ShroomSweeper GreatBay_SCIE

Inspiration

Mushroom, as a typical type of multicellular fungus, has been a foodstuff since civilizations first started. They appear in loads of delicious dishes across the world.

However, poisonous mushrooms are camouflaging themselves among other organisms with innocent looks. Today, mushroom toxin poisonings have evolved into larger problems. In the report "Mushroom Poisoning Outbreaks -- China, 2019" by CCDC (Chinese Center for Disease Control and Prevention)[1], it is pointed out that in the year 2019 alone, there are 276 cases of mushroom poisonings, involving 769 patients from 19 provinces. This takes up 50% of all food poisoning cases that year.

Let's take Yunnan Province in Southwestern China as an example. When summer comes, the humid weather condition boosts the growth of mushrooms on almost every mountain and meadow, and wild mushroom markets come alive. Almost every family keeps the habit of collecting and consuming wild mushrooms, but this act is extremely risky because many untrained people are not able to correctly distinguish poisonous mushrooms from others. Collecting and consuming wild mushrooms is a culture and a tradition in Yunnan and many other regions of the world, and because of this, large-scale poisonings happen every year. We interviewed Dr. Deng, a professional mycologist from the Guangdong Institute of Microbiology. She gives us an explanation of why people collect and eat wild mushrooms.

Interview with Dr. Deng >

There are 435 known species of poisonous mushrooms in China. They are distributed mostly in Southern Chinese provinces; Yunnan, Guizhou, Sichuan, Hubei, Hunan, Guangxi, and Guangdong, just to name a few. According to the report below, the death rate caused by mushroom poisonings ranges from 11.69% to 42.30%, in which the death rate caused by hepatotoxic toxins (amatoxins, to be specific) from mushrooms of the Amanita genus can be as high as 80-90%.

We have shown these two pictures to public audiences. Most of them cannot distinguish between the safe one and the dangerous one, due to a common saying that the more colorful a mushroom is, the more poisonous it is.

In fact, contrasting to common belief, the bright orange mushroom is Amanita caesarea, a common edible mushroom, and is considered quite a delicacy. The white one is Amanita exitialis, a mushroom containing amanitin, the toxin mentioned before, with an extremely high death rate.

Many of the sayings like this are wrong. Some people say that fully cooked mushrooms are edible; others say that toxins are detectable by boiling the mushrooms with silverware, or garlic, and checking if they turn black. The former method is only partially true because some toxins cannot be deformed by heat, and the latter one is utterly false but is spread widely among people.

Our project

The two existing methods for mushroom toxin detection are LC-MS (Liquid Chromatography-Mass Spectroscopy), which detects the toxin through molecular mass; and ELISA (Enzyme-Linked ImmunoSorbent Assay), which requires a relatively large number of binding and washing procedures to give a result that is readable only by a specialized device. Both of these approaches require professional operators and cost approximately an hour, hence it is not widely accessible by local mushroom pickers and the general public.

We worked on our project in the hope of developing a much more convenient device to help people directly test mushroom toxins in situ before eating.

We have reviewed a commonly-used assay method, LFIA[2] (Lateral Flow ImmunoAssay), which makes use of the principles of the ELISA technique mentioned before. If you have seen a pregnancy test stick, then it won't be hard to understand how LFIA works, because a pregnancy test is one of the most common LFIA devices.

On an LFIA test strip, the sample flows horizontally on the test pad, on which the antigens - the targeted molecules - bind with the antibodies on the test line and control line, resulting in a color change on the lines and therefore proving the existence of the target molecule. 2 kinds of testing methods, direct and competitive tests, can be used depending on the size of the target molecules.

Albeit LFIA's mature development as an approach for rapid testing of target molecules, there are still many major drawbacks to it, for example, the necessary use of animal-produced antibodies, whose production process is unstable and time-consuming. To replace this, we have discovered two ready substitutes - scFv and aptamers.

A comparison between antibodies (left) and scFvs (right)

scFv, short for single-chained fragment variable[3], is a kind of peptide chain that can bind to target molecules, just like antibodies. scFv can be mass-produced with Escherichia coli using general protein production and purification methods. There is no need for animal experiments, which is used in the production of normal antibodies. scFv has improved accuracy than general antibodies, which caters to our need, not to mention that they are cheaper and more convenient in production as well.

Because of the chemical similarities of scFvs and antibodies, it could directly be utilized in LFIA, replacing the antibodies.

Aptamers are oligonucleotides that form secondary structures, giving them the ability to bind targeted molecules, including ions or small molecules, and, in our case, amanitin.

Aptamers

Aptamers have many advantages compared to antibodies. For example, aptamers perform better in terms of thermostability and chemistry stability than antibodies - they can automatically fold back into original shape after deforming, making them easier to preserve, transport, and used in detection. They are also smaller in size while keeping a large surface area and therefore can carry out a series of reactions with target molecules. Moreover, they can be produced through PCR, which is faster and easier when compared to the production process of antibodies that depend on eukaryotic cells. They don't need epitomes on antigens to bind with target molecules, yet still have high specificity against them. Those outstanding properties make aptamers very competitive against antibodies in detection.

However, due to the fundamental structural differences between aptamers and antibodies/scFvs, modifications need to be done to the design of the lateral-flow test strip in order for aptamers to be used. This modified assay method is referred to as ALFA (Aptamer Lateral-Flow Assay), read as "alpha".

Now, our goals are quite clear - to create an scFv-based LFIA test strip and an aptamer-based ALFA test strip for the detection of amanitins in mushrooms.

To make the product more approachable to the public, it must be made more presentable: the result needs to be easily read, preferably visible and colorful, so that everyone can understand and use it, therefore maximizing our product’s practicality. To achieve this, we use gold nanoparticles[4] to create stark color changes. When gathered together, these gold particles will show a red color. They can be engineered to be attracted to aptamers that bind to toxins, and upon this, they will become dispersed and show no color anymore. In this way, a positive result (toxin exists) will give no color changes, and a negative result will, in contrast, show a red color.

The possibilities of aptamers, scFvs, and other newly-arisen technologies range far and wide from merely toxin detection. Aptamers and scFvs can also bind to a wide range of analytes. For instance, illegal drugs and can be applied to detection processes in both daily life and scientific research, against any possible target molecule that you could name. If we successfully produce a detection device with high accuracy and low costs, then it can be built upon, redesigned, and extended to other, more demanding areas, making far more contributions than just detecting toxins in mushrooms. This is the ultimate goal that we aim to achieve through this project - to explore new possibilities of synthetic biology.

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References