Periodic Reporting for period 1 - RESAS (Real time high sensitive food allergen sensor)
Okres sprawozdawczy: 2023-03-01 do 2025-02-28
Food allergies affect around 26 million Europeans and 32 million Americans. The severity of the allergic reaction may vary from less severe symptoms such as hives and digestive problems, to a quickly progressing and potentially life-threatening anaphylactic shock. Nowadays, food allergy is an incurable condition, so the most important preventive measure is complete abstinence from the allergen. Existing allergen detection methods are slow, cumbersome, and costly, and most of them are limited to work in a lab environment.
The RESAS project’s goal is to fabricate a device able to sense extremely low concentrations of food allergens within minutes. This is achieved by combining an ultra-highly sensitive biosensor substrate, an integrated optical transducer and an electrical signal processing unit. The proposed kind of sensor on the basis of integrated optics enables small size (~5 cm3), high repeatability and instant response, as well as potentially low-cost and mass production. Furthermore, the RESAS device has a removable biosensing substrate that can be replaced after use, allowing users to operate RESAS several times and for as many types of food allergens as needed. This approach of interchangeable bio substrates tackles one of the major limitations of most current biosensing technologies, which can be used only once and are restricted to one type of food allergen.
The RESAS project will significantly impact the food industry, as our food allergen testing method promises to be quick, effective, and inexpensive. Finally, a new class of portable test devices for millions of allergists and end-users will be created. A schematic view of the system can be seen in the attached picture.
Objective 1: Instantaneous identification of small concentrations of food allergens using a bio substrate
Rapid allergen detection can be life-saving. Thus, for allergy specialists and end-users, the long waiting time of current food allergen analysis methods is unacceptable. This has led to a growing demand for the improvement of detection methods where instant detection is required. The RESAS project allows instant detection of allergens thanks to the specialized biochemistry which allows allergens to bind to the bio substrate. These bound allergens cause changes in the surface refractive index (RI), which is detected by an optical sensor and ultimately measures the allergen concentration. It is aimed to obtain first results instantaneously and final read-outs within 5 min with a limit of detection less than 10 ng of allergen per gram of food sample.
Objective 2: Integration of optics and biosensing technology for food allergen detection in a handheld device
Bulky and expensive laboratory-scale devices for analysis are impracticable for large-scale use. Using the technological approach of biosensing with optics, a handheld device can be realized. RESAS uses SPR to detect changes in the RI caused by binding of allergens. Radio-over-fibre (RoF) technology is implemented to pass an RF modulated laser through the biosensing substrate. This allows to process the reflected laser signal in the electrical domain rather than the optical domain, causing an improvement in the signal-to-noise ratio and enabling highly integrated powerful signal processing to measure the allergen concentration.
Reduction in size to a handheld device is feasible thanks to the relaxation of the instrumentation demands; specifically, in the following:
- The laser source is directly RF modulated, this avoids the use of modulators e.g. MZM.
- The use of a waveguide to take advantage of the SPR effect, rather than conventional bulky prisms.
- No need of spectrometers, as the signal processing is in the electrical domain.
- Bulky and expensive laboratory equipment such as VNAs is used. Instead, simpler application of specific electronics is implemented.
Objective 3: Demonstration of sensing efficiency
The RESAS device is tested against the most reliable method for food allergen detection, this is the ELISA assay. These experiments consist in comparing RESAS with ELISA, how sensitive they are to small concentration of food allergens and how rapid results can be obtained. Initial experiments to demonstrate proof of concept senses the most abundant allergen in bovine milk which is αS1-casein. Cow’s milk dissolved in water at different concentrations will be used as samples for detection.
The RESAS project’s goal is to fabricate a device able to sense extremely low concentrations of food allergens within minutes. This is achieved by combining an ultra-highly sensitive biosensor substrate, an integrated optical transducer and an electrical signal processing unit. The proposed kind of sensor on the basis of integrated optics enables small size (~5 cm3), high repeatability and instant response, as well as potentially low-cost and mass production. Furthermore, the RESAS device has a removable biosensing substrate that can be replaced after use, allowing users to operate RESAS several times and for as many types of food allergens as needed. This approach of interchangeable bio substrates tackles one of the major limitations of most current biosensing technologies, which can be used only once and are restricted to one type of food allergen.
The RESAS project will significantly impact the food industry, as our food allergen testing method promises to be quick, effective, and inexpensive. Finally, a new class of portable test devices for millions of allergists and end-users will be created. A schematic view of the system can be seen in the attached picture.
Objective 1: Instantaneous identification of small concentrations of food allergens using a bio substrate
Rapid allergen detection can be life-saving. Thus, for allergy specialists and end-users, the long waiting time of current food allergen analysis methods is unacceptable. This has led to a growing demand for the improvement of detection methods where instant detection is required. The RESAS project allows instant detection of allergens thanks to the specialized biochemistry which allows allergens to bind to the bio substrate. These bound allergens cause changes in the surface refractive index (RI), which is detected by an optical sensor and ultimately measures the allergen concentration. It is aimed to obtain first results instantaneously and final read-outs within 5 min with a limit of detection less than 10 ng of allergen per gram of food sample.
Objective 2: Integration of optics and biosensing technology for food allergen detection in a handheld device
Bulky and expensive laboratory-scale devices for analysis are impracticable for large-scale use. Using the technological approach of biosensing with optics, a handheld device can be realized. RESAS uses SPR to detect changes in the RI caused by binding of allergens. Radio-over-fibre (RoF) technology is implemented to pass an RF modulated laser through the biosensing substrate. This allows to process the reflected laser signal in the electrical domain rather than the optical domain, causing an improvement in the signal-to-noise ratio and enabling highly integrated powerful signal processing to measure the allergen concentration.
Reduction in size to a handheld device is feasible thanks to the relaxation of the instrumentation demands; specifically, in the following:
- The laser source is directly RF modulated, this avoids the use of modulators e.g. MZM.
- The use of a waveguide to take advantage of the SPR effect, rather than conventional bulky prisms.
- No need of spectrometers, as the signal processing is in the electrical domain.
- Bulky and expensive laboratory equipment such as VNAs is used. Instead, simpler application of specific electronics is implemented.
Objective 3: Demonstration of sensing efficiency
The RESAS device is tested against the most reliable method for food allergen detection, this is the ELISA assay. These experiments consist in comparing RESAS with ELISA, how sensitive they are to small concentration of food allergens and how rapid results can be obtained. Initial experiments to demonstrate proof of concept senses the most abundant allergen in bovine milk which is αS1-casein. Cow’s milk dissolved in water at different concentrations will be used as samples for detection.
Waveguide design and fabrication
To provide an affordable device, it was decided to use a multimode polymer waveguide made of sole PMDS. Apart from its low cost, this polymer presents different advantage such as biocompatibility, being optically transparent for the intended wavelength of around 850 nm and its optical properties can be easily modified. For example, the base:agent ratio can be tuned to obtain a specific refractive index. This allows us to design a rectangular Waveguide using PDMS only, where the core has a RI of 1.424 and a cladding with RI of 1.417. Initial simulations proved that the PDMS waveguide functioned well using this difference in the refractive indexes of core and cladding. The design and determination of required RI contrast as well as influence of allergens to be detected have been simulated.
The fabrication consisted of a combination of photolithography and replica moulding techniques. The first step was to fabricate a replica of the core made by a photoresist using conventional photolithography, this serves as a master mould. PDMS (20:1) was then casted in the master mould, cured and peeled off; creating the lower part of the cladding with a microchannel with equal dimensions to the core. Next, a flat PDMS slab was used as the upper part of the cladding and covalently bonded to its counterpart with oxygen plasma. After these steps, we have a block of PDMS with an empty microchannel inside of it. In the last step, two micro holes were punched to create inlets; these inlets allow to pouring the PDMS (5:1) mixture to fill the microchannel and therefore creating the PDMS core. To work as a biosensor, a small section of the cladding is removed which left a small part of the core open to the air. Here, antibodies will be immobilized to create the allergen-antibody reaction.
Bio functionalisation
The air-exposed part of the core was functionalized using EDC/NHS coupling to covalently bind antibodies to the PDMS. As proof of concept, αS1-casein antibodies were used because is the most abundant allergen in cow’s milk. These antibodies specifically target αS1-casein allergen protein in the food. This protein-antibody binding creates a change in the refractive index of the PDMS core, which translates into attenuation of the waveguide output signal. For each different allergen to detect, a different PDMS waveguide needs to be functionalized. Consequently, we designed an optical setup that allows to change the waveguide accordingly.
Optical setup and Signal processing.
First, the optical waveguide itself has to be characterized. Two optical fibres are connected in both ends of the PDMS waveguide core. Working first with a red (i.e. visible) light source has the advantage that the setup can be optimized easily. Then, sources with the planned wavelength of around 850 nm (i.e. infrared) will be used. A spectrometer is available to monitor spectral effects (e.g. absorption).
The binding of allergens causes resonances / wavelength-dependent absorption. In order to sample the spectrum, several sources have to be used. The challenge here is to identify the individual sources in the combined received signal after the PDMS waveguide. Here, CDMA techniques known from communications are used. This novel principle is demonstrated already without the bio functionalized substrate in a proof-of-concept setup.
To provide an affordable device, it was decided to use a multimode polymer waveguide made of sole PMDS. Apart from its low cost, this polymer presents different advantage such as biocompatibility, being optically transparent for the intended wavelength of around 850 nm and its optical properties can be easily modified. For example, the base:agent ratio can be tuned to obtain a specific refractive index. This allows us to design a rectangular Waveguide using PDMS only, where the core has a RI of 1.424 and a cladding with RI of 1.417. Initial simulations proved that the PDMS waveguide functioned well using this difference in the refractive indexes of core and cladding. The design and determination of required RI contrast as well as influence of allergens to be detected have been simulated.
The fabrication consisted of a combination of photolithography and replica moulding techniques. The first step was to fabricate a replica of the core made by a photoresist using conventional photolithography, this serves as a master mould. PDMS (20:1) was then casted in the master mould, cured and peeled off; creating the lower part of the cladding with a microchannel with equal dimensions to the core. Next, a flat PDMS slab was used as the upper part of the cladding and covalently bonded to its counterpart with oxygen plasma. After these steps, we have a block of PDMS with an empty microchannel inside of it. In the last step, two micro holes were punched to create inlets; these inlets allow to pouring the PDMS (5:1) mixture to fill the microchannel and therefore creating the PDMS core. To work as a biosensor, a small section of the cladding is removed which left a small part of the core open to the air. Here, antibodies will be immobilized to create the allergen-antibody reaction.
Bio functionalisation
The air-exposed part of the core was functionalized using EDC/NHS coupling to covalently bind antibodies to the PDMS. As proof of concept, αS1-casein antibodies were used because is the most abundant allergen in cow’s milk. These antibodies specifically target αS1-casein allergen protein in the food. This protein-antibody binding creates a change in the refractive index of the PDMS core, which translates into attenuation of the waveguide output signal. For each different allergen to detect, a different PDMS waveguide needs to be functionalized. Consequently, we designed an optical setup that allows to change the waveguide accordingly.
Optical setup and Signal processing.
First, the optical waveguide itself has to be characterized. Two optical fibres are connected in both ends of the PDMS waveguide core. Working first with a red (i.e. visible) light source has the advantage that the setup can be optimized easily. Then, sources with the planned wavelength of around 850 nm (i.e. infrared) will be used. A spectrometer is available to monitor spectral effects (e.g. absorption).
The binding of allergens causes resonances / wavelength-dependent absorption. In order to sample the spectrum, several sources have to be used. The challenge here is to identify the individual sources in the combined received signal after the PDMS waveguide. Here, CDMA techniques known from communications are used. This novel principle is demonstrated already without the bio functionalized substrate in a proof-of-concept setup.
In order to provide a cost-effective solution required for a mass-market application, spectrometers cannot be used for the detection of the bio functionalized substrate induced resonances / wavelength-dependent absorption. The novel approach of applying methods from communications such as CDMA techniques to identify multiple fixed-wavelength sources to sample the spectrum have been developed. However, the system integration is more challenging than expected. Therefore, a longer run-time of the project would have been beneficial. Concluding, further research is needed to provide a system-level demonstration and trigger further steps such as commercialization.