Final Report Summary - OTASENS (Novel photosensor-based device for rapid and quantitative ochratoxin A determination in wine, beer and feed) Executive Summary:The OTASENS project aimed the protection of consumer health through the fabrication of an innovative, low cost, portable detection system able to monitor ochratoxin A (OTA) contamination in wine, beer and in cereal based feed in a rapid and reliable way meeting the SMEs requirements operating in the production and trading of some food and feed commodities.Two systems have been designed to achieve this objective:1. A SMART TLC detection system, where the a-Si:H photosensors coupled with a TLC plate for detection of OTA fluorescence under UV illumination.2. A micro-immunoassay detection system, where the a-Si:H photosensors are coupled with a surface properly treated to host an antibody-antigen reaction involving OTA molecules.Simple protocols have been studied and set up to extract OTA from the investigated food samples:wine, beer and cereals with an environmental friendly, rapid and reliable method. In this manner the above systems of analysis become systems easy to use, to allow people in general non-experts to perform analysis even in poorly equipped places.The SMART TLC system integrates in a compact and portable device all the elements for a quantitative OTA analysis: a commercial HPTLC plate for chromatographic separation, the photosensors for fluorescence detection; the electronics for the photosensor signal amplification and the microcontroller for measurement control, data acquisition and USB communication and software for user interface, data handling and analysis.The micro-immunoassay system integrates the microfluidic device with the photosensors for chemiluminescence detection; the electronics for the photosensor signal amplification and the microcontroller for measurement control, data acquisition and USB communication; a peristaltic pump, bubble detector and pinch valve manifold with drip chambers for fluid handling and control, and software for user interface, data handling and analysis and system maintenance and verification.Tests for the detection of OTA in wine, beer and grain samples have been performed demonstrating that both systems are able to determine whether a sample has OTA contamination above or below a pre-determined level. In particular, it has been settled that for the SMART TLC system it is enough sampling and analyzing 5.5 ml of red wine to establish if a sample is within the law limit in OTA concentration, while the immunoassay system is able to determine whether a sample contains a concentration of OTA above or below 1 ng/mL.The development of the whole project was truly a team effort that involved not only the teams at the two RTD performers (INESC MN and University of Rome "La Sapienza"), but valuable involvement of the equipment manufacturing SMEs (AUTOMATION and LUMISENSE) whose input helped us go from the laboratory prototype to the final prototypes presented in this summary. Also extremely valuable has been the input from all end user partners, EWOS, PATO and BIER, who helped by providing samples and giving useful suggestions and input on how the system would be used by end users. The other partner, CROATIAKONTROLA, has given a valuable contribution to perform comparison analysis with traditional laboratory methods.An important activity of dissemination has been carried out by the partners; in particular by AUTOMATION, who has presented the project in two trade fairs in Milan, EWOS, who has done advertising campaign to its customers, the RTD Performers who have shown the scientific part of the OTASENS project in different posters and communications in congresses and conferences, with the obvious limits of confidentiality regarding the details. Project Context and Objectives:The attainment of high quality food commodities is a societal requirement is a difficult challenge.The deterioration of food commodities by biotic factors is a serious and widespread problem. In particular, the contamination of different food and feed commodities by ochratoxin A (OTA) produced by several widespread fungi represents a serious health risk due to the high toxicity of OTA. This toxin causes nephrotoxic, genotoxic, immunosuppressive and carcinogenic effects on humans and animals. Quality loss is due not only to spoilage but also to the ability by different fungal species to produce mycotoxins (group 2B-IARC).It is therefore necessary to have a close surveillance of the presence of this mycotoxin in food, beverages and feed through efficient, reliable and rapid analytical methods. The availability of low cost analytical methods which join reliability and rapidity is highly desired by the small medium enterprises (SMEs) which produce those commodities, not only for an economical question, to reduce the analysis costs, but also for the necessity to observe the contamination imposed by European legislation and national The main objective of the project is to design a system that simplifies the technique of detection while improving significantly the detection limit of ochratoxin A (OTA) in wine, beer and in cereal based feed. This objective has been achieved through the fabrication of two types of innovative, low cost, portable detection systems, easy to use and able to monitor OTA contamination in different matrices, in a rapid and reliable way, as aimed in the project proposal.The two laboratory devices are complementary and operate on different principles: one of the devices is called "smart TLC detection system" and is based on a-Si:H photo-sensors coupled with a TLC plate able to detect OTA fluorescence under UV illumination; the other one is called "microimmunoassay Lab-on-a-Chip detection system" and is based on a-Si:H photosensors coupled with a surface properly treated to host an antibody-antigen reaction involving OTA molecules.In order to prepare the food and feed samples, like wine, beer and cereals, to analyze them, a proper protocol of extraction for OTA has been developed, easy to use also by non expert people.The foreseen performances in the proposal have been achieved; in particular:a) reduction for a factor 3-4 of the time for analytical procedures; really the time necessary to prepare samples for analysis is reduced to not more than half an hour, while for traditional technique, like for HPLC, the sample preparation requires about 2 hours, taking into account the calibration time.b) The ease of use even for people not familiar and not expert in the analytical methods.c) Reduction of the cost necessary for the analysis of a factor 5, also considered the cost of staff.d) Reduction of the amount of the utilized organic solvents of a factor 10.e) Early and reliable quantification of OTA contamination in the assayed samples at less than 0,5ng. (practical case 0,2ng).The development of all project was truly a team effort that involved not only the team at INESC MN (in particular, Géraud Moulas for the immunoassay prototype development and Pedro Novo for the immunoassay and measurement development) and the team at University of Rome "La Sapienza" (in particular, C. Bello for the extraction protocol implementation, M. Nardini for the development of the a-Si:H photosensor array and R. Scipinotti for the chromatographic chamber development of the SMART TLC prototype), but valuable involvement of the equipment manufacturing SMEs (AUTOMATION and LUMISENSE) whose input helped the RTD Performers to go from the 1st generation proof of concept prototype to the final prototypes presented in this summary. Also extremely valuable has been the input from end user partners such as EWOS, PATO and BIER, who helped by providing samples and giving suggestions and input on how the system would be used by end users (e.g. that a YES/NO result allowing a fast screening of samples was necessary, thus allowing a simpler system with faster response to be developed within this project).Traditional laboratory analysis techniques were used to comparison reliability of the new devices.These have been prepared and developed in their laboratories by CORATIACONTROLA.The success of the dissemination campaign carried out by various partners, both from the scientific point of view and from the point of view of applications, has encouraged the Consortium to investigate the possibility of further development of the prototypes; in particular this has encouraged some partners to explore the possibility of commercially exploiting the results obtained through their industrialization. This action requires a reasonable financial commitment, however, for which some partners have decided, in agreement with the RTD Performers, to submit to the European Commission a proposal for a strong demonstration activity, under the DEMONSTRATION program, in order to verify the repeatability and reliability of prototypes manufactured in the laboratory, before moving to a real production. In addition, the group of partners interested in this action intends to carry out a market analysis to verify its potential: all of these steps are essential in order to address the involvement of industry, whatever the form of funding available (the Community Funding, Venture capital, bank financing).Anyway a further campaign of divulgation will be carried out also after the end of the Project, because the Consortium partners expect long-term impacts of the OTASENS Project. These impacts could be generated in different categories: health impact, economy impact and socioeconomic impacts on society as a whole.As regards health, it is clear that increasing the number of controls on the commodities, benefits on their quality are a direct consequence; in economy, OTASENS may have impact on increased competitiveness both of commodities producers and equipment producers, by improving their production and by improving technology of method of analysis. A direct job creation is expected.Finally, on society as a whole, as direct consequence of improvement of the citizen health.Foreground is therefore capable of industrial or commercial application (even if it requires further research and development, and/or private investment); it will be protected in an adequate and effective manner in conformity with the relevant legal provisions, having due regard to the legitimate interests of all participants, particularly the commercial interests of the other participants.In order to allow further development of the invention, it will be kept confidential: filing of a patent (or other IPR) application (and consequently any dissemination or part of this), for instance, will be postponed to avoid possible negative consequences associated with premature filing (earlier priority and filing dates, early publication, possible rejection due to lack of support / industrial applicability, etc.). The filing of a patent for the new devices will be decided during 2012, overall if the proposal under the program "DEMONSTRATION" will be approved or if the partners will receive adequate financing by venture capitalAs conclusion of the activity of Project, two types of devices were manufactured, made with different technology: a pre-industrial micro-immunoassay detection system and a pre industrial SMART TLC. It is also summarized the procedure to extract OTA from the food and feed samples.The final prototype designed by INESC-MN is described in a detailed "Standard Operating Manual" included in a confidential report as Annex I. The modules of this system are: a) Computer controlled valve manifold and automatic liquid insertion; b) Bubble detector; (c) Photodiode initialization and calibration.It was presented to the SMEs, with the purpose both for demonstration and training, at the final project meeting in Rome on 25 November 2011. Videos showing how to use the system were presented at the final meeting and have been made available on the OTASENS website (see http://www.OTASENS.it online). Tests were carried out with complex matrices (wine, beer, grain extracts). Samples were prepared by EWOS, PATO and BIER. Comparison measurement were carried out by CROATIA with traditional techniques.The measured chemiluminescence intensity in the competitive immunoassay is inversely proportional to the free OTA concentration in solution. To take this prototype to a commercial product some improvements have been suggested to produce a pre-industrial device.Protocol of OTA extraction were set up by the Uniroma1 group and described in suitable confidential report.The final prototype designed by the other RTD Performers (Uniroma1, SMART TLC system) to detect Ochratoxin A (OTA) in grains, wine and beer has been achieved by coupling a HPTLC plate to amorphous silicon photosensors and electronic data acquisition. Different stages were performed to reach optimization of the device in a final prototype, as the previous RTD Performer has made.Also the SMART TLC system aims to detect the presence of Ochratoxin A (OTA) in cereals, wine and beer, however the operating principle is different from the immunoassay system. The detection is based on chromatographic separation of OTA molecules from complex matrices within a small and compact chamber suitable for thin layer chromatography.The sample extract to be analyzed and a known quantity of OTA standard, utilized as reference, are spotted over a HPTLC plate. During the chromatographic run, an UV radiation impinges on the stationary phase of the HPTLC plate inducing in an array of amorphous silicon photosensors a photocurrent due to two main contributions: 1. a background signal from the UV excitation light that passes through the wetted HPTLC plate; 2. a signal proportional to the concentration of OTA present in the sample under measurement due to the light of the excited OTA molecules. The light level resulting from the sample under measurement is compared to the light from the reference sample. The difference allows this prototype system to indicate whether or not the sample contains OTA at levels above or below the concentration allowed by European Regulations. The time evolution of the chromatographic run of the two spots are monitored measuring the photocurrent induced in two pairs of sensors. The sensor signals monitoring the OTA standard spot has been used as a reference. The final prototype designed by Uniroma1 is described in a detailed "Standard Operating Manual" included in a confidential report.The final prototype of the SMART TLC system for the detection of OTA in wine, beer and grains has been demonstrated, tested with complex matrices and proven to allow the determination whether a sample has OTA contamination above or below a pre-determined level. In particular, it has been settled that it is needed sampling and analyzing 5.5 ml of red wine to establish if a sample is within the law limit in OTA concentration.The final prototype system integrates in a compact and portable device all the elements for a quantitative OTA analysis: a commercial HPTLC plate for chromatographic separation, the photosensors for fluorescence detection; the electronics for both the photosensor signal amplification and the microcontroller for measurement control, data acquisition and USB communication and software for user interface, data handling and analysis. Comparison measurements were carried out by CROATIA with traditional techniques.The system was presented to the SMEs, with the purpose both of demonstration and training, at the final project meeting in Rome on 25 November 2011. Tests were carried out with complex matrices (wine, beer, grain extracts). Samples were prepared by EWOS, PATO and BIER. The group of Uniroma1 has developed a new extraction protocol for OTA from food samples. This protocol is described in a "Operating Manual" annexed to a confidential "2nd periodic Report".To take this prototype to a commercial product some improvements have been suggested to produce a pre-industrial device. A video showing the operation of the final prototype can be seen at the OTASENS website (see http://www.OTASENS.it online).During the whole Project an intense activity of dissemination has been performed through scientific publications, oral presentation to specialized Congresses, leaflets distributed in exposition fairs, etc. as reported in detail in the "Final Report", Chap. 4. Project Results:4.1.3. Description of the main S &T results/foregrounds.Figures and diagrams are reported in the attached files "Final Report" , "2nd Periodic Report" and Annexes.As conclusion of the activity of Project, two types of devices were manufactured, made with different technology: a pre-industrial micro-immunoassay detection system and a pre industrial SMART TLC.It is also summarized the procedure to extract OTA from the food and feed samples.4.1.3.1.Pre-industrial micro-immunoassay detection systemINESC-MN has fabricated a first pre-industrial prototype, where an OEM peristaltic pump was incorporated into the system allowing pumping of the fluids. The pump was connected to the outlet of the microfluidic platform and was operated in suction mode. This mode allows different channels to drive fluids under the same flowrate in several channels in parallel with a single pump.A second transimpedance amplifier circuit together with a second set of photodetectors were fabricated in order to monitor the signal emitted from the reference. Both software (user interface) and hardware (acquisition control) were implemented to allow parallel data acquisition via serial USB communication. The software includes a user-friendly operator mode.This section summarizes the development of a prototype system to detect Ochratoxin A (OTA) in grains, wine and beer using a micro-immunoassay Lab-on-a-Chip device coupled to fluid handling and electronic data acquisition.Research activity has been carried out along different stages:- Stage 1: 1st Generation Prototype- Stage 2: 2nd Generation Prototype- Stage 3: Final PrototypeStage 1: 1st Generation PrototypeThe system consists of the following components:1. hydrogenated amorphous silicon (a-Si:H) photodetector2. microfluidic channels3. fluid pumping4. electronic circuit for signal processing5. software and hardware for data acquisitionDuring this first stage, the fluid pumping was not integrated into the prototype and a commercial syringe pump was used to control fluid flow to the microfluidic structure. Also, the electronic circuit was in preliminary form pending final design details.The 1st generation prototype was presented to AUTOMATION at their site in Abbiategrasso (I) on 1 June 2011. Participating at this meeting were INESC MN (V. Chu, J.P. Conde, G. Moulas, P. Novo), UniRoma (D. Caputo, R. Scipinotti) and AUTOMATION (F. Pavanello, R. Arrigoni).During this presentation, with the scope demonstration, the system performed in an irreproducible manner and after extensive tests and discussion with AUTOMATION, suggestions were made to improve the system.Stage 2: 2nd Generation PrototypeIn the 2nd generation, an OEM peristaltic pump was incorporated into the system allowing pumping of the fluids. The pump was connected to the outlet of the microfluidic platform and was operated in suction mode. This mode allows different channels to drive fluids under the same flowrate in several channels in parallel with a single pump.A second transimpedance amplifier circuit together with a second set of photodetectors were fabricated in order to monitor the signal emitted from the reference. Both software (user interface) and hardware (acquisition control) were implemented to allow parallel data acquisition via serial USB communication. The software was improved to include a more user-friendly operator mode.The microfluidic channels were redesigned to allow a larger tolerance for alignment with the photodetectors. The widths of the microchannels were increased from 300 microns to 600 microns. An acrylic fixture that is precision machined allows the direct placement of the PDMS microfluidic structure on the detector. Both the microfluidics and the photodetector chip were redesigned to allow the simultaneous flow of the sample and reference in 2 parallel channels and their detection with the 2 sets of photodetectors.The following tests were performed using the above prototype system:(1) Measurement with adsorbed IgG-HRP detection successful(2) Measurement of 50 ng/mL of OTA standard in buffer detection successful(3) Measurement of extraction of Chianti spiked with OTA (extraction made by U. Roma) detection not successful due to an overwhelming effect of some compounds present in the matrix (namely the anthocyanins).(4) Measurement of barley extract (barley supplied by EWOS, extraction made by U. Roma) detection of possible OTA contaminationThese tests showed the correct functioning of the system by detecting chemiluminescence of HRP linked molecules in the presence of luminal.In both tests (3) and (4) extracts were supplied by the UniRoma group. The extract was diluted in phosphate buffered solution prior to insertion into the prototype. In the case of the red wine extract in (3), the extract was diluted to a concentration of 100 ng/mL prior to measurement. The red wine extract had too many anthocyanins that interfered with the immunoassay making it impossible to detect OTA. A new extraction technique has been developed to allow the removal of the interfering molecules. In the case of the barley extract (4), the extraction was made without spiking with OTA. However, there is a strong chance of the natural presence of OTA in these samples since the presence of another toxin DON had been previously confirmed. The result of the measurement suggested the presence of OTA in these samples.During a meeting held in Lisbon and after detailed discussions with all the partners, the following improvements were planned for the final prototype:(1) Include valving system to allow completely automatic operation;(2) Include test/calibration of the correct functioning of the photodetector, detection of bubbles in the tubes and control of the fluid flow;(3) Improve software to allow immediate data analysis and autosaving of data.Stage 3: Final PrototypeThe final prototype is summarized in this section and presented in a detailed "Standard Operating Manual" included in a confidential report. The final prototype improves the 2nd generation prototype described above.The improvements are described in the following sections.a) Computer controlled valve manifold and automatic liquid insertionA computer controlled valve manifold was developed during the final stage of the project to allow the automatic control of fluid flow in the final prototype system. To avoid air bubbles in the lines during changeover from one solution to another, a drip chamber system (inspired by the IV drips used to deliver fluids to patients in the hospital) was developed.Each of the solutions used in the immunoassay is inserted into their respective fluid reservoirs coupled to polyurethane tubing and a normally closed pinch valve. When a valve is opened, the fluid flows into an open drip chamber. The fluid in the drip chamber is then pumped through the microfluidic device. The solutions common to both the sample and reference channels (OTA-BSA, PBS, and secondary AB-HRP) share 2 drip chambers, one for each of these channels. The sample and reference solutions have their own drip chambers, as does the luminol for the final chemiluminescent detection.b) Bubble detectorA commercial bubble detector was integrated into the system at the outlet of the microfluidic device. This approach has the advantage of requiring only 1 detector for the entire system. The detector works with an IR emitter and a phototransistor and detects the changes in the transmitted light intensity reflected in a change of the phototransistor voltage.The system software monitors the phototransistor voltage and if a bubble is detected, gives a warning to the user. After a certain number of warnings, the measurement is stopped.(c) Photodiode initialization and calibrationWhen the system is initialized at the beginning of an assay, a procedure has been included to verify the correct functioning of the two photodiodes corresponding to the reference and sample measurements. The procedure involves measuring in the dark and at two light intensities (provided by an LED inside the box). This measurement also allows the calibration of the 2 diodes and to correct for any differences in photoresponse between the two. The result of this procedure is graphically displayed to the user to ensure that the measurement can proceed. Example of the output of the photodiode calibration procedure at two different light intensities. The red and blue curves show the response from the photodiodes measuring the reference and sample microchannels, respectively.The final prototype was presented at the final project meeting in Rome on 25 November 2011. A full description of the prototype is in the "Standard Operating Manual" included in a confidential report, as Annex I. The results of tests made with the prototype are summarized in the following section.Videos showing how to use the system were presented at the final meeting and it is available on the OTASENS website (see http://www.OTASENS.it online).4.1.3.1.1. Development of immunoassay protocol and measurement resultsThis section summarizes the development of the immunoassay protocol to be applied to the prototype and the results of the measurements of OTA using the microfluidic system developed in this project.Selection and Optimization of Immunoassay protocolA robust competitive ELISA immunoassay protocol for OTA detection has been developed.In this type of assay, the free OTA (or extracted OTA from a sample matrix) to be quantified is competing with the OTA-BSA for the anti-OTA-IgG during the molecular recognition step. Thus, the more "free OTA" in the solution, the fewer anti-OTA-IgG molecules will be available to bond with the OTA-BSA adsorbed in the channel walls. Since the chemiluminescence to be detected in the final step results from the HRP tagged to the anti-IgG, which recognizes the anti-OTA-IgG, this will result in a lower chemiluminescent signal. Thus, the detected light level is inversely proportional to the amount of "free OTA" in solution.The steps are (from left): adsorption of OTA-BSA on the surface of the PDMS microchannel; molecular recognition with competition between Anti-OTA IgG and the free OTA or extracted OTA from sample matrix; reaction with secondary antibody Anti-IgG-HRP; chemiluminescent reaction resulting from luminol HRP.Details of this protocol are reported in a confidential periodic report.In tests with complex matrices (wine, beer, grain extracts), there were two situations. In the first, the sample is known to be OTA-free. In this case the sample is spiked with OTA and the reference is the unspiked sample.In another case, the sample is of unknown OTA concentration (this case is similar to the real world application of the prototype) and the measurement should determine if the sample has OTA above or below a predetermined level (in practice, this should correspond to the European Union legal limit). In this case, the reference is defined as the sample spiked with certain known quantity of OTA that is above the predetermined level.Data analysisThe measured chemiluminescence intensity in the competitive immunoassay is inversely proportional to the free OTA concentration in solution. Two series of measurements were made to show reproducibility.The prototype system is designed to return a YES/NO answer corresponding to whether the amount of OTA detected is above or below a pre-determined level. By making a ratio of the measured signal intensities of the sample versus a reference, it can be determined whether the sample has OTA above, equal or below the reference sample within the margin of error of the measurement.The choice of the reference is an important consideration. The reference should have the same complex matrix as the sample to eliminate their effects on the result. In a sample of unknown OTA contamination, the reference should be spiked to a know concentration. If the measured sample shows a signal similar to the reference, the sample can be said to be contaminated with OTA. Sample signals above the reference suggest it is OTA free.Detection of OTA in single microchannel systemIn the first tests of the prototype system, a single microchannel was used and the sample and reference were measured sequentially and then compared. The results of the measurements of different concentrations of OTA of both standard buffer solutions and complex matrices. For OTA in phosphate buffered solution (PBS), shown with the black circles, the results show an inverse linear relationship between OTA concentration and the measured chemiluminescence signal, consistent with that observed by microscopy. The measurements of OTA in red wine, beer and white wine are less sensitive due to effects of the matrix. The measurements of red wine and beer were made after an extraction process whereas in the white wine, the measurement was made directly from the OTA spiked wine. The sensitivity of the measurements was particularly low in red wine probably due to the presence of anthocyanins. A simple and effective extraction process is being developed by the UniRomaa group and will be described in their part of this final report.The solid line represents the signal of the photodiode in the dark and the dotted line shows the background signal level of a control assay in which no anti-OTA antibodies were used (testing of non-specific adsorption of the HRP antibody).Detection of OTA in final prototype systemAs described in the previous section, the final prototype included a microfluidic chip with 2 microchannels that allow the simultaneous measurement of the sample and the reference. From the ratio of the signals measured in the two channels, the system can return a result indicating if the sample has an OTA concentration above or below a certain level.In the case of red wine, the original simple extraction process resulted in an extract that contained too many anthocyanins which interfered with the competitive assay. The measurements shown below were made on extraction with TLC, a more complex and expensive technique.Several samples of grain provided by EWOS, were also extracted (by the UniRoma group) and measured with the final prototype. These samples have not been measured for OTA so the contamination is unknown. However, EWOS knows that the samples are contaminated with DON, another toxin, which suggests that some level of contamination with OTA is likely.Since the sample has unknown concentration of OTA, the reference had to be spiked with a known concentration. For this assay, the reference was spiked with 100 ng/mL of OTA. The results of the preliminary measurements are shown in table below:Extract Signal ratio (a.u.)Barley 0.70Oat 0.76The signal ratio is the signal from the sample (not spiked with OTA) to the signal from the reference (spiked with OTA).Tests were also performed on red and white wine from FILIPA PATO. The red wine was FP Ensaios (2009) and the wine wine was FP Branco (2010). Because of the problems of interference from anthocyanins in red wine, a simple extraction process was developed by the group from U. Roma using a syringe filled with silica gel. The wine is passed through this gel to remove the anthocyanins.The plot shows the signal of the photodiode in the dark (at time 0), then the measurement of the chemiluminescence from the reference (blue) and OTA spiked sample (black) channels, then finally the 2 photodiodes were submitted to external light source showing their photoresponse.The tests on the wines from FILIPA PATO were performed by passing the wine through the silica gel extraction and then spiking them to a final concentration of 100 ng/mL of OTA. The ratio of the sample versus the reference (after correction for the calibration of the 2 different photodiodes) in this sample gives a value of 0.63 successfully detecting the presence of OTA.The measurement of both red and white wine from FILIPA PATO showed the successful detection of wine extract spiked with 100 ng/mL of OTA. The ratio of the signal from the sample versus the signal from the reference was less than 1 indicating the presence of OTA. In agreement with previous results, the signal ratio for white wine was less than the signal ratio for red wine indicating that the measurement is more sensitive when used with white wine than red.Filipa Pato extracted samples (extracted using Silica gel) Signal ratioRed wine 0.63White wine 0.35The results of the measurements on samples of SME project partners EWOS and FILIPA PATO were discussed with them.Conclusion and suggestions for improvementsIn conclusion, the final prototype of the micro-immunoassay system for the detection of OTA in wine, beer and grains has been demonstrated, tested with complex matrices and proven to allow the determination whether a sample has OTA contamination above or below a pre-determined level.The final prototype system integrates the microfluidic device with the photosensors for chemiluminescence detection; the electronics for both the photosensor signal amplification and the microcontroller for measurement control, data acquisition and USB communication; a peristaltic pump, bubble detector and pinch valve manifold with drip chambers for fluid handling and control, and software for user interface, data handling and analysis and system maintenance and verification.A video showing the operation of the final prototype can be found at the OTASENS website (see http://www.OTASENS.it online).To take this final prototype to a commercial product some improvements could be made including:(1) Fluid valving system: the current fluid valving system involving pinch valves and drip chambers is operational but still suffers from episodic formation of air bubbles which disturb or invalidate the measurements. Possible improvements to the system include:i. Using larger tubing at the sample inlet to the drip chamber;ii. Make hydrophilic treatments to the inlet reservoir and tubingsiii. Instead of using a single pump at the outlet of the microfluidic chip, using a small pump at the inlet of each solution;iv. Insertion of In-Line Microfluidic Bubble Trap instead of the drip chambers(2) Immunoassay: The current protocol involves the use of a secondary antibody reaction. An improved protocol involving the use of anti-OTA-HRP would allow the elimination of two of the current steps of the protocol simplifying and shortening the process and reducing the number of solutions that the system is required to handle.(3) Data statistics: For a commercial product, a microfluidic chip with multiple sample microchannels (i.e. 4 or 6) could be designed to allow simultaneous measurements for statistics to be made on the results.4.1.3.2.Fabrication of a smart TLC detection systemThis section describes the development of a prototype system to detect Ochratoxin A (OTA) in grains, wine and beer using a SMART TLC system, achieved by coupling a HPTLC plate to amorphous silicon photosensors and electronic data acquisition.In particular, the section will discuss the development of the prototype in stages from the first system design (Stage 1) to the final prototype.Stage 1: 1st Generation PrototypeThe system consists of the following main components:- hydrogenated amorphous silicon (a-Si:H) photodetector array- commercial High Performance Thin Layer Chromatography (HPTLC) plate- chromatographic chamber- an excitation source constituted by a lamp for biological analysis- a quartz window for the excitation light- a box for light shielding- electronic circuit for signal processing- software and hardware for data acquisitionIn this prototype:- the photosensors were arranged as a couple of 2x8 arrays;- the chromatographic chamber implemented a horizontal run;- the eluent was transported to the HPTLC plate through a piece of Whatman paper- the electronic circuit was connected to the glass substrate hosting the photosensor array by a flex.This prototype was presented to AUTOMATION at their site in Abbiategrasso (I) on 1 June 2011. Participating at this meeting were INESC MN (V. Chu, J.P. Conde, G. Moulas, P. Novo), UniRoma (D. Caputo, R. Scipinotti) and AUTOMATION (F. Pavanello, R. Arrigoni). In the following picture the principal parts of the prototype are shown.Complete horizontal chromatographic chamber. Electronic boards for data acquisition. The flex connects directly the electronic board to the sensor electrodes over the glass substrate.Reliable measurement from this system were not obtained. After extensive tests and discussion with AUTOMATION, the following suggestions were made to improve the system:(1) Change to connection between the electronic circuit and the photosensor.(2) The connection between the electronic circuit and the photosensors should be designed to avoid the need of precision alignment equipment or microscope.(3) The lamp should be substitute by a more compact illumination source light emitting diodes (LEDs).(4) Make more reliable the positioning of the carta bibula to transport the eluent to the HPTLC plate(5) The software for the data acquisition should have an operator mode as simple as possible, instructing the user and giving back the result. There should also be a maintenance mode that allows more user intervention and diagnostic of problems.Stage 2: 2nd Generation PrototypeThe 2nd generation prototype included:1. a "LINK" electronic board connecting the flex coming from the data acquisition board to a PCI module. This PCI module ensures a reliable and simple connection to the photosensor array;2. a new design of the photosensor array (2x2 matrix) to allow an easily connection to the LINK electronic board;3. a set of four LEDs with emission spectra centered at 340 nm, wavelength very close to the OTA absorption peak wavelength (330 nm), and aligned with the four photosensors of the array . LEDs are driven by an apt electronic circuit;4. a new vertical chromatographic chamber, composed by a base hosting the eluent for the chromatographic run and a cover hosting the PCI module connected to the photosensor array, the HPTLC plate and the LEDs;This version of the prototype was developed together AUTOMATION and tested on many samples both of OTA standard and extracts fortified with OTA. However, the signals resulting from the photodiodes did not show any presence of OTA. These results were deeply discussed and ascribed to two main issues:1. not hermetic seal of the chromatographic chamber. This results in a lower speed of the chromatographic run and in a different shape of the photocurrent signal that hides the OTA signal;2. a broad spectrum of the light emitted by the LED that is not sufficiently filtered by the kapton filter. This light produces a high background signal that hides the OTA signal.Stage 3: Final PrototypeThe final prototype is summarized in this section and presented in a detailed "Standard Operating Manual" included in a confidential report. The final prototype includes two main features in the 2nd generation prototype to address the issues aroused above:1. a sheet of white rubber in the base of the chromatographic chamber and over the PCI module to achieve a hermetic seal;2. a filter between the LEDs and the HPTLC plate to cut the visible spectrum of the LED light. This spectrum region does not excite the OTA molecules, while induces a large background signal in the photodiodes. The filter has been taken from an old lamp for biological analysis and integrated in the cover of the chromatographic chamber as shown in the following figures:Cover Filter Filter incorporated in the coverThanks to the filter integration the amplitude of the photocurrent signal induced in the a-Si:H photodiodes by the background light (emission light of the LEDs) decreases of two orders of magnitude and its shape became suitable for the detection of the OTA signal.In the final prototype the graphic interface between the operator and the computer has been also simplified to make the analysis to be done also by unskilled personnel.The following figure shows an overall view of the final prototype including the pasteur for inserting the eluent into the base and the filler cap.Pasteur Filler capThe final prototype was presented at the final project meeting in Rome on 25 November 2011. A full description of the prototype is in the "Standard Operating Manual" included in this report as Annex II. In Annex III is reported the detailed description of the fabrication of the a-Si:H photodiode array in the different prototypes. The results of tests made with the prototype are summarized in the following section.Videos showing how to use the system are available on the OTASENS website (see http://www.OTASENS.it online).SMART TLC operating principleThe SMART TLC system aims to detect the presence of Ochratoxin A (OTA) in cereals, wine and beer. The detection is based on chromatographic separation of OTA molecules from complex matrices within a small and compact chamber suitable for thin layer chromatography.The sample extract to be analyzed and a known quantity of OTA standard, utilized as reference, are spotted over a HPTLC plate..During the chromatographic run, an UV radiation impinges on the stationary phase of the HPTLC plate inducing in an array of amorphous silicon photosensors a photocurrent due to two main contributions:1. a background signal from the UV excitation light that passes through the wetted HPTLC plate;2. a signal proportional to the concentration of OTA present in the sample under measurement due to the light of the excited OTA molecules.The light level resulting from the sample under measurement is compared to the light from the reference sample. The difference allows this prototype system to indicate whether or not the sample contains OTA at levels above or below the concentration allowed by European Regulations. The reference sample is a solution containing OTA standard at the law limit.The time evolution of the chromatographic run of the two spots are monitored measuring the photocurrent induced in two pairs of sensors. The sensor signals monitoring the OTA standard spot has been used as a reference.In order to clarify how we achieve quantitative information from the SMART TLC system, in the following figure are reported the photocurrents measured when only OTA standard (4 ng) has been spotted on the HPTLC plate. No extract has been spotted. Therefore, sensors B and D monitor the effect of the only eluent, while sensors A and C the effect of eluent plus OTA.The initial increase of the photocurrent is caused by the progressive wetting of the silica when the eluent front moves along the plate. This phenomenon makes the silica gradually transparent to the UV radiation. The inflection point of each curve corresponds to the alignment of the eluent front with the center of each sensor, while the plateau correlates with the complete wetting of the silica over the sensor.The peaks, observed in the curves associated with sensors C and D and superimposed to the eluent signal, correspond to the OTA signal. The peaks occur when the chromatographic band related to OTA molecules is aligned with the sensor.The width of each peak is a function of the width of the chromatographic band and the transit time of each component over a sensor: for a given band width, the higher the velocity, the narrower the peak width.The peak height instead is connected to the intensity of the fluorescence signal and therefore gives quantitative information on the OTA presence. The value of photocurrent related to the OTA signal is semi-automatically extracted with the following algorithm:1. the beginning and the end of the peak signal is chosen by the operator;2. the equation of the line joining the beginning and the end of the peak signal is calculated automatically;3. this line is subtracted to the measured curve automatically;4. the peak of the new curve is the photocurrent value due to the OTA molecules automatically.The choice of the reference is an important consideration. For each sample of unknown OTA contamination, the reference is spiked to a quantity equal to the law limit. If the measured sample shows a signal peak equal or above to the reference, the sample can be said to be contaminated with OTA, otherwise it is OTA free.4.1.3.2.1 Measurements results obtained with the SMART TLCIn this section we describe the results obtained with the final prototype on real samples extracted following the extraction procedure developed in this project.As first step the reproducibility of SMART TLC system has been tested performing two series of measurements at different OTA standard quantities.Results are summarized in this figure, where we report the photocurrent peak value due to OTA fluorescence, achieved following the algorithm described above, as a function of OTA quantity. A very good linearity (R=0.999) is obtained in the investigated range for both series of measurements with standard deviation below 5 %.As demonstration activity, during the last OTASENS meeting held in Rome on 25 November 2011 at the Department of Electronic Engineering, University of Roma "La Sapienza", the following tests on food samples were accomplished using the SMART TLC system:1. Concurrent measurement of extract of fortified "Ceres" beer and OTA standard. In particular, measurements were performed spotting on the HPTLC 2μl of beer extract diluted in methanol and 2 l of OTA standard as reference. Both spots contained 4 ng of OTA. Results are reported in the following figure, where the closed circles refer to the OTA signal coming from the extract, while the open circles refer to the standard. We observe that in both photocurrent time evolutions it is possible to clear distinguish the signal due to the OTA molecules2. Concurrent measurement of fortified "Chianti" red wine and OTA standard. In particular, measurements were performed spotting on the HPTLC 2μl of the red wine extract diluted in methanol and 2 l of OTA standard as reference. Both spots contained 4 ng of OTA.Results are reported in the following figure, where the closed circles refer to the OTA signal coming from the extract, while the open circles refer to the standard. We observe that in both photocurrent time evolutions it is possible to clear distinguish the signal due to the OTA molecules.3. Concurrent measurement of fortified wheat (wheat supplied by EWOS) and OTA standard. In particular, measurements were performed spotting on the HPTLC 2μl of wheat extract diluted in methanol and 2 l of OTA standard as reference. Both spots contained 4 ng of OTA. Results are reported in the following figure, where the closed circles refer to the OTA signal coming from the extract, while the open circles refer to the standard. We observe that in both photocurrent time evolutions it is possible to clear distinguish the signal due to the OTA molecules.Quantitative detection of OTA in extract of red wineThe extraction procedure was applied to "Chianti" red wine fortified with different amount of OTA. Measurements with the SMART TLC system were performed spotting on the HPTLC 2μl of red wine extract diluted in methanol. Results are reported in the following figure, where we observe that the system is able to detect OTA in the limit of 0.2 ng both in the extract and in the standard. Taking into account the extraction efficiency, the dilution of the proposed extraction procedure and that the law limit for OTA in red wine is 2 ppb, we deduce that it is necessary to analyze only 5.5 ml of red wine to determine if the sample is above or below the law limit.Conclusion and suggestions for improvementsIn conclusion, the final prototype of the SMART TLC system for the detection of OTA in wine, beer and grains has been demonstrated, tested with complex matrices and proven to allow the determination whether a sample has OTA contamination above or below a pre-determined level. In particular, it has been settled that it is needed sampling and analyzing 5.5 ml of red wine to establish if a sample is within the law limit in OTA concentration.The final prototype system integrates the in a compact and portable device all the elements for a quantitative OTA analysis: a commercial HPTLC plate for chromatographic separation, the photosensors for fluorescence detection; the electronics for both the photosensor signal amplification and the microcontroller for measurement control, data acquisition and USB communication and software for user interface, data handling and analysis.A video showing the operation of the final prototype can be found at the OTASENS website (see http://www.OTASENS.it online).To take this final prototype to a commercial product some improvements could be made including:(1) Automatic spotting of the extract and OTA standard on the HPTLC plate: during the development of the 2nd generation prototype we made two holes in the upper cover of the chromatographic chamber as houses for a syringe to dispense automatically and in the exact points the solution. However, this system did not work in the correct way, because the tip of the syringe was not suitable to make a well defined spot on the HPTLC plate. Possible improvements to the system include:i. making houses in the upper part of the chromatographic chamber cover to host the tips used for biological pipette;ii. automatic dispensing of the solution drops through a small pump(2) Data analysis: The current data analysis involves a semi-automatic process, where the operator chooses the beginning and the end of the photocurrent shape due to OTA molecules. Improved software could select these two points basing on a derivative process of the measured curve. This new protocol of data analysis will simplify the analysis process, making it independent on the operator.4.1.3.3 A new extraction protocol for OTA from food samplesIn the Annex to the "2nd Periodic Report" the development of novel extraction procedures for ochratoxin A (OTA) from wine, beer and grain is described. It gives a sample suitable for the detection and quantification by the novel device fabricated. The protocol is summarized in the next section.4.1.3.3.1 Development of a novel extraction method for liquid matrices (Red Wine and Beer).1st Step: efficient and environmentally safe extraction mixture for ochratoxin A.In order to achieve this objective, as scheduled at the beginning of the project, different novel extraction methods of ochratoxin A (OTA) from the food matrices considered in the project are being evaluated. Three different liquid-liquid extraction methods from wine samples were compared:System 1. extraction by a chloroform-formic acid mixture in a 9:1 v/v ratio;System 2. extraction with an ethyl acetate-formic acid mixture in a 9:1 v/v ratio;System 3. extraction with ethyl acetate-hexane-formic acid mixture in a 8:1:1 v/v ratio.System 1 was employed as a reference method because is commonly used in literature as extraction method of ochratoxin A from wine and other grape products (Zimmerli, 1996). Systems 2 and 3 are methods developed in our laboratories in order to realize simple, easy to use and environmental friendly extractive systems avoiding unsafe solvents and expensive systems such as immunoaffinity cleanup (Leitner, 2002). Ethyl acetate is a low toxic solvent whose degradation produces ethanol and acetic acid certainly much less toxic than chlorinated solvents. In order to improve the solubility of OTA in the organic solvent, formic acid was added, either in chloroform or in ethyl acetate. Formic acid allows the protonation of the carboxylic and hydroxyl group of the molecule increasing its lipophilic character. In fact the intrinsic acidity of wine (pH 3.5 - 4) was not sufficient to quantitatively protonate ochratoxin A, so formic acid was employed increasing OTA uptake by organic solvent. Hexane was employed in system 3 to further increase the lipophilicity of the extractive system. Samples extracted with the above described mixtures were analysed by HPLC and recoveries are summarized in table 1.2nd Step: testing solvent extraction mixturesOnce all the solvent extraction mixtures developed in step 1 revealed high extraction capacity, we focused our attention on the mixture composed of ethyl acetate acidified with formic acid with lower acidity (1% v/v instead of 10% in system 2). This extraction mixture will be indicated as ETHYFORM. The goal was to verify if this extraction mixture (modified system 2) could be applied successfully to a wide range of different matrices such as red wines, beers and grains (wheat, oat, barley and malt).Red Wines and Beers.Several red wines and beers (purchased in local markets) were fortified with ochratoxin A and extracted with ETHYFORM mixture (ethyl acetate acidified with 1% of formic acid). Extracted samples were analysed by HPLC. Results are depicted in table 2.Grains.Several grounded samples of Wheat, Oat, Barley and Malt (provided by EWOS (Norway) and Giesinger Biermanufaktur (Germany)) were fortified with ochratoxin A and extracted with ACEWATER mixture (60% of acetonitrile in water acidified with 1% of formic acid).ETHYFORM mixture was unsuccessfully applied to the extraction of OTA from grains (wheat, oat, barley, malt), because of the poor recovery percentage (about 15-20%). Results are summarized in table 3.3rd Step: improvement of the operational extraction procedures.An improvement of the extraction procedures was developed in order to simplify the manual operations. In order to perform the extractions as simply as possible, syringes were used instead of test tubes. The idea was to have systems in which it could be directly performed liquid liquid extraction avoiding the use of pipettes to withdraw the appropriate volume of liquid (syringes are sized their own). The proper volume of wine (or beer), and the proper weight of grains is placed into a bottom capped syringe, proper volume of extraction mixtures (ETHYFORM for liquid and ACEWATER for solid matrices) is added and the syringe is closed with the plunger. After vigorous mixing, organic phase containing OTA can be easily removed from syringe. Such procedures, with the use of the syringe innovation, has allowed greatly simplify the manual operations for the sample preparation without affecting the recovery percentage.The same procedures was applied successfully to the extraction of solid matrices (wheat, barley, oat and malt). Grounded samples were placed into the bottom capped syringe and ACEWATER solution was added. After vigorous mixing, organic phase containing OTA can be easily removed from syringe.Resolution of a problem for INESC-MN.In the determination of OTA in red wine by the device developed by INESC-MN, a problem, probably due to the presence of anthocyanins in red wine (red coloured molecules), occurred. Purification of anthocyanins in red wine extracts was performed by thin layer chromatography (TLC) and purified samples was given to INESC-MN. Since successfully purification of anthocyanins by TLC is a tedious and time consuming procedures, we developed a simple procedures to remove anthocyanins from red wine extracts. Anthocyanins in a wide range of pH are positively charged and could interact with negatively charged (in a wide range of pH) silica gel surface. A bottom capped syringe, filled with silica gel, was developed; the ETHYFORM red wine extracts are passed through the syringe by pushing the plunger, and the colorless solution was collected in a test tube.Colourless extracts were analysed by HPLC-DAD and the presence of anthocyanins in the solution was not detected.Analyses were performed by High Performance Liquid Chromatography Diode Array Detector (HPLC-DAD, Agilent) by using a C18 column with 3 μm of particle size and 2.0 mm of internal diameter and 150mm of length (YMC-TRIART). Gradient elution of increasing concentration of mobile phase B in mobile phase A, at flow rate of 0.2mL/min was employed, using water acidified with formic acid (1% v/v) as mobile phase A and acetonitrile acidified with formic acid (1% v/v) as mobile phase B. Quantification of OTA in fortified wine sample was performed by the external standard method using a three point regression graph of the UV-visible absorption data collected at the OTA specific wavelength of 333 nm. A calibration curve was built by analysing three different concentrations of OTA standard: 10, 50 and 100ng; the correlation coefficient (R2) of the curve was 0.999. Concerning the wine samples, 30 μL of each sample were injected in HPLC system.1st step Experimental:3 samples of 4mL of homemade red wine fortified with 200ng of ochratoxin A were extracted with 4mL of the different solvent mixtures (1, 2 and 3). The extractions were repeated twice and the collected fractions were dried and dissolved in 100 μL of methanol in order to have a presumed concentration of 2ng/μL of OTA in each sample. The extracted samples were analysed following the protocol written in Annex 1.2nd step Experimental Procedures applied to Red Wine and Beer.4 mL of liquid samples (red wine and beer) fortified with 200ng of ochratoxin A were placed in a test tube and 4 mL of extraction mixture (ethyl acetate acidified with 1% of formic acid: ETHYFORM) were added. Mixture was mixed for 30 seconds and after a short time two phases were separated. The upper one, slightly red, is the organic phase, the lower one (deep red if wine is extracted or yellow pale if beer) is the aqueous matrix solution. Just for beer a degassing step should be performed prior extraction. Organic phase was removed by pipette and placed into another test tube. Extraction was repeated twice and the collected fractions were dried with an airflow and the residue dissolved in 100 μL of methanol to have a presumed OTA concentration of 2ng/μ in each sample. The samples were analysed following the protocol written in Annex 1. In table 2 results in terms of OTA recovery percentage are summarized, extraction by ETHYFORM showed a good percentage of recovery.2nd step Experimental Procedures applied to grains.5 grams of grounded solid materials fortified with OTA were placed in a test tube and 10mL of a mixture composed of 60% of acetonitrile and 40% of water and acidified with 1% of formic acid (ACEWATER) were added. Mixture was vigorously mixed for 2 min, the upper liquid phase was removed and placed in another tube. Extraction was repeated twice, the collected fractions were dried by airflow and the residue dissolved by 200μL of methanol.Potential Impact:A list of the principal dissemination activities is shown below and reported in the deliverables n. 2 and n.10: One goal of the research project named "OTASENS", funded by the European Commission under Seventh Framework Programme, Area "Research for SMEs", is to facilitate the take-up of results achieved by the SMEs, not only those involved in the Consortium, but other European SMEs operating in the same fields.In order to realize these activities a proper web-site has been realized. As indicated in the project, the Partners will be charged specifically to disseminate the results carried out through workshops, conferences, meetings, authorized publications and seminars.Tables for these activities are collected in the Deliverable 2 and 10.In the attached Final Report all required information are includedThe plan for the use and dissemination of the foreground will be as following steps:- presentations to internal and external conferences, publications, seminars and workshops to disseminate the knowledge related both to the risks connected to fungal contamination and the advantages coming from new developed technology.- development of a website, updated each three months- participation at shows, exhibitions, fairs.In the first period of the Project ( 9 months) the RTD Performers have presented project background in workshops and conferences (see Table, next page), while each SMEs has operated to diffuse knowledge about to the risks connected to fungal contamination fund in food, feed, biers and wines. AUTOMATION has presented a poster connected to the OTASENS Project at the exhibition held in (Rho, Milan, Italy) on November 2010 and November 2011.Copy of the poster shown at the above exhibition and leaflet are included and added to the website.Copies of articles on local newspapers are included too.Different sectors other than the ones directly involved in the project may be interested on applying the new method resulting from the Project. Namely the sectors of the flour and meal producers for human food, the sector of dried fruits and the coffee traders, and consequently the sectors of bread production and coffee roasting. In these cases the sample preparation protocol could be modified for customers following regulations indicated on Commission Regulation (EC) No 401/2006 of 23 February 2006, laying down the methods of sampling and analysis for official control of mycotoxins in foodstuffs.Preliminary contacts have been taken with European Association for Animal Production and with some Analysis laboratories.Contacts with the Agriculture Minister are in being taken up.For the second period of the Project, the Consortium's partners have tentatively organized workshops and contacts with researchers and food/feed producers in order to attract attention to the projectThe following workshops and meetings have been held (see for details Deliverable n.o 10) :Address of the project public websiteThe address of the project public website is http://www.otasens.itList of Websites:http://www.otasens.it Related documents Final Report - OTASENS (Novel photosensor-based device for rapid and quantitative ochratoxin A determination in wine, beer and feed)
