (Falcon) and we stored liposomes.
Evaluation of the linearity of the optical response of the fluorescent probe
The linearity of the optical response of the fluorescent probe was evaluated by measuring the fluorescence of standard solutions of the fluorescent probe, umbelliferone, in EtOH abs in a reference fluorimeter (Fluoromax Horiba) and in the optical unit of the AQUALITY prototype.
The concentrations of the standard solutions were chosen in order to explore the same range of concentrations of umbelliferone embedded in liposomes in the conditions tuned for the experiments with bacteria. In the Fluoromax the emission (em=453 nm, ex=365 nm) was collected at 90° with respect to the incident beam, i.e. according to the geometry allowed by the features of the instruments, whereas in the AQUALITY optical unit the linearity of the optical response was evaluated collecting the fluorescence of umbelliferone at both 90° and 180°.
Evaluation of the fluorescence of liposomes in the absence/presence of bacteria (blank)
In the case of tests in absence of bacteria, the contamination of the pipeline water, buffer and liposomes was evaluated by streaking on non-selective agar plates for 24h.
In the case of tests in presence of bacteria, the concentration of bacteria before the tests was evaluated by a turbidimetric method and, after the tests, the real concentration of bacteria was evaluated by inoculating an agar plate and counting the colonies.
TESTS IN ABSENCE OF BACTERIA
The fluorescence of umbelliferone embedded (7%) in the mixed liposomes was investigated at a final concentration 0.02 mM of total lipids starting from different concentrated solutions to evaluate the presence of artifacts in the emission of fluorescence probe deriving from potential phenomena of the concentrated solutions, such as liposome clustering.
In fact, the most suitable concentration for liposome storing was found 5 mM in total lipids, however the final set up of the device could have required the addition of larger volumes of liposome solution and hence a more dilute stock solution. Dilution of 1 mM and 5 mM stock solutions of umbelliferone containing liposomes to the final 0.02 mM concentration gave the same value of fluorescence. This result indicates that dilution of different stock solutions to a final 0.02 mM concentration in total lipids did not influence the fluorescence of the probe.
Evaluation of the time for spontaneous mixing: tests were carried out to verify the dependence of signal intensity on the addition time. Actually it was found that it is necessary to mix the solution upon dilution of the small volume of liposome stock solution to the final volume in order to read properly the emission signal. This involved the additional set up of a mixing procedure in the AQUALITY device.
TESTS IN PRESENCE OF BACTERIA
The fluorescence of umbelliferone embedded (7%) in the mixed liposomes upon the interaction with bacteria was investigated at a final concentration 0.02 mM of total lipids starting from different concentrated solutions to evaluate the potential arising of artifacts due to concentration.
Dilution of 1 mM and 5 mM stock solutions of umbelliferone containing liposomes to the final 0.02 mM concentration gave, in the presence of bacteria, the same value of fluorescence. This result confirms that the emission of umbelliferone is independent from the initial concentration of liposome suspension.
Evaluation of the liposome/optical unit detection limit
Other fluorescence experiments, aimed at finding the detection limit of the AQUALITY optical sensor were carried out at different concentration of bacteria. Because of the difficulty of preparing and counting a solution of 1CFU/ml, the minimal concentration used to test the optical unit was 10CFU/ml. In this condition the optical unit measured ~900 F.U.
PHASE 2 – THE SYSTEM AS A BLACK BOX
Once each single component was tested separately, new tests with the entire system were performed.
These tests was needed to verify that every single operation ran with the others in the correct way. Therefore, these were a verification that the hydraulic system worked properly. These were necessary also to verify that the phases of loading and discharging of all liquids in tanks ran properly.
All the operations worked properly.
However for verifying the robustness of the results, several tests were done, first using water without bacteria and then water with a known value of CFU/ml over the threshold. These two tests typologies were repeated more times in order to verify also that the cleaning activities were executed well and no bacteria remained in the hydraulic circuit: for this purpose, two cleaning activities were performed and set in the control software. As a conclusion, it was important to include also a third test case after the positive test.
Finally, for the reason above, the three more representative test cases executed in sequence and continuously repeated during the black box tests, are reported in the table below. In addition, a fourth test case has been added to the list. This test case takes into consideration a human error: if the user does not load the correct tank with the liposomes solution, the software, just before the optical analysis phase, must display a warning message in the HMI in order to advise the user of the lack of liposomes in the system.
The result of a complete automated tests is reported in the log file, generated by the software. In figure 23 the content of log files of two explicative tests (positive and negative).
During the tests, the user interface showed the state of each pump and valve during the process execution. Precisely, the user interface visualizes the command that the control system sends to the specific valve/pump. Therefore, there was the possibility to check if the physical state of a pump /valve matched with the state displayed in the user interface.
In conclusion, the hydraulic system worked properly and the results reported were coherent with the input.
At the end of these activities the prototype was finally ready to be installed on site (Norway) for the final tests and validation.
Pilot Projects
The prototype was finally ready to be installed on site (Norway) for the final tests and validation. The pilot project will be set up for demonstration purposes in Norway at ROROS water treatment plant. In this task TI will take care of the installation of the AQUALITY online monitoring system at ROROS plant. TI will be the responsible, in this task, of realizing the report on the installation and will take care of validating the system and of granting the possibility, for ROROS, to remotely monitor its facilities through AQUALITY technology. The activity includes test run and training session as well as preparation of instruction manual for the operator of the AQUALITY prototype.
The system was assembled in the basement of the water plant, were all the electrically powered units were placed on a small table (picture below). The three cables, running between the hydraulic unit and the control unit, were quickly connected into their sockets (BNC cable, SMA cable, and a 40 pin plug). The power supply was connected to a nearby 230V two pin grounded socket. Even though the installation took place in a basement, the 3G internet antenna only need to be placed close to the basement ceiling to obtain a good signal strength. The antenna was connected to a dedicated cabinet, which included power supply and adapter. A standard Ethernet cable was used to wire the signal to the control unit. The setup is shown in the picture 24.
The AQUALITY prototype was tested over a 3 weeks period and results from the testing of the AQUALITY system was compared with waters samples taken at the same location and time as the running of the AQUALITY system. These water samples was then sent to an accredited laboratory for analyses. Based on the experience and positive results from the installation and testing, further recommended actions in the process toward a fully commercial product are presented.
In conclusion of the pilot Projects, the Consortium can state that the different tasks have been successfully completed.
Finally, the AQUALITY project focuses on an evaluation of users’ acceptance and this activity has been performed in close cooperation between Teknologisk Institutt AS (TI) and Rørosmeieriet (ROROS). The system validation includes identification of key provisions according to Norwegian legislation (“Drikkevannforskriften”) with regards to water quality and monitoring program. Existing practices and costs for water quality monitoring at Rørosmeieriet and Røros Water Plant have been also identified and described, including investment costs of equipment, installation costs including purchase of equipment and rental costs as well as operational costs. These costs are then discussed and evaluated compared against the costs of existing water monitoring program of Rørosmeieriet and Røros Water Plant and the performance of the AQUALITY system is evaluated compared with regards to compliance with the provisions in the Norwegian legislation (“Drikkevannforskriften”).
Potential Impact:
In order to assess the economic perspective of the AQUALITY device and its potential impact and use, a dedicated study has been performed. At the beginning, the water monitoring market has been studied with its trends and requirements. Then, the results of the AQUALITY project and the maturity of the technology in general has been assessed. Finally, a strategic plan for the development of the final product from the prototype has been assessed.
Market trends and requirements
On the basis of the application scenarios identified in the early stages of the project’s lifecycle, although several applications could be possible for the AQUALITY technology, the Consortium focussed the priority on the scenario Public & Private Utilities. This scenario, in fact, best represents the target industrial sector for the project, and best fits with the AQUALITY water analysis method, which, according to the technical discussions following the realization of the application scenario description, is intended for industrial processing more than for the drinking water market.
For this reason a deep market investigation in the field of the water monitoring has been performed. The key facts about the state of art of this sector have been turned out to be:
1. Water supply agencies are responsible at all times for the quality and safety of the water they produce and supply. That’s why, surveillance and quality control are mandatory for such organisations and entities (surveillance meaning an investigative activity aiming at identifying and evaluating potential health risks associated with the supplied water);
2. Infectious diseases caused by pathogenic bacteria, viruses and parasites are the most common and widespread health risk associated with drinking-water. The public health burden is determined by the severity and incidence of the illnesses associated with pathogens, their infectivity and the population exposed; microbial drinking water safety is not related only to a faecal contamination;
3. Preventive management is a preferential approach to ensuring drinking water safety, and should take into account the characteristics of the water supply from catchment and source to its use by consumers;
4. One core objective of surveillance is to assess the quality of water supplied by the supply agency and at the point of use;
The sector is also largely dependent on testing laboratories. The market is highly integrated with a few firms offering whole services for monitoring and testing at in-house labs. There is a need for miniaturisation of testing technologies to reduce the large costs associated with laboratory testing of samples: a trend also highlighted by data analysers. Overall, the water testing market can be divided into three distinct categories:
Low-end equipment - Test kits for low-end equipment include hundreds of variations of colorimetric devices, handheld electronic analysers, and spectrophotometers.
In-line equipment - These include pH, dissolved oxygen and turbidity measures . Water utilities and industrial processes are increasingly using in-line monitoring to maintain high standards of quality by changing their focus from the detection of issues in quality to preventing them.
High-end equipment - consists of organic analysis (gas Chromatography and Liquid Chromatography) and metal analysis (Inductively Coupled Plasma) which are applied with Mass Spectrometry technologies.
In Europe, Germany was the leading exporter (38% share) in 2010, while France and the UK exported similar levels. A third cluster of exports included Sweden, the Netherlands, Italy, Austria and Ireland. The USA is the clear market leader in the supply of instrumentation for water monitoring and testing and is the dominant supplier of high-end equipment.
Approximately 25% of testing in the water sector is analysed by large multinational firms who supply laboratory services. This market is dominated by a small group of major players, 2 of which are from the EU. Coupled with a leading EU presence in the low-end equipment market demonstrates EU strengths overall in the global market for water monitoring and testing.
Innovation in the water monitoring sector is incremental, largely due to the resistance of users to adopt new technologies and the overall maturing of the market. Scope for innovation exists in the need for the miniaturisation of equipment that is able to analyse data onsite and produce real time results. These types of technology have the potential to generate huge cost savings by eliminating the amount of testing required in laboratories.
In conclusion, markets are demanding easy to use, low cost and automated devices comparable to the currently available solutions. The AQUALITY device meets these market demands through an incremental innovation. Obviously, further optimization of the system will be necessary after the end of the project to make it marketable.
Here below, a short list of the SMEs’ market objectives is included. After, their potential market for the AQUALITY device is reported.
SMEs’ Market objectives
ENSATEC
• Ramp-up of the AQUALITY device at ENSATEC in order to target industries of any size, differing the commercial offer.
• Target the water monitoring market with the optimization of the system for industrial processes.
• Create marketing analysis to enter the market: the first step is the Spanish market in which ENSATEC is able to exploit the exclusivity.
• Investigation of new applications/markets for the Opto-US Unit.
• Investigation on the Liposomes market.
ARGUS
• Optimize the system to address the market of the portable devices.
• Optimize the algorithm for processing the data and the HMI (copyright on the algorithm is under discussion).
• Unlock additional markets by offering the device in a price-sensitive segment.
• Investigation of new markets for the Opto-US unit.
• Create in collaboration with ENSATEC a market strategy to access the German market with the Opto-Us unit (in which ARGUS has the exclusivity).
OPTO
• Ramp-up of the OPTO-US Unit in order to target different markets.
• Identification of the most profitable markets and set up of the marketing strategy.
• Creation of the marketing strategy (in collaboration with ENSATEC) for the European markets of the water monitoring.
• Address 20% of the quality control modules (for which we have a patent pending).
IRBM
• Optimization of the liposomes formulation, targeting the EU water directive (in order to reach the requested detection limit).
• Identification of a correct marketing strategy (in collaboration with ENSATEC) for the correct commercialization of the whole system (IRBM is the exclusive supplier of the liposomes to ENSATEC).
• Investigation on the Liposomes market with the purpose to find other channel of exploitation.
ROROS
• Application of the system in its processes for free.
• After a period in which the system will be tested (12 months), if concrete savings for water and wastewater monitoring are registered and the safeguard of the water throughout the process is actually increased, ROROS will evaluate the possibility to install other systems along the process, exploiting the special conditions of the 25% off on the price.
SMEs’ Potential market/s
ENSATEC is the exclusive installer of the Online System in Spain. The first launch in the market will be done exploiting this country since it is the geographical segment in which ENSATEC is more confident. Therefore, taking into account only the Spain, at an average approx. 60 units sold 3 years after the market launch, it is estimated a revenue of approx. 1.2 M€.
ARGUS is the exclusive installer of the Online System in Germany. Therefore, taking into account only the German segment, the average number of until sold could be estimated at approx. 110 units (3 years after the market launch). It is estimated a revenue of approx. 1.5 M€. Also, the liposomes will be supplied by ENSATEC to Argus, applying a minor markup and permitting the competitive commercialization in Germany.
IRBM sells the liposomes to ENSATEC. This will create revenues of approx. 1.6 M€. In addition, IRBM is targeting also other markets for the liposomes directly to end users in the biological research markets.
OPTO sells the opto-US unit to ENSATEC. This will create revenues of approx. 165 K€. In addition, OPTO is targeting the market of the small and medium ultrafiltration units which is growing very fast thanks to the membrane technology getting mature. In those channels, the target sales price could be around 800 euro. The projection for the 2017/2018 is about 900 units sold which will create revenues of around 720 Keuro.
Economic assessment
With the aim to develop an economic analysis, which supports assumptions made on demands in the short-and medium-term, the SMEs involved provided market data and data of potential end users. Based upon these data and under consideration of the results returned from benefit-oriented cost-effectiveness evaluation, the predicted future sale of AQUALITY system has been identified.
For an integrated evaluation, both quantitative and qualitative cost-benefit analysis was performed. In addition, the costs linked to the implementation of the AQUALITY device, losses due to incidents related to water quality, and costs of illness treatment were considered to verify the potential cost saving with implications of AQUALITY.
The unique selling point of the system is being the first online microbiological testing system for water analysis available on the market. The AQUALITY system uses luminescent liposomes for continuous monitoring of microbiological contamination in industrial process water, industrial sewage, drinking water and surface water. It has the capability of protecting chemical, pulp and paper, microelectronics, metal-working, pharmaceutical, food and drinking water companies against microbiological contamination entering their industrial process water and ending up into their products.
The AQUALITY device are able to detect a very wide range of microbiological contaminants. It needs few hours of maintenance per month and in case of alarm, the results will be automatically sent to the staff responsible of the water quality check. It is a robust and easy to use system upgradable in its functionalities.
The following features characterize the final product:
Simplicity: An easy-to-use water analysis product on the market. Absence of time consuming sample and reagent preparation, multi-step test procedures, and clean up.
Speed: Fastest microbiological testing on the market.
Accuracy: Quick, clean and accurate.
Cost-effectiveness: Time saving, money saving, lower cost per completed test.
The system has a very wide range of potential customers from water quality analysis laboratories, and to all industries requiring huge quantities of high water quality for their processes. Therefore, the device has a great potential to get good market recognition. However, estimation for future sales is tightly depending on the SMEs activities to introduce the AQUALITY system to the thirsty but extensive market. For the first three years, it is predicted that more than 400 devices will be sold by the SMEs in the project.
Result of our assessments shows that AQUALITY has a great potential to increase economic benefits of the SMEs and end-users. In addition, installing AQUALITY leads to several qualitative benefits such as producing safer products that leads to have a better publicity.
Exploitation Plan
Talking about the exploitation strategy, it is a fact that the main output of this research project is a prototype and, as a such, it needs further optimization, re-engineering and tests in order to have final product ready to go to the market.
In fact, the possibility to launch the system in the market requires a severe testing phase after the end of the project. This is needed both to overcome the performance of the systems already available but also to fulfill the requirements of the Water directive. In this sense, the SMEs have already taken contacts with the policy makers, in order to make them available of the new solution.
The SMEs have put in place a post-project plan that is divided in three main phases: the first phase is the phase in which the full commitment will be held for reaching the full compliance with the EU Water directive (detection limit able to detect even 1CFU). Taking into consideration the results achieved by the research project, we can now state that the limit is not far to be achieved. However, a re-engineering phase and new dedicated tests will be necessary. In fact, even if a good level has been reached with the prototype, a deeper investigation and optimization will be required to pass from the prototype to a commercial product.
Likely, not only the compliance with the directive will be necessary but also the adaptation to new customer requests. This brings to the second phase. After the re-engineering process, field test will be necessary. The new contacts with the customers will be also exploited to set up the marketing plan.
Finally, the commercialization can start only after this period and not before a marketing revision of the design. The final product, in fact, even if in line with the technical performance, will need a revision under the marketing point of view. The market requires specific conditions that should be taken into consideration before the official launch. This third phase will start during the second semester of the 2016, when the release of the device is stable.
A launch master plan will be prepared defining the sequence of market regions approached with the technology. For each region a detailed launch plan is to be developed to ensure efficient introduction of the technology. The launch plan has to include details like:
1. marketing mix (4 Ps: product, price, promotion, place);
2. identification of opinion leaders and reference accounts;
3. publications;
4. participation in congresses and exhibitions;
5. advertising.
This approach permits to accurately estimate the date in which the product not only will be ready from the technical side but also it will be customized with an ad hoc marketing campaign. It is, in fact, essential that the launch in the market is associated with a solid marketing and advertising phase. At the beginning, the target segments will be addressed (Spain, Germany above all). Only after a semester, the full market can be targeted.
Since the most interesting result of the project is the integrated and automated device for on-line installations, a tentative marketing mix analysis has been performed at this stage in order to identify the main features of the future strategy.
Dissemination activities
As planned by the Consortium members, the dissemination activities have been structured in 2 main stages, as reported below.
By starting early to disseminate the research of AQUALITY, and by providing with some advance notices about the project, the Consortium intended to strengthen the impact of such activity. The objective of this stage of the dissemination was that of presenting to the target groups that the project exists and of giving them an idea of what it is about and tries to achieve, and this can be done even at an early stage of development.
This stage has been the “core” of the First Period of the project. However, the activities foreseen in this stage have been intensified and completed also during the Second Period of the Project’s lifecycle.
The objective of this stage was that of showing what the AQUALITY technology can offer to the interested companies and organisations, the preliminary outputs/outcomes that can be relevant in solving specific problems or needs of the water supply sector.
In the second period of the Project, the dissemination activities have followed the provisions of the second stage.
The scientific community, including technical staff of companies or the R&D divisions of water supply companies, researchers and people interested in technological innovation in the field of water monitoring and analysis, have been the main target of our communication. Then, stakeholders and potential end-users have been the main focus with a communication strategy aims at creating awareness in the general public about the main outcomes of the AQUALITY project.
In the table below, which show the key means of dissemination as hypnotized in the P1, we have reported those means that actually have been used in the course of the project lifecycle.
Stage 1 To raise awareness on the features of the new AQUALITY technology
- Newsletters
- Press releases
- Articles on online magazines
- Project website, etc.
- Social Networks
- SMEs’ official websites
- Specialized portals
- Contact with other EU project and relevant sharing of lesson learnt
Stage 2 To provide in-depth knowledge about the AQUALITY technology - Relevant event in the target sectors, at both national or international level
- Contacts with stakeholders, regulators and policy makers in the water monitoring sector
- Continuous contact with the end users with specific updates on the status and progress of the project
- Seminars/workshops or demo events to be organised with selected groups of stakeholders
- Scientific publications and articles on magazines, journals, web portals
- Dissemination materials, such as brochures and posters
- Presentations
- Video clip
- Wiki page
List of Websites:
The AQUALITY website is one of the vehicles for the dissemination of the project; this online tool is in fact the main interface towards the external audience and the stakeholders. The website provides basic information about the project in a very immediate way, it gives an overview on the relevant events in the addressed market and on the intended technological approach, and it allows to establish contacts with the interested companies and persons reading about the project through the contact form.
The domain of the AQUALITY website has been registered at: www.aquality-project.eu.
The contents are online since March 2012, thus it is important to highlight that the decision of the Consortium to create this dissemination tool in advance with respect to what was planned was taken with the strategic objective of starting disseminating from the very beginning of the project.
Diseases caused by bacteria, viruses, fungi and other parasites are major causes of death, disability, and social and economic disruption for millions of people. Water is an important environmental source and agent for transmission of many of these pathogens. The AQUALITY solution is an online water monitoring device for microbiological contamination analysis, that allows industries and environmental protection agencies to replace the routine activities of sampling and laboratory testing of pathogens. The new system is able to real time monitor the quality of industrial process water and effluents basing on an opto-ultrasonic device and on a lipid-based diagnostic kit. To realize the device, the focus of the first project period was on the conceptual design and development of the single components. During the last phase of the second project period, the AQUALITY consortium dedicated its efforts to the system integration and verification.
Starting from the analysis of the User and System requirements, the technical specification of all the components have been defined on the base of the requirements and specification set up by the beneficiaries and end users. The final architecture of the AQUALITY system has been drawn. The main components of the system are: the mechanical layout; the Ultrasound Unit; the Optical Unit and the Control system. Thanks to the effort dedicated to this delicate phase, the following steps of design, development and integration preceded with less risks and uncertainties, permitting to recover the extra time invested in this phase and, above all, permitting to have an integrated device able to measure and detect bacteria.
The part of the AQUALITY project concerning liposomes was driven by the CNR and involved efforts in three main activities: synthesis of lipid components (positively charged lipids) to engineer liposomes and make them more sensitive to the interaction with bacteria; formulation and characterization of liposomes and evaluation of the liposome/bacterium interaction. Two liposomes formulations have been developed, composed of the natural lipid DOPC, a cationic surfactant and the fluorescent probe umbelliferone, suitable to be efficient nanosensors to be integrated in the AQUALITY device.
The system integration was led by LABOR, with the support of CNR and USOUTH. The main output of the activities performed was the integrated AQUALITY device. The system can be considered made up by two main subsystems: the hydraulic and the electronic (control) with their external interfaces which permit the integration of the two subsystems in the complete prototype. After the system integration, the system has been tested at lab scale. The purpose of the initial AQUALITY prototype system tests is to verify the overall system functionality and to perform tests with calibration beads. As first step, each single component was tested separately. Then, new tests with the entire system were performed. These tests were needed to verify that every single operation ran with the others in the correct way. Therefore, these were a verification that the hydraulic system worked properly. These were necessary also to verify that the phases of loading and discharging of all liquids in tanks ran properly. In conclusion, the hydraulic system worked properly and the results reported were coherent with the input.
Finally, the prototype was finally ready to be installed on site (Norway) for the final tests and validation. The pilot project will be set up for demonstration purposes in Norway at ROROS water treatment plant. In this task TI will take care of the installation of the AQUALITY online monitoring system at ROROS plant. TI will be the responsible, in this task, of realizing the report on the installation and will take care of validating the system and of granting the possibility, for ROROS, to remotely monitor its facilities through AQUALITY technology. The activity includes test run and training session as well as preparation of instruction manual for the operator of the AQUALITY prototype. The results of the testing and operation of the AQUALITY system for a 3 weeks period has been evaluated and the results from of the microbiological parameters is compared to water samples analyzed at an accredited laboratory. In addition, the system validation includes identification of key provisions according to Norwegian legislation (“Drikkevannforskriften”) with regards to water quality and monitoring program. Existing practices and costs for water quality monitoring at Rørosmeieriet and Røros Water Plant have been also identified and described.
Project Context and Objectives:
Diseases caused by bacteria, viruses, fungi and other parasites are major causes of death, disability, and social and economic disruption for millions of people . Despite the existence of safe and effective interventions, many people lack access to needed prevention methods and treatment. The lost productivity, missed educational opportunities and high health care costs caused by infectious diseases directly impact families and communities. Infections are prevalent in developing countries, where co-infection is common.
Water is an important environmental source and agent for transmission of many of these pathogens. Water-related pathogens that have emerged or re-emerged recently include Cryptosporidium, Legionella, Escherichia Coli 0157, Rotavirus, Hepatitis E virus, and Norovirus (formally Norwalk virus) (WHO, 2003).
It is well known that many industries require huge quantities of water for their processes and need to be supplied by water with specific characteristics, since water quality may seriously affect production units and quality of finished products. Currently, the identification and quantification of pollutants in water are mostly carried out manually through sampling and subsequent laboratory analysis (off-line analysis. These methodologies of work involve some significant costs in terms of displacement to sampling points, reagents and specialized personnel dedicated to the operation, leading to time consuming and economically challenging approaches, causing the number of analyses performed to be kept at the bare minimum.
The industry therefore is calling for novel, cost-effective solutions to meet these new challenges: there is an urgent need for rapid methods for detecting the major waterborne pathogens, which can be implemented both online and at laboratory scale; this achievement would represent a huge competitive advantage for the enterprise proposing it and would open up a significant international market.
We propose to develop an online water monitoring device for microbiological contamination analysis, that allows industries and environmental protection agencies to replace the routine activities of sampling and laboratory testing of pathogens. The new system will be able to real time monitor the quality of industrial process water and effluents basing on an opto-ultrasonic device and on a lipid-based diagnostic kit. The novelty of our approach is the use of engineered liposomes for detecting bacteria in water: these are nanoparticles formed by a lipid bilayer enclosing an aqueous compartment displaying features that can be different (pH, ionic strength, composition) with respect to the bulk. We will load liposomes with a chromophore and will engineer them in order to make them specifically react with target bacteria; this is the simple operating system of the AQUALITY system, which is completed by an ultrasonic unit to concentrate bacteria and an optical unit for detecting the sample colour change following to the interaction between liposomes and bacteria.
Therefore, the tangible outcomes of AQUALITY project will be:
o A new method for rapidly detecting the presence of bacteria strains in water and wastewater, that relies on the interaction of liposomes with bacteria without specific antigen- antibody interactions;
o The implementation of an opto-ultrasonic unit to concentrate the bacteria and detect the sample color changes;
o The implementation of this method in a new device, which will be integrated in a real industrial production line (online mode) at an end-user facility.
THE LIPOSOMES
Liposomes are nanoparticles formed by a lipid bilayer enclosing an aqueous compartment displaying features that can be different (pH, ionic strength, composition) with respect to the bulk. Because of their morphology, since their discovery in 1964 [J. Mol Biol 1964, 8, 660-668] liposomes have been exploited as models of biological membranes.
The use of liposomes as sensor elements for the detection of different species has been widely described and is inspired to some functions of biological membranes [J. Am. Chem. Soc. 2011, 133, 9720; J. Am. Chem. Soc. 2008, 130, 5010; Trends Anal. Chem. 2005, 24, 9; Sens. and Actuators B 2005, 107, 82; Anal. Chim. Acta 2006, 556, 127; Biotechnol. Prog. 2006, 22, 32; Biosensors and Bioelectronics 2007, 22, 2848]; actually the basic concept is mimicking the role of biomembranes in signal transduction and information processing, based on the reorganization of the lipid components upon the interaction with defined entities (bio molecules, ions, bacteria, viruses….). The interaction of bacteria with the lipid membrane of defined liposomes might trigger processes such as lipid reorganization or membrane disruption, or induce changes in the electrical properties of the liposome surface, the kind of perturbation depending both on lipid composition and on the interacting entity. The changes induced on the lipid bilayer by the interaction with bacteria might in turn affect the optical features of a proper dye included in the liposomes and thus be revealed as an optical response.
The initial goal of the project was developing engineered liposomes capable of interacting specifically with target bacteria, namely E. Coli, S. Aureus, E. Faecalis, and loading their internal aqueous compartment with a proper dye (pH indicator, chromophore), so that its released into the bulk upon disruption of liposomes due to their specific interaction with bacteria, would have produced an optical signal (changes in the absorbance or emission) (Figure below).
Figure 1 - Interaction of liposomes with bacteria.
However, as the project developed it became evident that the lipid bilayer stability, necessary for storage, was in contrast with the attitude to disrupt upon interaction with bacteria, and that specificity toward target bacteria could detract efforts from other features such as high sensitivity and fast response. Therefore we changed means of detection of the perturbation caused by the interaction of liposomes with bacteria, and chose a chromophore sensitive to changes in the electrical features of liposome surface, exploiting the fact that the interaction of positively charged liposomes with bacteria (that are negatively charged) changes dramatically the electrical features of liposome surface. Further we set as a goal the development of aspecific liposomes, i.e liposomes capable of giving an optical signal (due to the change of surface potential) upon interaction with various bacteria.
THE ULTRASOUND UNIT
A major obstacle in the implementation of rapid pathogen detection methods in drinking water is the low concentration of microorganisms in samples, requiring laborious methodological steps to increase the microbial concentration to a level sufficient for analysis1.
Concentration of bacteria in microfluidic systems has been achieved by different means, including electrical forces2-5, hydrodynamic effects6, evaporation7,8, and ultrasound-enhanced sedimentation9.
The use of ultrasound (US) in microfluidic environments has recently emerged as a non-invasive way of manipulating cells and particles for a range of applications10, such as sample enrichment11,12, intra-cellular drug delivery13-16, sensing and bio-detection17-19, sample filtration20-22, cell and particle sorting23-25 and trapping26,27. Continuous-flow concentration of particles by acoustophoresis is usually performed by generating an ultrasonic standing wave (USW) within a fluid chamber, causing suspended particles to focus in a confined liquid volume under the action of primary acoustic radiation force28. Either one-dimensional (1-D) or two-dimensional (2-D) focusing can be achieved, depending on the properties of the acoustic field. In order to obtain a significant increase in particle concentration, the fluid flow must be split into the particle-rich and the particle-depleted fractions28,29 at finely controlled flow rate ratios. Based on this functioning principle, concentration of a range of particles including bacterial spores, biological cells and polystyrene spherical micro-beads 11,24,30-35 has been demonstrated.
Manipulating bodies as small as bacteria by means of acoustic radiation forces is however a considerable physical challenge27. This is mainly due (i) to the fact that the acoustic primary radiation force scales with the cube of particle radius29, and to the increased particle susceptibility with respect to hydrodynamic drag force resulting from acoustic streaming or thermal convection36,37. It has been found that the critical particle size at which a transition to drag-dominated behaviour occurs is ~1 μm, at operating frequencies of approximately 2 MHz36,38. For this reasons, only few studies have demonstrated concentration of flowing particles with diameter <2μm32,33 and, to the best of our knowledge, continuous-flow concentration of bacteria by acoustophoresis has not been previously demonstrated. Recently, Hammarström et al. reported on highly efficient trapping of E. coli by half-wave resonance in glass microcapillaries27; however they employed 12 μm diameter seed particles to increase the trapping efficiency through the use of secondary, inter-particle radiation forces.
In this project we initially set ourselves the goal of producing a 4x bacterial concentration with a half-wave device, however as the project developed it became evident that higher concentration factors would be required to meet system performance requirements. In order to meet these higher concentration factors we developed a unit based around a thin-reflector acoustic mode.
Project Results:
System Requirements
The main aim of the work package “System Requirements” was to perform a study of the industry addressed in the AQUALITY project. The intention of the study was that of comprehending the actual characteristics of the target industrial scenario and of identifying the relevant aspects to be reached during the project.
To do this, the consortium identified and reported in a document the main information related to water monitoring technologies actually in use across Europe together with the main EU regulations in the fields of water control and quality assessment.
Three possible scenarios have been taken into account and described accurately in our reports:
• Scenario 1: Food & Beverage Industry
• Scenario 2: Private and Public Utilities
• Scenario 3: Bathing Waters
The AQUALITY online monitoring device is mostly intended for industrial processing waters. Thus, the choice of the application scenario for the project has been made consistently with the objective of the project of reaching the water suppliers, and the industrial processors making great use of water of high quality.
On the basis of the application scenarios, the user requirements have been collected and organized. However, stated that several applications could be possible for the AQUALITY technology, the Consortium focussed the priority on the second identified application scenario, that of Public & Private Utilities. This scenario, in fact, best represents the target industrial sector for the project, and best fits with the AQUALITY water analysis method, which, according to the technical discussions following the realization of the application scenario description, is intended for industrial processing more than for the drinking water market.
The project partners were involved in the definition of all the user requirements of the project. This activity, carried out as an iterative process, led to the identification of slightly different bacteria to be detected by the system, respect to what was originally planned. Furthermore, the functionalities, the alarms and interface requirements, the size and properties of the device have been indicated in this data collection.
After the user requirements collection, the system requirements have been identified, collected and organized. The goal was obviously that of assessing a complete, consistent and clear set of requirements through which the different components of the system could be outlined.
The techniques used for the identification of the AQUALITY device requirements have been the following:
- Plenary meeting (kick-off meeting in Rome) for performing a brainstorming session,
- Interviews in the target industry performed at a Consortium level and at a restricted external group of stakeholders/potential end-user in the sector, in Italy and Norway,
- Use of a supporting model for requirements collection and sharing of the chosen (IEEE) shell structure with the explanation of how these should be filled in,
- Exchange of further contributions via email/ phone with the Consortium members,
- Iterative revision of the document for updates and improvements.
At the end of these activities a complete set of system requirements was available for the further technical specifications of all the system component.
Hardware development
Starting from the analysis of the User and System requirements, the technical specification of all the components have been defined on the base of the requirements and specification set up by the beneficiaries and end users.
The final architecture of the AQUALITY system has been drawn, and the responsibilities of each partner in the design and implementation of the specific units have been highlighted; the final version of the architecture can be considered the results of a close collaboration among the partners (RTDPs in particular).
The main components of the system are:
1. The mechanical layout;
2. The Ultrasound Unit;
3. The Optical Unit;
4. The Control system.
The technical specification of the single components has been a fundamental phase. Thanks to the effort dedicated to this delicate phase, the following steps of design, development and integration preceded with less risks and uncertainties, permitting to recover the extra time invested in this phase and, above all, permitting to have an integrated device able to measure and detect bacteria.
The Mechanical Layout
The design activity of the mechanical layout has been performed using 3D CAD technology by LABOR, and the images of the digital mock-up are very well representative of the prototype.
The results of the design activities are of three different types:
• manufacturing drawings of the parts to be prototyped in mechanical workshop,
• assembly drawings, which serves as a guide for assembling and installing the system,
• BOM (bill of material), to source parts and off the shelf components.
The device consists of a parallelepiped, 410mm width X 330mm depth X 252mm height, with the inlet filter mounted on the right side, for a total width of 550mm, and handles on top cover, for a total height of 301mm. The main section and components are outlined. The structure of the AQUALITY is supported by four identical plastic legs (represented in black), which are connected and fixed in position by the internal main plate and the six covers.
The top cover is equipped with two robust handles, for easy handling and transportation of the device, and a window, which can be removed, without any additional tools, by means of four knurled head screws, to replace the consumables inside the device (i.e. filling the liposome storage tank), to make fine regulations (i.e. adjusting the pressure of the CO2 regulator), or to allow monitoring the internal of the device during testing.
The Ultrasound Unit
The picture 3 of the attachment shows electrical impedance plots for Half-wave (HW) (a) and Thin-reflector (TR) (b) devices. Impedance minima were identified at fR = 1.43 MHz (HW) and fR = 831 kHz (TR), for water-filled devices under static fluidic condition.
Final verification of the resonant frequency was carried out using a digital oscilloscope, and adjusted resonant frequencies of 1.46 MHz and 826 kHz were determined for HW and TR device, respectively.
Assessment of devices functioning was also performed qualitatively, by in situ microscope observation of bacteria behaviour in the absence and in the presence of US. In a series of experiments, the total flow rate was conveyed into OBR and bacteria flow behaviour in the outlet slot was visualized. In figure 4, (a) shows images of bacteria flowing into the slot (i.e. in a direction perpendicular to the image plane) of a HW device, both in the presence and in the absence of US (fR = 1.46 MHz; 2.5Vpp; QIN = 20 ml/h). Acoustic focusing of E. coli in a narrow fluid layer located approximately at the centre of the slot can be appreciated, whilst bacteria distributed uniformly when US was deactivated. This was confirmed quantitatively by performing a z-scan analysis in the fluid chamber. Results show that the large majority of bacteria (~90%) are focused within a ~30 μm thick fluid layer centred on the chamber axis (b).
The picture 5 (a) shows microscope images of bacteria flowing on the reflector surface of the TR device (QIN = 20ml/h and QBR/QIN = 0.1). It can be observed that the acoustic field was capable of effectively constraining the bacteria in close proximity or in contact with the glass reflector. (b) shows microscope images of bacteria flowing into QBD slot, in the presence (left) and in the absence (right) of US. Microscope focus was set at the very bottom (in z-direction) of the slot, and a nearly bacteria-free slot was observed when US was activated signifying that most of E. coli were forced to flow above the slot towards OBR (c). (d) shows a microscope image of bacteria flowing into OBR, where the presence of lateral modes can be appreciated.
Ultrasonic bacterial manipulation: E. coli vs S. epidermidis
1. Half-wave device
The figure 6 (a) shows the % number of bacteria along the z-coordinate, determined from z-scan analysis (taking successive images at varying focal positions) at a fixed x- and y-position (QIN = 20 ml/h; f = 1.46 MHz; 3.1Vpp). Representative results for both E. coli and S. epidermidis are reported, demonstrating that the HW device is capable of effectively manipulating both bacterial species. The average performance is illustrated in (b), where the average % number of bacteria located in a 40 μm thick fluid layer (centred on the chamber mid-plane) is reported. Device performance slightly reduced when the inlet flow rate was increased from 10 ml/h to 20ml/h, with no statistical difference between E. coli and S. epidermidis. Figures c-e and f-h show representative microscope images of E. coli and S. epidermidis, respectively; taken at z = 100, 190 and 300 μm (where z = 0 μm corresponds to the carrier surface and z = 390 μm corresponds to the reflector surface ).
2. Thin-reflector device
The figure 7 (a) shows the % number of bacteria along the z-coordinate, determined from z-scan analysis at a fixed x- and y-position (QIN = 20 ml/h; f = 826 kHz; 22Vpp). Representative results for both E. coli and S. epidermidis are reported, demonstrating that the TR device is capable of effectively manipulating both bacterial species. The average performance is illustrated in (b), where the average % number of bacteria located in a 10 μm thick fluid layer (from the reflector surface) is reported. As for HW device, no statistically significant difference between the two bacterial species was detected, at both inlet volumetric flow rates applied. The figures c-e and f-h show representative microscope images of E. coli and S. epidermidis, respectively; taken at z = 0, 50 and 100 μm (where z = 0 μm corresponds to the macor surface and z = 100 μm to the reflector surface).
Minimization of bacteria attachment on the reflector surface in TR device
Bacterial loss due to adhesion to the glass reflector surface was a major cause of low performance in initial devices. A number of coatings were evaluated to solve this problem. For each surface experiments were carried out with the aim of quantifying the total % loss of bacteria due to attachment on the glass surface. The table 1 shows the % loss of bacteria, for both silane- and PMOX-coated glass reflectors. In the preparation of silane coatings, two different strategies were investigated, namely (i) silane was dissolved in anhydrous dimethylformamide (DMF) (as described in Section 5.5.6) and (ii) silane was dissolved in hexane. The latter approach was associated with reduced formation of undesired patterns on the glass surface, due to the higher solubility of silane in hexane compared to DMF.
Results show that silane dissolved in DMF provided the best performance (i.e. lowest % number of bacterial loss), for both bacterial species. This is coherent with in situ results illustrated in Section 6.5.1. PMOX demonstrated to be an effective repellent for E. coli, but was less effective in preventing attachment of S. epidermidis (~9 % loss in bacteria). Silane dissolved in hexane performed less effectively compared to the other surface treatments, with % bacterial loss of ~22 % (E. coli) and ~15 % (S. epidermidis). Results from these studies confirm the suitability of hydrophobic silane (dissolved in DMF) as a bacterial repellent coating in TR devices. However, it is worth mentioning that there appears to be a time evolution of the attachment process which could lead to different results depending on the duration of the experiment. This, and other aspects of bacterial adhesion in the presence of US, is currently under investigation in our laboratories.
Performance assessment of TR device
Figure 8 shows the concentration increase achieved with TR device, at QBR/QIN ranging from 0.0025 to 0.2. Experimental values are compared with theoretical values calculated assuming that all bacteria flow into OBR. The percentage difference between experimental and theoretical values is reported.
Results show that the TR device is capable of achieving significant increase in bacterial concentration and thus it may represent a powerful technological platform in pathogens analysis systems for water quality examinations. A maximum 60-fold concentration increase was achieved at QBR/QIN = 0.005. However, by comparing experimental with theoretical results, it can be observed that there is still a large margin for performance improvement. Notably, device performance was observed to degrade with reducing QBR/QIN, as it can be observed in Figure 8 where theoretical and experimental results are plotted together. This is likely due to a significant number of bacteria escaping the bacteria-rich layer, caused by insufficient radiation force to constrain the bacteria in a fluid layer as thick as the characteristic size of the bacteria itself.
While the device was observed to perform very effectively in the central region of the chamber, a loss of performance was observed in close proximity to the channel side walls, which may be the cause of the observed degradation of device performance with reducing QBR/QIN. Other sources of performance degradation may include bacteria sedimentation in the syringe or attachment on the tubing inner surfaces, particularly in the long-term. Given that E. coli and S. epidermidis displayed a similar behaviour when exposed to US in both HW and TR resonators, we expect that analogous concentration increase will be achieved with S. epidermidis.
Effect of initial bacterial concentration on TR device performance
Figure 9 shows the increase in bacterial concentration (θ) achieved using the TR device (at a fixed QBR/QIN = 0.05) at varying initial concentrations of bacteria in the inlet suspension (CIN), corresponding to 10, 102, 103 and 104 CFU/ml.
Results show that device performance slightly decreases with reducing the initial bacterial concentration from 104 CFU/ml (θ = 14.31) down to 103 CFU/ml (θ = 12.07). Notably, TR performance remains substantially invariant with further decreasing bacterial concentration (i.e. θ = 11.23 at CIN = 10 CFU/ml), and likely no statistical difference in performance subsists at these lower inlet concentrations. Increased performance at the higher CIN may be attributed to the effect of acoustic secondary radiation forces, which becomes stronger when the distance between bacteria reduces (conforming to higher bacterial concentration). However, it should be noted that approximations in the calculation of device performance at the lower CIN may have caused marginal underestimation of TR performance. Overall, TR device is capable of achieving significantly high concentration increase under a wide range of bacterial concentrations in the inlet suspension.
Comparison between HW and TR devices
Table 2 shows the concentration increase achieved with using TR and HW resonators at QIN = 20 ml/h and QBR/QIN = 0.2 0.1 and 0.05. Results show that at the higher QBR/QIN, the two resonators performed similarly (percentage difference in concentration increase: 17.4%). However, with reducing QBR/QIN, the TR configuration displayed a significantly improved performance compared to the HW configuration. The concentration factor for the HW device reached a maximum of 3.39 at QBR/QIN = 0.1 and reduced to 2.98 at QBR/QIN = 0.05; likely due to an increased number of bacteria escaping the top and bottom outlets of the device.
Results show that the TR device is capable of achieving significantly higher concentration increase (~1 order of magnitude) compared with traditional HW configurations. The latter performed similarly to previously reported half-wave or quarter-wave devices32,33.
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The Optical Unit
Taking into consideration the Technical specification for all the components the Optical Unit has been developed by LABOR. The Optical unit is connected and driven by and electronic Control Board: a microcontroller based board which controls the light source and the detection unit has been implemented. The picture is the CAD design of the positioning of the Optical Unit inside the system.
The unit is made up by 5 main sections:
1. Light source: an emitting diode; this is to obtain the peak of emission of umbelliferone, the marker used by CNR as fluorescent dye;
2. Two optical fiber (source side and detection side): the first one placed on the source side is for transmitting the irradiation from the light source to the optical chamber and the second one placed on the detection side is for the signal detection device;
3. Optical chamber: this is made up by a steel cuvette square-based in order to permit an angle of 90° between light source and detection, except for the optical windows which will be made of optical glass to perform a fluorescence analysis;
4. Two optical filters: these are necessary in order to focus the signal on the selected wavelengths;
5. Light detector: Photomultiplier module which takes in input the output signal from the Optical chamber and communicates the measurement to the electronic control system.
The Control system
The main sections of the AQUALITY control system are the electronics, the firmware and the software.
The control system developed for the AQUALITY process is a particular hardware and software configuration controlling the entire functioning of the AQUALITY system. More in detail, the control system is made up by:
An Electronic main board (which can execute commands given by a running software on the central unit): microcontroller based circuit, that runs a dedicated firmware;
A central unit (industrial panel PC) which runs automation software which manages all the operations needed in the process, with an human machine interface (HMI) which shows the process status and the results to the user.
The firmware of the AQUALITY system is the part of the program embedded in electronics card that allows the software to use it as passive command executor. The control system has pumps valves, an optical unit, temperature sensors and heater unit actuator. Since the card simply actuates commands, it must have all the operative links for all single input or output connection. The serial interface has the role of centralize all the peripherals which are cross-present through all the pinout layout.
The control software of the AQUALITY automation system handles all the logic operations by controlling the operations of the electronic subsystem to the microcontroller via a serial protocol. LABOR has defined a communication protocol with a set of intuitive controls. The software also decodes the string communicated by the microcontroller to obtain information about the status and operation of the system, and in particular of all the valves and the pumps in the hydraulic subsystem and the temperature sensor. The latter is the only transparent value for the end user and displayed in a box in order to allow the monitoring of the internal temperature of the system. All other values of the string are used automatically by the software, in order to ensure the correct sequence of operations and reduce the human error. The home screen of the interface appears as shown in the picture 11.
Nanosensors Development
The part of the AQUALITY project concerning liposomes involved efforts in three main activities:
- Synthesis of lipid components (positively charged lipids) to engineer liposomes and make them more sensitive to the interaction with bacteria.
- Formulation and characterization of liposomes.
- Evaluation of the liposome/bacterium interaction
Synthesis of lipid components.
The ten cationic lipid components reported in Figure 2 were synthesized. Compounds 1 and 2 were chosen based on the results of a study where they were included in formulations to deliver a drug to MRSA [C. Bombelli et al. Mol. Pharm. 5, 672-679, (2008)], compounds 3 were chosen because structurally related to compounds 1 and 2, whereas compounds 4 and 5 were chosen because unsaturated lipids are reported to interact with bacterium wall.
Formulation and characterization of liposomes.
A number of liposomes formulations composed of different ratios of one of the cationic components and a phospholipid both in the presence and in the absence of cholesterol were prepared and evaluated for their capability of retaining in the internal aqueous compartment a water soluble dye (calcein or phenol red). This evaluation allowed selecting a number of stable formulations that were further investigated for the interaction with bacteria.
The idea was that interaction of the lipid nanoparticles with bacteria could disrupt the lipid bilayer thus allowing the release of the dye from liposomes. Calcein is a fluorescent molecule generally used to evaluate the extent of release of liposome content into the bulk medium; its fluorescence is quenched when it is confined at high concentration in the aqueous compartment of liposomes, but quenching ceases in dilute conditions. Therefore its release from liposomes and its dilution in the bulk is accompanied by the appearance of a fluorescent signal. In the case of phenol red the appearance of an optical signal is triggered by the change of pH when moving from the aqueous compartment of liposomes to the bulk medium.
We evaluated the optical response of calcein or phenol red loaded liposomes to the interaction with bacteria. We tested three different bacterial strains: Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 28212 and Stafilococcus aureus ATCC 29213. Unfortunately none of the selected formulations gave the expected optical signal. The expected optical signal was not observed or was very slow to appear.
It was then necessary to understand whether the lack of an optical response upon the addition of different bacteria to the liposome suspensions had to be ascribed to the lack of interaction, to the nature of formulations or to the selected chromophore. The occurrence of interaction could be investigated by Z potential measurements.
Z potential ( is the difference of potential between the dispersion medium and the stationary layer of fluid attached to a nano- or microparticle in solution (picture below). Bacteria and liposomes are characterized by a certain determined by the surface charge and by the ions associated to the surface. The interactions of particles characterized by different values of will give a new value of as shown in the picture below.
measurements were carried out on liposomes selected on the basis of the previous experiments, i.e those formulated with cationic lipids 1b, 4 and 5. In themeasurements the concentrations of bacteria and liposomes were chosen to obtain an acceptable ratio between the surface area of liposomes and bacteria. The values obtained in the case of the liposome suspensions in the presence of bacteria demonstrated that the interaction actually occured. Therefore the absence of an optical response in liposomes loaded with a dye indicated that the selected formulations are too stable to disrupt in the presence of bacteria.
It was therefore decided to include in the lipid bilayer of liposomes a chromophore whose optical features depend on the electrical features of liposome surface, i.e. 4-heptadecyl-umbelliferone (from now on umbelliferone) and is generally used to measure the surface potential. [N.J. Zuidam, Y.Barenholz Biochim. Biophys. Acta 1997, 1329, 211-222].
The formulations that according to all previous investigations were evaluated as the most promising, i.e. 7/3 DOPC/4c and 7/3 DOPC/1b (DOPC=1,2-dioleoyl-sn-glycero-3-phosphocholine), were formulated with 3-7% of umbelliferone in the presence and in the absence of cholesterol (the presence of cholesterol could further stabilize the formulation). The following investigations were carried out on the engineered liposomes:
- a preliminary evaluation of the occurrence of a fluorescent signal upon the interaction with bacteria, to verify if it was worthwhile to follow the ‘surface potential strategy’. Actually the addition of bacteria to umbelliferone loaded liposomes changed its fluorescence.
- evaluation of their stability as fluorescent devices (leakage of the fluorescent probe upon storage), to check the stability of the liposome sensors to storage. This allowed to set the best experimental conditions (lipid formulations, lipid/umbelliferone ratio, concentration, pH).
- assessment of stability/morphology in the presence of umbelliferone by dynamic laser light scattering (DLS) experiments. In the preparation of liposomes it is possible to impose a defined size to the lipid nanoparticles; after preparation the size is measured by DLS to check if liposomes feature the expected size and are mono dispersed, this being also an evaluation of their stability.
- interference by ions commonly present in water with the probe fluorescence. In fact, the actual water to be analyzed might contain ions that could change the fluorescence features of umbelliferone included in the liposome formulations. Therefore, in order to identify any possible interference with our detection system, we investigated the fluorescence of umbelliferone included in the liposome lipid bilayers in the presence of a number of the ions present in all Berlin water wells for drinking water (a 2011document given by ARGUS, one of the partners of the project). We considered concentration ranges between the maximum value actually found in water and the maximum value admitted by the law. It was observed that: i) the fluorescence of liposome included umbelliferone decreases of~ 20% in the presence of 150 mg/L calcium; ii) the fluorescence of liposome included umbelliferone increases of ~300% in the presence of 140 mg/L carbonate.
- evaluation of the stability to contamination. We had to find the best preparation conditions to guarantee sterile conditions for the different liposomes formulations, thus avoiding contamination of liposome suspension by environmental bacteria during preparation. We found that the extrusion of liposomes directly in sterile plastic tubes used as furnished by the producer (Falcon) is the best procedure, and no differences between 4°C and room temperature storage were observed.
Evaluation of the liposome/bacterium interaction
And finally we were ready for a detailed investigation of the interaction of the two selected formulations with three different bacteria namely E. Coli, S. Aureus, E. Faecalis. The investigation was aimed at finding the best conditions to have a fluorescent signal from the umbelliferone included in the liposome formulations upon the interaction of liposomes with low concentrations of bacteria. We set up the conditions of the fluorescence experiments to meet engineering requirements and explored different concentrations of 7/3 DOPC/4c and DOPC/1b liposomes, containing different amounts of umbelliferone (3%, 6%, 7%), in the presence and in the absence of cholesterol (3% and 5%), in the absence and in the presence of added salts.
Fluorescence experiments in Phosphate Buffer Saline (PBS). First experiments were carried out in the buffer used in all previous experiments (PBS). The best results obtained are reported in Figure 14 that shows the decrease of fluorescence observed when different bacteria were added to the 7/3 DOPC/4c (Figure 14a) and 7/3 DOPC/1b (Figure 14b) formulations. Both formulations gave a change of fluorescence signal upon the interaction with 102 CFU/mL bacteria solutions.
Fluorescence experiments in Rome pipeline water. Analogous experiments were carried out in a medium more similar to the actual media to be analysed by the AQUALITY device, i.e. a pipeline water (from one of Rome pipelines). As shown in Figure 15, in pipeline water, due to presence of different salts, an increase of the fluorescence of umbelliferone was observed. This result, confirmed by analogous experiments in samples of water enriched with mixtures of salts indicate that the response of the lipid sensors to the presence of bacteria depends also on the medium and that a calibration has to be done on each specific implant.
In order to simulate a real situation, we carried out experiments in pipeline water cointaining at the same time the three bacteria, e. coli, s. aureus and e. faecalis. The total concentration of bacteria was set to 102 CFU/mL, as in previous experiments, in order to maintain constant the ratio between liposomes and bacteria. The results are reported in Figure 6. As expected, also in the presence of the mixture, liposomes formulated with the surfactant 4c display a higher sensitivity.
In conclusion our liposomes sensors are able to detect three different bacteria, i.e. e. coli, s. aureus and e. faecalis, at a concentration of 102 CFU/mL. This means that by combining our liposomes with the opto-ultrasonic device the AQUALITY device will be able to detect these bacteria at 10 CFU/mL.
It is important to note that though during the development of the project it was decided to skip over the specificity of liposome sensors toward defined bacteria in order to focus our efforts on important features such as high sensitivity and fast response, the different response obtained from the two selected formulations suggests that specificity might not be a too far goal to accomplish.
System Integration
The system integration was led by LABOR, with the support of CNR and USOUTH. The main output of the activities performed is the integrated AQUALITY device.
At a glance, the system can be considered made up by two main subsystems: the hydraulic and the electronic (control) with their external interfaces which permit the integration of the two subsystems in the complete prototype.
PHASE 1 - THE HYDRAULIC SYSTEM
The design activities regarding mechanical layout have been included in the design of the thermostated box for containing the hydraulic system.
All the single components have been placed in the hydraulic system.
The hydraulic subsystem has the following electronic interfaces towards the control unit:
sma connector;
BNC connector;
40 plugs connector: this is an industrial connector and is used in AQUALITY for connecting the hydraulic system with the control one. It includes cables for connecting the sensors, the pumps and the valves.
PHASE 2 - THE CONTROL UNIT
The electronic board, was mounted on a specific plate. On the reverse side of the plate were installed the power supply modules for the electronics. This plate was housed into the box, previously prepared.
The panel PC was housed in the case cover and connected to the electronics.
PHASE 3 - INTERGRATION OF THE TWO SUBSYSTEMS
The integration of the two subsystem (hydraulic and control) has been designed to be easy: 3 connectors interface the 2 cases.
In the picture 18, the complete and final prototype is showed.
Generally speaking, systems integration is potentially the most problematic phase in a research project, since all the sections (previously tested stand-alone) have to “talk” together. In the particular case of the AQUALITY project, the system integration went smoothly.
After the system integration, the system has been tested at lab scale. The purpose of the initial AQUALITY prototype system tests is to verify the overall system functionality and to perform tests with calibration beads.
These tests were performed at lab scale, just before the installation on the target site. From the technical point of view, these tests were the most important because, in a lab, we were able to detect the device functioning in a corrupted environment (which means water with bacteria). Due to the dangerously of the bacteria involved, obviously, it was not possible to check the functioning of the device in a water municipality (where the probability to detect bacteria is unlikely and it is not possible to flow bacteria in the main pipeline). For the reason above, particular attention has been paid in the management of these tests and the necessary timeframe has been dedicated to them in order to check the real detection power of the system, in case of bacteria.
The tests has been performed in two different phases:
• Phase 1: tests were performed on the single components before testing the entire system functioning; in particular the following components were tested: Piezoelectric pumps, the Ultrasonic unit and the Optical unit.
• Phase 2: Description of the tests performed on the entire system, as a black box.
Here below a summary of the two phases is reported.
PHASE 1
1 Piezoelectric Micro Pumps
The pumps were tested with a range of driving signals in order to find the right control signals to produce the needed flow rate.
The experimental setup for the pumps calibration is represented in the picture 19. It consisted of a very simple arrangement. The backer in the foreground contained the water to be injected in the pumps. The calibrated column in the foreground contained the water in output from the pumps.
From the analysis of the values obtained, we observed the most suitable value in line with the flow rate specification established and calibrated the pumps on that value.
Other tests have been carried on in order to optimize the amplitude and frequency rate to drive the pump with the higher possible frequency value.
2 Us Unit
The ultrasonic unit was tested with the piezoelectric pumps characterized in the previous section, in order to evaluate the resonance frequency of the transducer and the concentration performance in the real working conditions.
Resonance frequency
The resonance frequency of the transducer is a critical parameter for the good concentration performance. During the AQUALITY preliminary tests, LABOR and USOUTH agreed to use an oscilloscope.
The value of the resonance frequency can be different depending on the flow rate of the liquid which flows into the US unit and some parameters of fabrication (such as the thickness of the silane glass).
In the case shown in the figure above (representing one of the preliminary tests), the resonance frequency was 785 KHz.
Bacteria count
In order to evaluate the performance of the system, a solution of bacteria at known value was prepared and was measured by bacteria count on plates. Then, this sample was flowed into the unit. The volume of the water collected after concentration was counted again.
This test was repeated 3 times. In the first two repetitions, a concentration increasing approximately of 2x was observed.
In the last repetition, no concentration was observed, but a check of the unit revealed a crack on the silane glass: so this case was not considered as valid.
A 2x concentration is smaller than the values obtained by studies in Southampton (8x), but this can depend on the different operating conditions of the integrated system in respect the lab test for the single component (such as flow rate and frequency).
3 Optical Unit
Validation of the optical response of fluorescently labelled liposomes with other pathogens.
Before performing the final tests on the prototype, a detailed investigation was carried out to explore the behaviour of the previously selected liposome formulations in the presence of two other bacterial strains, Clostridium perfringens (ATCC 13124) and Salmonella thyphimurium (ATCC 14028).
Liposomes containing a fixed amount of umbelliferone (7%) were used at fixed total lipid concentration.
The fluorescence experiments were carried out in the emission mode, collecting the fluorescence of umbelliferone. Experiments were carried out in a medium similar to the actual media to be analysed by the AQUALITY device, i.e. a pipeline water (from one of Rome pipelines). These tests were carried on by the CNR. The results indicate that variation of the fluorescence of umbelliferone can be observed upon the addition of bacteria to liposomes.
In order to simulate a potential real situation, CNR carried out experiments in pipeline water containing at the same time S. thyphimurium , E. coli, S. aureus and E. faecalis. The total concentration of bacteria was set as in previous experiments, in order to maintain constant the ratio between liposomes and bacteria. As expected, also in the presence of the pathogen mixture, liposomes formulated with the surfactant display a higher sensitivity.
Tests on the AQUALITY optical unit
The experiments described below concern the set-up of the optical unit of the AQUALITY device and were carried out both off line and on line. The tests in the presence of bacteria were carried out in the presence of the following pathogen strains: E. faecalis, S. aureus and E. coli. These tests were carried on by LABOR and the CNR.
Liposome preparation
To guarantee sterile conditions for the different liposomes formulations, we performed liposome extrusions in 15 ml sterile plastic tubes (Falcon) and we stored liposomes.
Evaluation of the linearity of the optical response of the fluorescent probe
The linearity of the optical response of the fluorescent probe was evaluated by measuring the fluorescence of standard solutions of the fluorescent probe, umbelliferone, in EtOH abs in a reference fluorimeter (Fluoromax Horiba) and in the optical unit of the AQUALITY prototype.
The concentrations of the standard solutions were chosen in order to explore the same range of concentrations of umbelliferone embedded in liposomes in the conditions tuned for the experiments with bacteria. In the Fluoromax the emission (em=453 nm, ex=365 nm) was collected at 90° with respect to the incident beam, i.e. according to the geometry allowed by the features of the instruments, whereas in the AQUALITY optical unit the linearity of the optical response was evaluated collecting the fluorescence of umbelliferone at both 90° and 180°.
Evaluation of the fluorescence of liposomes in the absence/presence of bacteria (blank)
In the case of tests in absence of bacteria, the contamination of the pipeline water, buffer and liposomes was evaluated by streaking on non-selective agar plates for 24h.
In the case of tests in presence of bacteria, the concentration of bacteria before the tests was evaluated by a turbidimetric method and, after the tests, the real concentration of bacteria was evaluated by inoculating an agar plate and counting the colonies.
TESTS IN ABSENCE OF BACTERIA
The fluorescence of umbelliferone embedded (7%) in the mixed liposomes was investigated at a final concentration 0.02 mM of total lipids starting from different concentrated solutions to evaluate the presence of artifacts in the emission of fluorescence probe deriving from potential phenomena of the concentrated solutions, such as liposome clustering.
In fact, the most suitable concentration for liposome storing was found 5 mM in total lipids, however the final set up of the device could have required the addition of larger volumes of liposome solution and hence a more dilute stock solution. Dilution of 1 mM and 5 mM stock solutions of umbelliferone containing liposomes to the final 0.02 mM concentration gave the same value of fluorescence. This result indicates that dilution of different stock solutions to a final 0.02 mM concentration in total lipids did not influence the fluorescence of the probe.
Evaluation of the time for spontaneous mixing: tests were carried out to verify the dependence of signal intensity on the addition time. Actually it was found that it is necessary to mix the solution upon dilution of the small volume of liposome stock solution to the final volume in order to read properly the emission signal. This involved the additional set up of a mixing procedure in the AQUALITY device.
TESTS IN PRESENCE OF BACTERIA
The fluorescence of umbelliferone embedded (7%) in the mixed liposomes upon the interaction with bacteria was investigated at a final concentration 0.02 mM of total lipids starting from different concentrated solutions to evaluate the potential arising of artifacts due to concentration.
Dilution of 1 mM and 5 mM stock solutions of umbelliferone containing liposomes to the final 0.02 mM concentration gave, in the presence of bacteria, the same value of fluorescence. This result confirms that the emission of umbelliferone is independent from the initial concentration of liposome suspension.
Evaluation of the liposome/optical unit detection limit
Other fluorescence experiments, aimed at finding the detection limit of the AQUALITY optical sensor were carried out at different concentration of bacteria. Because of the difficulty of preparing and counting a solution of 1CFU/ml, the minimal concentration used to test the optical unit was 10CFU/ml. In this condition the optical unit measured ~900 F.U.
PHASE 2 – THE SYSTEM AS A BLACK BOX
Once each single component was tested separately, new tests with the entire system were performed.
These tests was needed to verify that every single operation ran with the others in the correct way. Therefore, these were a verification that the hydraulic system worked properly. These were necessary also to verify that the phases of loading and discharging of all liquids in tanks ran properly.
All the operations worked properly.
However for verifying the robustness of the results, several tests were done, first using water without bacteria and then water with a known value of CFU/ml over the threshold. These two tests typologies were repeated more times in order to verify also that the cleaning activities were executed well and no bacteria remained in the hydraulic circuit: for this purpose, two cleaning activities were performed and set in the control software. As a conclusion, it was important to include also a third test case after the positive test.
Finally, for the reason above, the three more representative test cases executed in sequence and continuously repeated during the black box tests, are reported in the table below. In addition, a fourth test case has been added to the list. This test case takes into consideration a human error: if the user does not load the correct tank with the liposomes solution, the software, just before the optical analysis phase, must display a warning message in the HMI in order to advise the user of the lack of liposomes in the system.
The result of a complete automated tests is reported in the log file, generated by the software. In figure 23 the content of log files of two explicative tests (positive and negative).
During the tests, the user interface showed the state of each pump and valve during the process execution. Precisely, the user interface visualizes the command that the control system sends to the specific valve/pump. Therefore, there was the possibility to check if the physical state of a pump /valve matched with the state displayed in the user interface.
In conclusion, the hydraulic system worked properly and the results reported were coherent with the input.
At the end of these activities the prototype was finally ready to be installed on site (Norway) for the final tests and validation.
Pilot Projects
The prototype was finally ready to be installed on site (Norway) for the final tests and validation. The pilot project will be set up for demonstration purposes in Norway at ROROS water treatment plant. In this task TI will take care of the installation of the AQUALITY online monitoring system at ROROS plant. TI will be the responsible, in this task, of realizing the report on the installation and will take care of validating the system and of granting the possibility, for ROROS, to remotely monitor its facilities through AQUALITY technology. The activity includes test run and training session as well as preparation of instruction manual for the operator of the AQUALITY prototype.
The system was assembled in the basement of the water plant, were all the electrically powered units were placed on a small table (picture below). The three cables, running between the hydraulic unit and the control unit, were quickly connected into their sockets (BNC cable, SMA cable, and a 40 pin plug). The power supply was connected to a nearby 230V two pin grounded socket. Even though the installation took place in a basement, the 3G internet antenna only need to be placed close to the basement ceiling to obtain a good signal strength. The antenna was connected to a dedicated cabinet, which included power supply and adapter. A standard Ethernet cable was used to wire the signal to the control unit. The setup is shown in the picture 24.
The AQUALITY prototype was tested over a 3 weeks period and results from the testing of the AQUALITY system was compared with waters samples taken at the same location and time as the running of the AQUALITY system. These water samples was then sent to an accredited laboratory for analyses. Based on the experience and positive results from the installation and testing, further recommended actions in the process toward a fully commercial product are presented.
In conclusion of the pilot Projects, the Consortium can state that the different tasks have been successfully completed.
Finally, the AQUALITY project focuses on an evaluation of users’ acceptance and this activity has been performed in close cooperation between Teknologisk Institutt AS (TI) and Rørosmeieriet (ROROS). The system validation includes identification of key provisions according to Norwegian legislation (“Drikkevannforskriften”) with regards to water quality and monitoring program. Existing practices and costs for water quality monitoring at Rørosmeieriet and Røros Water Plant have been also identified and described, including investment costs of equipment, installation costs including purchase of equipment and rental costs as well as operational costs. These costs are then discussed and evaluated compared against the costs of existing water monitoring program of Rørosmeieriet and Røros Water Plant and the performance of the AQUALITY system is evaluated compared with regards to compliance with the provisions in the Norwegian legislation (“Drikkevannforskriften”).
Potential Impact:
In order to assess the economic perspective of the AQUALITY device and its potential impact and use, a dedicated study has been performed. At the beginning, the water monitoring market has been studied with its trends and requirements. Then, the results of the AQUALITY project and the maturity of the technology in general has been assessed. Finally, a strategic plan for the development of the final product from the prototype has been assessed.
Market trends and requirements
On the basis of the application scenarios identified in the early stages of the project’s lifecycle, although several applications could be possible for the AQUALITY technology, the Consortium focussed the priority on the scenario Public & Private Utilities. This scenario, in fact, best represents the target industrial sector for the project, and best fits with the AQUALITY water analysis method, which, according to the technical discussions following the realization of the application scenario description, is intended for industrial processing more than for the drinking water market.
For this reason a deep market investigation in the field of the water monitoring has been performed. The key facts about the state of art of this sector have been turned out to be:
1. Water supply agencies are responsible at all times for the quality and safety of the water they produce and supply. That’s why, surveillance and quality control are mandatory for such organisations and entities (surveillance meaning an investigative activity aiming at identifying and evaluating potential health risks associated with the supplied water);
2. Infectious diseases caused by pathogenic bacteria, viruses and parasites are the most common and widespread health risk associated with drinking-water. The public health burden is determined by the severity and incidence of the illnesses associated with pathogens, their infectivity and the population exposed; microbial drinking water safety is not related only to a faecal contamination;
3. Preventive management is a preferential approach to ensuring drinking water safety, and should take into account the characteristics of the water supply from catchment and source to its use by consumers;
4. One core objective of surveillance is to assess the quality of water supplied by the supply agency and at the point of use;
The sector is also largely dependent on testing laboratories. The market is highly integrated with a few firms offering whole services for monitoring and testing at in-house labs. There is a need for miniaturisation of testing technologies to reduce the large costs associated with laboratory testing of samples: a trend also highlighted by data analysers. Overall, the water testing market can be divided into three distinct categories:
Low-end equipment - Test kits for low-end equipment include hundreds of variations of colorimetric devices, handheld electronic analysers, and spectrophotometers.
In-line equipment - These include pH, dissolved oxygen and turbidity measures . Water utilities and industrial processes are increasingly using in-line monitoring to maintain high standards of quality by changing their focus from the detection of issues in quality to preventing them.
High-end equipment - consists of organic analysis (gas Chromatography and Liquid Chromatography) and metal analysis (Inductively Coupled Plasma) which are applied with Mass Spectrometry technologies.
In Europe, Germany was the leading exporter (38% share) in 2010, while France and the UK exported similar levels. A third cluster of exports included Sweden, the Netherlands, Italy, Austria and Ireland. The USA is the clear market leader in the supply of instrumentation for water monitoring and testing and is the dominant supplier of high-end equipment.
Approximately 25% of testing in the water sector is analysed by large multinational firms who supply laboratory services. This market is dominated by a small group of major players, 2 of which are from the EU. Coupled with a leading EU presence in the low-end equipment market demonstrates EU strengths overall in the global market for water monitoring and testing.
Innovation in the water monitoring sector is incremental, largely due to the resistance of users to adopt new technologies and the overall maturing of the market. Scope for innovation exists in the need for the miniaturisation of equipment that is able to analyse data onsite and produce real time results. These types of technology have the potential to generate huge cost savings by eliminating the amount of testing required in laboratories.
In conclusion, markets are demanding easy to use, low cost and automated devices comparable to the currently available solutions. The AQUALITY device meets these market demands through an incremental innovation. Obviously, further optimization of the system will be necessary after the end of the project to make it marketable.
Here below, a short list of the SMEs’ market objectives is included. After, their potential market for the AQUALITY device is reported.
SMEs’ Market objectives
ENSATEC
• Ramp-up of the AQUALITY device at ENSATEC in order to target industries of any size, differing the commercial offer.
• Target the water monitoring market with the optimization of the system for industrial processes.
• Create marketing analysis to enter the market: the first step is the Spanish market in which ENSATEC is able to exploit the exclusivity.
• Investigation of new applications/markets for the Opto-US Unit.
• Investigation on the Liposomes market.
ARGUS
• Optimize the system to address the market of the portable devices.
• Optimize the algorithm for processing the data and the HMI (copyright on the algorithm is under discussion).
• Unlock additional markets by offering the device in a price-sensitive segment.
• Investigation of new markets for the Opto-US unit.
• Create in collaboration with ENSATEC a market strategy to access the German market with the Opto-Us unit (in which ARGUS has the exclusivity).
OPTO
• Ramp-up of the OPTO-US Unit in order to target different markets.
• Identification of the most profitable markets and set up of the marketing strategy.
• Creation of the marketing strategy (in collaboration with ENSATEC) for the European markets of the water monitoring.
• Address 20% of the quality control modules (for which we have a patent pending).
IRBM
• Optimization of the liposomes formulation, targeting the EU water directive (in order to reach the requested detection limit).
• Identification of a correct marketing strategy (in collaboration with ENSATEC) for the correct commercialization of the whole system (IRBM is the exclusive supplier of the liposomes to ENSATEC).
• Investigation on the Liposomes market with the purpose to find other channel of exploitation.
ROROS
• Application of the system in its processes for free.
• After a period in which the system will be tested (12 months), if concrete savings for water and wastewater monitoring are registered and the safeguard of the water throughout the process is actually increased, ROROS will evaluate the possibility to install other systems along the process, exploiting the special conditions of the 25% off on the price.
SMEs’ Potential market/s
ENSATEC is the exclusive installer of the Online System in Spain. The first launch in the market will be done exploiting this country since it is the geographical segment in which ENSATEC is more confident. Therefore, taking into account only the Spain, at an average approx. 60 units sold 3 years after the market launch, it is estimated a revenue of approx. 1.2 M€.
ARGUS is the exclusive installer of the Online System in Germany. Therefore, taking into account only the German segment, the average number of until sold could be estimated at approx. 110 units (3 years after the market launch). It is estimated a revenue of approx. 1.5 M€. Also, the liposomes will be supplied by ENSATEC to Argus, applying a minor markup and permitting the competitive commercialization in Germany.
IRBM sells the liposomes to ENSATEC. This will create revenues of approx. 1.6 M€. In addition, IRBM is targeting also other markets for the liposomes directly to end users in the biological research markets.
OPTO sells the opto-US unit to ENSATEC. This will create revenues of approx. 165 K€. In addition, OPTO is targeting the market of the small and medium ultrafiltration units which is growing very fast thanks to the membrane technology getting mature. In those channels, the target sales price could be around 800 euro. The projection for the 2017/2018 is about 900 units sold which will create revenues of around 720 Keuro.
Economic assessment
With the aim to develop an economic analysis, which supports assumptions made on demands in the short-and medium-term, the SMEs involved provided market data and data of potential end users. Based upon these data and under consideration of the results returned from benefit-oriented cost-effectiveness evaluation, the predicted future sale of AQUALITY system has been identified.
For an integrated evaluation, both quantitative and qualitative cost-benefit analysis was performed. In addition, the costs linked to the implementation of the AQUALITY device, losses due to incidents related to water quality, and costs of illness treatment were considered to verify the potential cost saving with implications of AQUALITY.
The unique selling point of the system is being the first online microbiological testing system for water analysis available on the market. The AQUALITY system uses luminescent liposomes for continuous monitoring of microbiological contamination in industrial process water, industrial sewage, drinking water and surface water. It has the capability of protecting chemical, pulp and paper, microelectronics, metal-working, pharmaceutical, food and drinking water companies against microbiological contamination entering their industrial process water and ending up into their products.
The AQUALITY device are able to detect a very wide range of microbiological contaminants. It needs few hours of maintenance per month and in case of alarm, the results will be automatically sent to the staff responsible of the water quality check. It is a robust and easy to use system upgradable in its functionalities.
The following features characterize the final product:
Simplicity: An easy-to-use water analysis product on the market. Absence of time consuming sample and reagent preparation, multi-step test procedures, and clean up.
Speed: Fastest microbiological testing on the market.
Accuracy: Quick, clean and accurate.
Cost-effectiveness: Time saving, money saving, lower cost per completed test.
The system has a very wide range of potential customers from water quality analysis laboratories, and to all industries requiring huge quantities of high water quality for their processes. Therefore, the device has a great potential to get good market recognition. However, estimation for future sales is tightly depending on the SMEs activities to introduce the AQUALITY system to the thirsty but extensive market. For the first three years, it is predicted that more than 400 devices will be sold by the SMEs in the project.
Result of our assessments shows that AQUALITY has a great potential to increase economic benefits of the SMEs and end-users. In addition, installing AQUALITY leads to several qualitative benefits such as producing safer products that leads to have a better publicity.
Exploitation Plan
Talking about the exploitation strategy, it is a fact that the main output of this research project is a prototype and, as a such, it needs further optimization, re-engineering and tests in order to have final product ready to go to the market.
In fact, the possibility to launch the system in the market requires a severe testing phase after the end of the project. This is needed both to overcome the performance of the systems already available but also to fulfill the requirements of the Water directive. In this sense, the SMEs have already taken contacts with the policy makers, in order to make them available of the new solution.
The SMEs have put in place a post-project plan that is divided in three main phases: the first phase is the phase in which the full commitment will be held for reaching the full compliance with the EU Water directive (detection limit able to detect even 1CFU). Taking into consideration the results achieved by the research project, we can now state that the limit is not far to be achieved. However, a re-engineering phase and new dedicated tests will be necessary. In fact, even if a good level has been reached with the prototype, a deeper investigation and optimization will be required to pass from the prototype to a commercial product.
Likely, not only the compliance with the directive will be necessary but also the adaptation to new customer requests. This brings to the second phase. After the re-engineering process, field test will be necessary. The new contacts with the customers will be also exploited to set up the marketing plan.
Finally, the commercialization can start only after this period and not before a marketing revision of the design. The final product, in fact, even if in line with the technical performance, will need a revision under the marketing point of view. The market requires specific conditions that should be taken into consideration before the official launch. This third phase will start during the second semester of the 2016, when the release of the device is stable.
A launch master plan will be prepared defining the sequence of market regions approached with the technology. For each region a detailed launch plan is to be developed to ensure efficient introduction of the technology. The launch plan has to include details like:
1. marketing mix (4 Ps: product, price, promotion, place);
2. identification of opinion leaders and reference accounts;
3. publications;
4. participation in congresses and exhibitions;
5. advertising.
This approach permits to accurately estimate the date in which the product not only will be ready from the technical side but also it will be customized with an ad hoc marketing campaign. It is, in fact, essential that the launch in the market is associated with a solid marketing and advertising phase. At the beginning, the target segments will be addressed (Spain, Germany above all). Only after a semester, the full market can be targeted.
Since the most interesting result of the project is the integrated and automated device for on-line installations, a tentative marketing mix analysis has been performed at this stage in order to identify the main features of the future strategy.
Dissemination activities
As planned by the Consortium members, the dissemination activities have been structured in 2 main stages, as reported below.
By starting early to disseminate the research of AQUALITY, and by providing with some advance notices about the project, the Consortium intended to strengthen the impact of such activity. The objective of this stage of the dissemination was that of presenting to the target groups that the project exists and of giving them an idea of what it is about and tries to achieve, and this can be done even at an early stage of development.
This stage has been the “core” of the First Period of the project. However, the activities foreseen in this stage have been intensified and completed also during the Second Period of the Project’s lifecycle.
The objective of this stage was that of showing what the AQUALITY technology can offer to the interested companies and organisations, the preliminary outputs/outcomes that can be relevant in solving specific problems or needs of the water supply sector.
In the second period of the Project, the dissemination activities have followed the provisions of the second stage.
The scientific community, including technical staff of companies or the R&D divisions of water supply companies, researchers and people interested in technological innovation in the field of water monitoring and analysis, have been the main target of our communication. Then, stakeholders and potential end-users have been the main focus with a communication strategy aims at creating awareness in the general public about the main outcomes of the AQUALITY project.
In the table below, which show the key means of dissemination as hypnotized in the P1, we have reported those means that actually have been used in the course of the project lifecycle.
Stage 1 To raise awareness on the features of the new AQUALITY technology
- Newsletters
- Press releases
- Articles on online magazines
- Project website, etc.
- Social Networks
- SMEs’ official websites
- Specialized portals
- Contact with other EU project and relevant sharing of lesson learnt
Stage 2 To provide in-depth knowledge about the AQUALITY technology - Relevant event in the target sectors, at both national or international level
- Contacts with stakeholders, regulators and policy makers in the water monitoring sector
- Continuous contact with the end users with specific updates on the status and progress of the project
- Seminars/workshops or demo events to be organised with selected groups of stakeholders
- Scientific publications and articles on magazines, journals, web portals
- Dissemination materials, such as brochures and posters
- Presentations
- Video clip
- Wiki page
List of Websites:
The AQUALITY website is one of the vehicles for the dissemination of the project; this online tool is in fact the main interface towards the external audience and the stakeholders. The website provides basic information about the project in a very immediate way, it gives an overview on the relevant events in the addressed market and on the intended technological approach, and it allows to establish contacts with the interested companies and persons reading about the project through the contact form.
The domain of the AQUALITY website has been registered at: www.aquality-project.eu.
The contents are online since March 2012, thus it is important to highlight that the decision of the Consortium to create this dissemination tool in advance with respect to what was planned was taken with the strategic objective of starting disseminating from the very beginning of the project.
