BIOFOS

BIOFOS: Micro-ring resonator-based biophotonic system for food analysis
[November 2013 – January 2017]

 Project Overview

 

BIOFOS has offered a forum, driven by the end-users of the project, for identifying the needs of stakeholders from different sectors of the food industry. The outcomes of extended surveys done within the duration of the project verified that the actual specifications of BIOFOS system are in line with the stakeholder’s requirements.

On the biological platform four aptameric sequences against OTA, AFM1, AFB1, and Copper ions were characterized and three new aptamers against Lactose, Penicillin and Phosmet were developed through the process of Capture-SELEX. In parallel, the use of the two-strand approach, managed to successfully immobilize all aptamers of interest onto functionalized Si3N4 surfaces at optimal concentrations, increasing thus the analyte binding and sensitivity of the final integrated biosensing platform was developed. Finally, a high number of regeneration cycles (30) have been achieved, with minimal losses in the binding affinity of the aptamers after each successive regeneration cycle catering for a truly reusable sensor platform.

On the photonics platform, emphasis was given on the design of the individual structures, MRR chips, Y-splitters, MMI splitters and various grating design for all the production runs. These designs where used in several mask designs which resulted in total five (5) production runs to produce passive and hybrid MRR chips and testing structures to optimize the sensor chips during the project. After the passive chip production run we have produced the G1 Hybrid chip run which contained MRR sensor chips and testing structures with gratings used for hybrid integration of an 850 nm VCSEL and a 12x photodiode array on the sensor. After the two G1 runs (Hybrid and passive) another two runs were performed with the second generation of passive and hybrid chips. VCSELs and Photodiodes were bonded directly to chip with the use of Au/Au thermo-compression bonding technique and attached a FPC cable to the side of the chip using anisotropic tape. This resulted in completely functional hybrid chip which can be directly inserted into the cartridge/fluidic handling system developed in BIOFOS.

On the nanochemical platform of the sensor different methods and approaches for the functionalization of the sensor substrate and the immobilization of the aptamers on the sensing surface of the optical sensing chip were employed including, novel laser-based immobilization approaches, alkenes based surface modification by photoactivation for site-specifically modification of the sensor sensing surface, and polymer-based layer functionalization approaches. Within the course of the project, the application of laser-based approach, resulted in the efficient immobilization of the aptamers on functionalized surfaces developed both by Wu, Surfix and BRFAA and was employed for the bio-modification of the final optical chips used for validating the developed system. Moreover, the functionalization of an azide, synthesis of zwitterionic monomer for ATRP was synthesized and up-scaled it to gram scale. This resulted in the successful preparation of an antifouling zwitterionic copolymer coating bearing a variable amount (5-15%) of clickable moieties for introduction of aptamers using established click chemistry protocols (i.e. the so-called SPAAC reaction). As a result of the work achieved within the framework of these activities, a PCT patent was filed by ICCS/NTUA (in collaboration with Surfix and WU, as co-inventors) on 15.12.2016.

On the microfluidic platform the activities were devoted to the design and development of the fluidic sample pretreatment units for oil, milk and nut extract samples, the development of the microfluidic analysis cartridge, the development of the regeneration module and the electronic platform. During the project’s lifetime, the pretreatment protocols for the three selected food types i.e. nuts, olive oil and milk have been established. They can be performed without the need of laboratory equipment. Maximum a blender for nuts and a vortex for olive oil is required. The common pretreatment steps of the three different food types have been combined into the automated pretreatment unit. This unit offers to either clean the sample by filtering or concentrating the sample by using solid phase extraction. An especially for BIOFOS developed pump allows to pump a number of different aggressive solvents which are required for the solid phase extraction. For additional exploitation, this pump module was additionally converted into a standalone dispensing pump. The development phases of the pretreatment unit started with a bread board setup and finalized with its integration into the BIOFOS-system. Washing solutions allow to clean reuse the pretreatment unit.

The integration of the BIOFOS-system was implemented with the focus put on user friendliness. Bottles and sensor are protected within the housing and provide enough liquid for at least 30 measurements. The implemented touch screen offers the operator images and commands to guide him through the sample taking, sample treatment and sample adding into the machine. The pretreatment results were successful for the three food categories. In the framework of system integration, all components for the final system were integrated successfully and the BIOFOS prototype has been extensively tested for the pretreatment module, the electronics and user interface and software. The pretreatment results were successful for the three food categories. The detection unit was successfully validated with good specificity for the case of Copper.

BIOFOS gained remarkable visibility through a variety of dissemination actions and prestigious publications (including the Langmuir), and succeeded in defining concrete exploitation plans by all partners. The foreground knowledge produced in the last period of the project and the filling of the two PCT patents by the BIOFOS partners gives the credential for a potential exploitation of the project results.

 

 

Summary of project context and objectives

 

With a market share of 14.5 % of the total manufacturing turnover (€917bn for the EU-27), the food industry is the second largest sector in the manufacturing industry of EU, consisting of about 310,000 enterprises. With 4.8 million employees, the food industry’s share of employment is about 14% of the total manufacturing sector, of which 62% are employed in SMEs (undertakings of less than 250 persons) representing virtually the total number of enterprises. Despite the fact that Europe’s food and drink sector has always been a pillar of the EU economy, current methodologies for detection of food contamination based on heavy analytical tools cannot guarantee a safe and stable food supply. The reasons are the complexity, the long time-to-result (2-3 days) and the cost of these tools, which limit the number of samples that can be practically analyzed at food processing and storage sites. The need for screening tools that will be still reliable but simple, fast, low-cost, sensitive and portable for in-situ application is thus urgent.

BIOFOS was conceived in 2013, with the aim to develop a simple, fast, low-cost, sensitive, portable and reliable, screening tool for in-situ detection of food contaminations in nuts, olive oil and milk and also for the quantitative detection of lactose in milk. In order to achieve these targets, BIOFOS combined the most promising concepts from the photonic, biological, nanochemical and fluidic parts of LoC systems, aiming to overcome limitations related to sensitivity, reliability, compactness and cost issues.

Specifically, BIOFOS relies on the ultra-low loss TriPleX photonic platform in order to integrate on a 10×10 mm2 chip 7 micro-ring resonators, a VCSEL and 12 Si photodiodes and achieve a record detection limit in the change of the refractive index of 5·10-8 RIU. The reusability and high specificity of the system is supported by the use of aptamers as biotransducers, while advanced surface functionalization techniques have been developed and used for their immobilization on the sensor surface. New microfluidic structures introduced for the sample pre-treatment and the regeneration process.

The main objectives of the BIOFOS project are listed below.

  • Development of monolithic photonic circuits for label‐free, high‐throughput optical biosensing at 850 nm based on 1:8 MMI couplers and 8‐fold MRR arrays for operation.
  • Hybrid integration of actives elements (VCSEL and photodiode arrays) on TriPleX boards using flip‐chip bonding and butt‐coupling techniques.
  • Design and production of aptamers for highly‐selective detection of food contaminants and lactose in milk.
  • Development of effective techniques for functionalization of sensor substrates and immobilization of biomediators (aptamers).
  • Development of robust techniques for regeneration of biomediators (aptamers) and reusability of sensor arrays.
  • Development of microfluidics for solid and liquid phase treatment.
  • System integration in a cost‐effective, reusable LoC system and system validation in real settings.

Main Scientific and technical (S&T) results and foregrounds

 

To accomplish its objectives, BIOFOS based the organization of the necessary management and RTD around 9 work-packages (WPs) as shown in Figure 1 below.

Work-package 3: Innovation chain, stakeholders’ requirements & specifications, System design and methodology for integration and packaging processes

Activities within WP3 were aiming in defining the innovation chain of BIOFOS system, and the stakeholder’s requirements involved in the relative food sectors and enable the definition and design of the system specifications and packaging methodologies of the system, as well as evaluation of the system through system level modelling and simulations.

During the course of the project, WP3 has offered a forum, driven by the end-users of the project, for identifying the needs of stakeholders from different sectors of the food industry. The outcomes of extended surveys done within the first year of the project, and updated in the following ones, verified that the actual specifications of BIOFOS system are in line with the stakeholder’s requirements. In addition, during the project’s lifetime, in the framework of WP3, the system specifications of BIOFOS components and systems, the packaging methodologies and integration methodologies have been defined taking into account the specific characteristics of the different integration platforms, and simulation studies regarding the system performance of BIOFOS devices and systems have been performed. WP3 has also taken care of a smooth transition of all these activities into WP8 at the final stages of the project.

Work-package 4: Design, fabrication and regeneration of biomediators

WP4 activities are devoted to the selection, characterization and design of aptameric sequences against specific targeted analytes which are commonly found as contaminants in food samples, their chemical modification, and identification and definition of the most efficient processes for their regeneration.

Within the BIOFOS project, UPVD selected all known aptameric sequences against OTA, AFM1, AFB1, and Copper ions and characterized them in terms of successful immobilization on functionalized surface while new aptamers against Lactose, Penicillin and Phosmet were developed through the process of Capture-SELEX. In parallel, BRFAA, by making use of the two-strand approach, manage to successfully immobilize all aptamers of interest onto functionalized Si3N4 surfaces at optimal concentrations, increasing thus the analyte binding and sensitivity of the final integrated biosensing platform. Finally, a high number of regeneration cycles have been achieved, with minimal losses in the binding affinity of the aptamers after each successive regeneration cycle catering for a truly reusable sensor platform.

The work achieved within the framework of WP4 activities, resulted in two publications in peer-review articles: 1. ‘Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor’, N. Paniel, G.Istamboulié, A. Triki, C. Lozano, L. Barthelmebs, T.Noguer. Talanta 162, 232 (2017) and 2. ‘Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk’, G. Istamboulié, N. Paniel, L. Zara, L. Reguillo Granados, L. Barthelmebs, T. Noguer. Talanta, 146, 464-469 (2016).

 

Work-package 5: Photonic platform of the biosensor system

WP5 activities are related to the design, production and optical characterization of the Micro ring resonator (MRR) chips. ICCS/NTUA has performed during the project simulations in several stages during the production to optimize the MRR chip design with respect to the Y-splitters, MMI splitters and various grating design for all the production runs. These designs where used in several mask designs developed by Lionix. With these designs LioniX has performed in total five (5) production runs to produce passive and hybrid MRR chips and testing structures to optimize the sensor chips during the project.

At the start of the project LioniX has produced G0 chips which were based on a known design and where used for the first experiments and was followed by a passive chip production run G1 which contained the first series of testing structures and the G1 generation of the BIOFOS MRR sensor. These where all optically tested by SAXION and ICCS/NTUA and used in WP6 and WP8 for functionalization and “bio” testing.

After the passive chip production run we have produced the G1 Hybrid chip run which contained MRR sensor chips and testing structures with gratings used for hybrid integration of an 850 nm VCSEL and a 12x photodiode array on the sensor. These where all characterized by ICCS/NTUA and SAXION. After the two G1 runs (Hybrid and passive) another two runs are performed with the second generation of passive and hybrid chips which were also characterized by ICCS/NTUA and SAXION and used in WP6 and WP8. For the Hybrid chips LioniX has also developed techniques to Flip-Chip the VCSELs and Photodiodes directly to chip with the use of Au/Au thermo-compression bonding technique and attached a FPC cable to the side of the chip using anisotropic tape. This resulted in completely functional hybrid chips which can be directly inserted into the cartridge/fluidic handling system developed in WP7.

Work-package 6: Functionalization of the sensor surface/immobilization of the biomolecules

WP6 is devoted to the nanochemical platform of the sensor. Different methods and approaches for the functionalization of the sensor substrate and the immobilization of the aptamers on the sensing surface of the optical sensing chip are employed including, novel laser-based immobilization approaches, alkenes based surface modification by photoactivation for site-specifically modification of the sensor sensing surface, and polymer-based layer functionalization approaches.

Within the course of the project, a new approach based on the Laser Induced Forward Transfer technique, namely known as LIFT, has been applied successfully for the bio-modification of the sensor surfaces with high spatial control, by ICCS/NTUA. The application of laser-based approach, resulted in the efficient immobilization of the aptamers on functionalized surfaces developed both by Surfix and BRFAA and was employed for the bio-modification of the final optical chips used for validating the developed system. Furthermore, the ability of the LIFT technique to deposit aptamers onto non-functionalized surfaces has been shown, which could be useful if further reductions in cost and time-to-product. In parallel, an elegant approach, to exclusively modify silicon nitride while backfilling the surrounding silicon oxide so that it acts as a repellent and prohibits non-specific binding, has been successfully developed by Surfix. WU, on the other hand, synthesized an azide modified zwitterionic monomer for ATRP and upscaled it to gram scale. This resulted in the successful preparation of an antifouling zwitterionic copolymer coating bearing a variable amount (5-15%) of clickable moieties for introduction of aptamers using established click chemistry protocols (i.e. the so-called SPAAC reaction).

Finally, BRFFA has successfully developed an alternative protocol to functionalize silicon nitride based on silanes and studied and optimized two additional functionalization approaches, namely PAMAM dendrimers and polysaccharide meshes, to load more aptamers onto the surfaces and thus ultimately lower the detection limits and the sensitivity of the sensing platform.

As a result of the work achieved within the framework of WP6 activities, a PCT patent was filed by ICCS/NTUA (in collaboration with Surfic and WU, as co-inventors) on 15.12.2016, entitled “Method for activating click reactions through laser induced forward transfer of molecules” describing a novel method that has been developed within the BIOFOS project through the combination of the laser-based immobilization approach with click chemistry reactions. This work resulted also to a publication: ‘Direct creation of biopatterns via combination of laser-based techniques and Click-chemistry’, M. Chatzipetrou, M. Massaouti, G. Tsekenis, A. K. Trilling, E. van Andel, L. Scheres, M. M. J. Smulders, H. Zuilhof, I. Zergioti, Langmuir, 33(4), 848 (2017). In addition, the work of WU related to the work on the development of clickable and fully zwitterionic antifouling polymer brushes on stoichiometric silicon nitride resulted in a scientific Publication: ‘Efficient and tunable three-dimensional functionalization of fully zwitterionic antifouling surface coatings’. Stefanie C. Lange, Esther van Andel, Maarten M.J. Smulders, Han Zuilhof, Langmuir 2016, 32 (40), 10199, which was selected as cover and highlighted as ACS Edistor’s choice.

 

Work-package 7: Microfluidics and system electronics

WP7 activities are devoted to the design and development of the fluidic sample pretreatment units for oil, milk and nut extract samples, the development of the microfluidic analysis cartridge, the development of the regeneration module and the electronic platform.

During the project’s lifetime, in the framework of WP7, pretreatment protocols for the three selected food types i.e. nuts, olive oil and milk have been established. They can be performed without the need of laboratory equipment. Maximum a blender for nuts and a vortex for olive oil is required.

The common pretreatment steps of the three different food types have been combined into the automated pretreatment unit. This unit offers to either clean the sample by filtering or concentrating the sample by using solid phase extraction. A especially for BIOFOS developed pump allows to pump a number of different aggressive solvents which are required for the solid phase extraction. For additional exploitation, this pump module was additionally converted into a standalone dispensing pump. The development phases of the pretreatment unit started with a bread board setup and finalized with its integration into the BIOFOS-system. Washing solutions allow to clean reuse the pretreatment unit.

The pretreated sample is analyzed within the detection unit. The detection unit consists of a glass based microfluidic chip with an integrated rotary valve. This microfluidic chip provides a fluidic connection to the bio-optical-chip which can be inserted and later be removed via a clamping mechanism. Via the rotary valve the loaded sample can be pushed over the bio-optical chip. If required a dilution can be performed within the implemented mixing structure. Finally, regeneration buffers can be used to regenerate the bio-optical chip to perform at least 30 measurements. To speed-up the development process, a bread board system was build and following it an enhanced version was integrated into the BIOFOS-system.

To control the individual units, electronics were developed which orchestrated pumps, valves, VSCEL, photo diodes, peltier element and much more. A simple user interface guides the operator via images and commands from the sample preparation to the result.

 

Work-package 8: Performance evaluation, and system integration and validation in real settings

Work Package 8 is devoted to the characterization and evaluation of the performance of the optical chips, the integration of the system components in a single box, the definition of the testing procedures and finally to the validation of the BIOFOS system performance.

After the individual units were developed in WP7, the integrated design of the BIOFOS-system was started. The focus was put on user friendliness.

Bottles and sensor are protected within the housing and provide enough liquid for at least 30 measurements. The implemented touch screen offers the operator images and commands to guide him through the sample taking, sample treatment and sample adding into the machine.

The validation of the BIOFOS prototype has been performed for the pretreatment module, the electronics and user interface and software. The pretreatment results were successful for the three food categories. The breadboard detection unit was successfully validated with good specificity for the case of Copper.

Work-package 9: Exploitation, standardization & dissemination.

The major objectives of WP9 were to:

  • Plan the exploitation of the project foreground knowledge.
  • Prepare techno-economic studies on the deployment of BIOFOS technology (and the relevant solutions along the innovation chain) in biosensing systems for the food sector with emphasis on the three food systems under consideration (milk, olive oil, nuts).
  • Generate and protect intellectual property (patents portfolio) in order to set the basis for potential commercialization of products relevant to the project outcomes.
  • Continuously monitor intellectual property generated world-wide and assess the possible threats for BIOFOS exploitation plan.
  • Coordinate activities towards contributions to standardization bodies.
  • Interact with other EU and national projects.
  • Disseminate project results to the scientific/technical community and towards the general public.

 

In overall, the major outcomes achieved in WP9, during the whole period of the BIOFOS project can be summarized in the following bullets:

 

  • Extensive document on market outlook, system applicability and techno-economic analysis.
  • Cost analysis of the BIOFOS prototype and the cost per analysis.
  • Extensive overview of competing developments (companies, R&D literature).
  • Identification of many potential business cases as a foundation for further developments.
  • Follow-up projects, proposals and plans.
  • Business case description of the milk contamination test at the delivery at the dairy processing plant.
  • Standardization Plan and Agency (NEN-NL) as a highly interested partner in an IA proposal consortium.
  • Extensive patent searches resulting in a positive evaluation in connection with Freedom-to- Operate.
  • PCT patent application on laser surface functionalization by ICCS/NTUA (co-inventors Surfix, WU).
  • Patent application by CSEM.

 

 

Impact

 

BIOFOS exploitation plan relies on the fact that four different technological platforms have been developed in parallel. BIOFOS has developed biological and nanochemical protocols for the successful immobilization of Biorecognition elements on the Si3N4 surface. It has also developed integrated hybrid photonic chip and microfluidics and pretreatment modules. The individual platforms are expected to give disruptive solutions on the sensors applications.

The achievement of the challenging objectives of BIOFOS brings Europe at the forefront of photonic sensors component market. The individual platforms and the integrated BIOFOS prototype are of TRL 4-5 and through the exploitation of the project results, BIOFOS partners will be in the position to offer a technology solution with unmatched and directly transfer this technology to food and to other sectors, such as the health sector. The ICT project BIOCDx (http://biocdx.eu/) is under this direction.

 

The Project’s Public web-site: (http://www.ict-biofos.eu/)

 

The BIOFOS website (http://www.ict‐biofos.eu) is updated on a regular basis with publications, press releases, news about recent achievements within BIOFOS. Apart from the public area of the web‐site, the private area continued to be the point of reference for partners regarding the exchange of working documents, reports (deliverables), presentations (such as the PPR2 final presentation, presentations of the SC meetings etc.), meeting minutes etc.

The visibility of the BIOFOS website exhibits a continuous increase as shown in Figure 8, where the evolution of the number of unique visitors from the beginning of the web‐site operation till March 2017, is presented.

The BIOFOS Consortium