Team:Strasbourg/Entrepreneurship
Entrepreneurship
Research phase
We came with the idea of an allergen detection kit while discussing the problem of detecting female hormones that pollute water. At that time, one of the team members, allergic to peanuts, was talking about her allergies with another member. It was questioned whether there were any commercially available rapid allergen detection kits. This was the first phase of investigation of our project. Without knowing it, we had decided to revolutionize allergen detection. Identifying the competition is essential before starting to launch a project. Currently, the main qualitative allergen detection techniques are based on immunochromatographic tests that work in much the same way as pregnancy tests. The ELISA technique is widely used: it uses antibodies that specifically recognize a particular epitope on an antigen, here the allergen. In addition, there is one kit already on the market and other being developed that allow the user to get the result of the test on his smartphone. The only kit available to purchase was created in San Francisco. It is a gluten and peanut detection kit based on the use of antibodies. In the same vein, other kits are still in development such as the Ally kit, which detect lactose and is developed by a student from Brunel University, London [1]. In addition, another kit named iEAT (integrated Exogenous Antigen Testing system) was proposed by Harvard Medical School to detect food allergens by chemical technique and data analysis [2]. These different techniques are rather expensive and are not available for some potential users who cannot necessarily afford to spend large amounts of money to test their food. For example, the NIMA kit is sold in the United States for $229.00 for the device alone, on top of which $30.00 for 6 tests must be added [3]. The fact that the user can buy the refills aside, making the device reusable is interesting, but it still remains a barrier in terms of price. The Ally test is expected to be cheaper but is not yet commercialized. The reusable device could cost $30.00 and the back strips for the test would be very cheap. However, this test only detects lactose. The idea for our test would be therefore to take this concerns into account to offer a reliable, inexpensive, reusable and connected test.
Figure 1. Allergens detection kits: the NIMA kit, the Ally kit and the iEAT kit (from left to right)
Identifying Targets
When you are subject to severe allergy, you may hesitate twice before going to the restaurant or eating at a friend's house, and risking to unknowingly ingest an allergen. The risk is real : every 3 minutes in the world, someone is hospitalized for an allergic reaction .
As we would like answer the concerns on the mislabelling of allergens on food products found in supermarkets and restaurants, our new detection kit is primarily intended for consumers who would like to ensure the composition of their dishes. Among the people we interviewed, 45% think that allergens are mislabelled on packaging and 82% think that they are mislabelled in restaurants. These numbers reinforce the need for a solution to this lack of guidance. Upon interviewing allergic people, we found that the proportion of people who had an allergic reaction after a meal outdoors is 75%: it is thus very important to act on this problem. In addition to being used by consumers, this kit could be accessible to the agri-food industries when preparing their products. Indeed, they often use the statement "possible traces of …" on their packaging at the end of the list of ingredients contained in the product. To verify this, one of the team member checked four menus prepared in her kitchen, produced in four different countries: prepared lentils from France, Asian noodles from Thailand, a poutine sauce from Quebec and a vegan freeze-dried sauce for lasagna from Germany. All the four products randomly selected from a kitchen indicate traces of food allergens. Interestingly, vegan guaranteed lasagna sauce is one of the products that contains the most traces of food allergens.
Figure 2. Identifying AptaTest targets: allergic consumers and agri-food industries.
This statement naturally leaves allergic consumers in doubt as to whether or not they can consume the product without risking a severe allergic reaction. This is why our kit can also be used by industries to check the presence or absence of an allergen at the end of the product production. Some techniques are already used to detect allergens in food, such as ELISA assay or mass spectrometry, but they are unfortunately time-consuming, expensive and they require to be performed by qualified workers on specific laboratory equipments. These numerous disadvantages often lead the manufacturer to not control the presence of the allergen in his products, which poses a problem for allergic consumers.
Modeling for design
We assumed that we wanted a simple, aesthetic kit, not too bulky and lightweight to be transportable in a handbag for example, and that it could be refilled for several uses. It is also important that our kit is easy to use for the consumer and not too expensive. In addition, it was necessary to find a system enabling the adaptation of our detection kit to different allergens, in order to have one specific kit per food allergen. Lastly, it is essential that our kit is reliable, so that users can have complete trust in the results.
Figure 3. Key points of the AptaTest
Many brainstorming sessions were conducted to finalize the concrete process to detect allergens and the scientific design of the device.
First of all, we imagined a kit with a size close to a medicine package. The dimensions were about 9x6x2cm. The test is composed of several compartments (Fig.4). First, you must open a trapdoor in (1) in which you must put a small piece of the food you want to test. The compartment (2) corresponds to a lysis chamber composed of a buffer to facilitate the extraction of allergen molecules. It is necessary to have lysis, solubilization, buffering and dilution of the sample. The compartment (3) is important because it contains a solution that neutralizes the pH and solubilizes the allergens in the buffer. The module (4) corresponds to a trapdoor mechanism that opens only when pressed. This allows the buffer containing the allergen from the sample to pass through a compartment containing bacteria. These bacteria are modified and express in their genome a system that is able to recognize the allergen and produce a colored response. The idea of the system is to produce a sufficiently strong color signal when the desired allergen is present in the sample. When the allergen is absent, there is no color signal. This signal is visible through the small window (7) inserted in the kit. Compartment 6 contains enriched medium that must be added to the bacteria.
Figure 4. First kit we imagined
- Add Food
- Close Cap (Gate 1) (1)
- Wait 5 minutes in Lysis Room (2)
- Wait 2 minutes in Neutralization Room (3)
- “Clic” to open Gate 2 (4)
- Operate a 90° rotation of the device
- The liquid will flow into the chamber containing the bacteria (5)
- Allergen of interest is now in contact with the bacteria
- Addition of medium contained in the compartments (6)
- In presence of allergen of interest = DETECTION by a colour change visible through the window (7)
After several feedbacks and discussions, we tried to optimize our kit. The first concern was that since the kit is placed flat on a table, it can prevent the sample from flowing out. The loss of a certain volume of sample that has not flowed properly would create difficulties in detection: if the allergen concentration is not sufficient at the time of testing, it may not work. This is why we thought about designing a vertical rather than a horizontal prototype. From this reflection was born the AptaTest.
Device conception
To meet our expectations as much as possible, we have given a lot of thought to the design of the object: its shape, its capacity, its appearance, the elements needed for optimal detection and the scenario of use. Our complete system consists of:
- Detection device separated into several compartments:
- The input with mechanical grinding
- The lysis chamber
- The neutralisation chamber
- The bacterial chamber
- The output with security system
- A box containing the kit
- An instruction manual for the kit indicating the colour of the positive signal for the presence of the allergen and the negative signal for the absence of the allergen.
- A mini cleaning kit to destroy possible bacteria releases (which should not happen)
Figure 5. All components of the AptaTest kit
First we drew the technical plans of the object. Our prototype is about 15cm high and 4-5cm wide. The compartments can be rotated by a simple rotary movement of a module by the user (see notice). Each compartment is connected to the upper and lower compartments by small holes. On each module, there is an edge on the outside and a seal that allows the kit to remain waterproof. When two edges are aligned, then both compartments communicate, it means that the contents of the upper compartment will flow into the lower compartment.
Figure 6. Cut view of the different elements of the AptaTest kit
Figure 7. Technical drawings of the AptaTest and lysis chamber.
The first module at the top of the kit is composed of two parts: a high grinder and a low grinder. These parts are manufactured with many tips, which allow the food sample to be mechanically ground thanks to fast rotating movements. The crumbled food passes through small holes drilled in the lower part of the grinder into the next module.
Figure 8. First module: grinder in which the food sample is mechanically ground.
The second module corresponds to a lysis chamber that will chemically lyse the sample in order to extract the molecules of interest, i.e. allergens. The module is manufactured so that the solubilized sample flows easily into the lower compartment.
Figure 9. Second module: Lysis chamber
We carried out a lot of research to identify the different lysis buffers that could be used to degrade food. Depending on the allergen to be extracted, the buffer can be optimized. The final goal is always to lyse the sample, solubilize the sample, buffer it, and eventually dilute it. For example, for a peanut allergen (e. g. Ara H1, Ara H2, Ara H3, Ara H3, Ara H3, etc.), the extraction buffer may include PBS with NaCl (e. g. NaCl 1M), Tris (e. g. 20 mM Tris), milk (e. g. skimmed milk powder), tricin, and any other buffer components that may be suitable to facilitate extraction.
Finally, in the alternative buffers that we can find for other allergens, we often find BSA (e. g. 0.33 mg/ml BSA, pH 8.6), PBS (e. g. 1xPBS and 1%T20 or 1xPBS and 2%T20), phosphate (e. g. 50 mM phosphate, pH 7), borate buffered salt solution (BBS) and Tris buffer for a dairy derived allergen (e. g. lactose, casein). It is also possible to find a parvalbumin extraction solution for a fish-derived allergen, an ara-h2 extraction solution for a nut-derived allergen, an egg protein extraction solution for an egg-derived allergen (e. g. ovomucoid protein, ovalbumin protein, ovotransferrin protein, lysozyme protein), a tropomyosin extraction solution for an allergen derived from molluscs or crustaceans, and/or any other appropriate extraction solution for any other allergen [4].
Of course, tests must be done to determine the lysis time in the second module. Indeed, depending on the type of food and the quantity and allergens that compose it, the lysis time will have to be adapted. We hope that this time will not exceed 4-5min. It is also possible to shake the kit slightly to allow faster sample lysis and thus optimal allergen extraction. Indeed, the higher the concentration of allergen in the sample, the more reliable the result obtained.
Then, once the sample has been lysed, a rotation between modules 2 and 3 is necessary, allowing the solution containing the sample to flow into module 3. This module is designed in the same way as the lysis chamber. Module 3 is a neutralization chamber that contains a solution mixture to adjust the pH and solubility of the previously extracted sample if necessary. As mentioned above, the neutraliszation solution required will be different depending on the biochemical properties of the allergen. Tests are also necessary to determine the required neutralization time and the optimal solution volume.
Figure 10: Third module: Neutralization chamber
Between compartments 2-3 and 3-4 there are filters to retain any residues of unleaded food samples. These filters also prevent bacteria from moving up into the upper compartments at the time of mixing required for lysis.
Figure 11: Exploded view of the kit and filter details
After the sample is well solubilized and the pH is optimal, a final mechanical rotation between the upper compartments and the bacterial chamber allows the solution containing the allergen to be brought in contact with the bacteria that have the detection system. The module is transparent, which allows the user to see the colored signal appear.
Figure 12: Bacterial chamber
When the allergen comes in contact with the aptazyme, then the bacterium will transcribe or not a reporter gene (depending on the construction) which will result in a colored or uncolored signal. The long-term idea is to integrate an electronic system into the object in order to accurately measure the intensity of the colored signal and establish thresholds that must be exceeded for the test to be significantly positive or negative.
The volume and concentration of our bacteria is also a key parameter and must be determined. For this reason, we chose to test different concentrations and different culture volumes to realize a growth kinetic in order to optimize the life chamber of the bacteria in our kit to obtain a reliable and interpretable signal for a certain amount of allergen. In addition, it is necessary to test the different storage conditions of the kit. When the bacteria are at 4°C their metabolism is really slowed down. Indeed, the optimal growth temperature of E. coli is 37°C. The kit must therefore be stored at 4°C to prevent the culture space from being filled too quickly by dividing bacteria. Indeed, if the kit is left at room temperature, the bacteria will multiply and arrive in the stationary phase and then they will die. If they die, the detection system will be lost. It is therefore necessary to control this parameter. We have started to test these different parameters. We noticed that the OD600nm of our bacteria stored at 4°C in liquid LB medium remained stable for 12 hours (data not shown) while the OD of our bacteria stored at room temperature and 37°C increased rapidly. The ideal solution would therefore be to store the kit at 4°C and take it out of the fridge a little bit before use so that the bacteria can resume their division.
In our test it is essential to have controls. We imagined adding in parallel with our bacterial chamber another chamber that recognizes a control allergen with the same bacteria. This control would confirm that the bacteria detect the allergen and that the signal obtained or not is not due to chance or a problem with the bacteria. If the control is valid, then the test can be interpreted.
For our test to be totally reliable, many parameters must be considered. Afterwards, a long optimization phase is necessary to determine the correct lysis and neutralization solutions, to determine the necessary bacterial concentration in our kit, as well as the conditions of culture and storage of the bacteria. It is also necessary to test the controls and allergen quantity threshold that our kit can recognize.
Scenario
The manual we have created allows the user to use the detector properly and, above all, to do so safely. Indeed, the test contains biological material, it must be handled with care.
Figure 13: AptaTest notice
For our logo, we have chosen the name AptaTest, "Apta" referring to aptazyme and "test" for detection. The logo was designed by one of our members. We tried as much as possible to design a box for our kit that would be attractive for the consumer. We kept the colors of the logo on the box as well as on the object. We also asked through a survey if the detection kit could interest future users, and we had 70% positive responses in France and 80% positive responses from iGEM teams around the world. In addition, most of the experts we interviewed were in favour of having access to such a kit on the market.
Figure 14: Design of the AptaTest boxes
Communication
References