Team:GreatBay SCIE/Hardware
"We cannot send every mushroom sample to detection centers to find out whether they are toxic or not. It is too inconvenient and expensive," a mushroom collector said.
"There is not a single device in the academic field now that can detect mushroom toxins in a rapid, cheap and convenient way, " said Dr. Deng, the mycologist from Guangdong Institute of Microbiology who we visited for our Human Practices activities.
What our stakeholders said matches our research findings, which show that existing methods of toxin detection are too expensive and complicate as shown in the table below:
Current methods of amanitin detection. [1]
Therefore, we intend to develop an easy, cheap, rapid, and portable mushroom toxin detector that can benefit our stakeholders. In order to be clear about our stakeholders' expectations about the hardware, we conduct interviews and questionnaires to find out their needs and wants.
Visit our Integrated Human Practices page >
These are some of the results from our questionnaires.
The priority of qualities in detection for Mushroom Collectors
The priority of qualities in detection for researchers
The rank of the respondents' priority (from high to low) is accuracy - portability - detection time - price - safety - appearance. Thus, when we are designing our product, we try to make sure that our hardware can identify the toxic mushrooms correctly. Also, we are thinking about how to minimize the weight and size of the detector to achieve portability.
The time and cost that our stakeholder accepts
Moreover, the results show that our stakeholders may prefer a detection time within 30 minutes and a price below 100 RMB (about $15 USD).
After making clear about our stakeholders' needs and wants, we begin to design our project in detail!
Our original design is inspired by the traditional construction of LFIA (Lateral Flow ImmunoAssay). Different from the sandwich assay used in the pregnancy test kit, we selected a competitive test principle since amatoxins are small in size for enough binding sites at the upper and lower surface. Basically, the sample is extracted by shaking with extraction buffer (mainly water) provided in the toolbox, which is added to the sample pad. Then, the toxin flows through the NC membrane and bind the aptamer-functionalized Au particle in the conjugate release pad. The competition occurs at the test line which is composed of immobilized amatoxins that competes with aptamers for the free toxin in the sample. As the toxin concentration in the sample increases, fewer aptamers would be available to bind the test line toxin and this is displayed as disappearing of the red color of Au nanoparticle at the test line, and Vice versa. The control line is complementary to 20 base pairs at 3’ end of aptamers on Au particle, which always binds enough aptamers functioned Au particle to report a red color to display a normal function of the construction.
ALFA test strip design 1
We conducted a pilot test on Au particles' reporting on NC membrane, albeit without lateral flow. Basically, the toxin is conjugated with BSA to strengthen immobilization on the membrane, mixed with CBS (carbonate-bicarbonate saline buffer). The aptamer functioned Au nanoparticle successfully reports a red color in the sample loading region, specifically to alpha amatoxin, indicating a possible binding followed by aggregation of Au particle.
Au Nanoparticle conjugation.
However, after contacting one of our stakeholders who is a doctor dealing with mushroom poisoning (see in Integrated Human Practices). She suggests that the limit of detection should be further reduced in order to detect the toxin in blood and urine samples under clinical situations. Hence, we innovated another construction, incorporating the signal amplification method of DNA amplification on aptamers. Among all the amplification methods, we selected RPA(recombinase polymerase amplification" since it's isothermal with the optimum working temperature at room temperature. Also, its amplification time is within 10 minutes, which is much more rapid than other amplification methods such as PCR. This matches the expectations from our stakeholders about a rapid mushroom toxin detector. More importantly, RPA can be done in outdoor conditions and only a few reagents are needed --- this meets the requirement of "portability" from our stakeholders.
In this design, the immobilization pad of toxin is prior to the test line, allowing a pre-detection competition and amplification of bound aptamers. Briefly, the sample would first meet the lyophilized aptamer on the conjugation pad. After the conjugation, the excess aptamer is left to bind the immobilized toxin just as in method 1 above. Here, instead of reporting directly at the immobilized pad, the aptamer is amplified and then flows to the test line, captured by a complementary strand of single-strand DNA. This strand is complementary to the tail added to aptamer during the amplification, in which we designed C3 spacer to obscure elongation and reserve the ssDNA tail, from our specific RPA primers. Another tail reserved is complementary to the reporter probe(also an ssDNA) adsorped at the surface of Au nanoparticle, and thus the aptamer amplicons are sandwiched between Au particle and complementary strand, reporting a red color. If the free toxin in the sample drains the aptamer, no amplicon would form and there's an absence of red color, indicating a positive result, and vice versa.
ALFA test strip design 2
Competitive test with aptamer amplification is done in the lab to prove the utility of this novel concept, by competition of toxin on magnetic beads and free toxin in the sample for aptamer binding. See more on our Results page.
Originally, we aimed to separate the competitive test and amplification with the lateral flow of aptamer amplicon according to previous literature yet after we showed Dr. Deng (a mycologist from Guangdong Institute of Microbiology who needs to collect wild mushrooms) how to use our hardware, she mentioned that it would be better if we could facilitate the entire design on a single component, using the microfluidic methodology to avoid cross-contamination.
Therefore, we adopted her suggestions and utilized microfluidic sliding chip methodology which is compatible with our lateral flow design, without significant change in our designing initiatives.
Overall there are 2 chambers alongside the lateral flow strip on the layer above the strip. Firstly, as the competition between the free toxin and the immobilized toxin is complete at the immobilized pad(5 minutes specified in the instruction), the user is instructed to slide the upper layer rightward, which allows a mass flow of water in the chamber on the left to flush aptamer-toxin complex directly to the absorbent pad at the end. Then, the user would be informed to further slide the upper layer leftward, adding the RPA-Au particle mixture to the pad from the chamber on the right. We have found a kit combining RPA and Au reporting, which allows a concurrent binding of reporter probe on Au particle with tailed amplicon. Then the user would wait for about 10 minutes without any further motion of the external chips for the result.
We 3D printed the skeleton of the sliding chip to test the microfluidic motion resulted from sliding as mentioned in the instruction. As expected, the sidetrack successfully flushes the mixture to the absorbent pad and the RPA-Au particle mixture is transferred to the immobilization pad subsequently.
Sliding Chip Design Model.
This arrangement is not only user-friendly but also feasible due to the separation of reagents, thus avoiding cross-contamination and reducing the background noise of the signal. The prototype of an easy, rapid, cheap, and portable mushroom toxin is made, but we still need to receive feedback from our potential users to further improve our hardware. Then, we asked the mushroom collectors for their feedback about our product. They also considered it to be easy to operate and useful, however, they suggested that it would be even better if we could have a toolbox to put all the devices together for enhancing portability. Therefore, we made a toolbox as shown below The toolbox contains essential and basic tools to facilitate the detection, including a flask for toxin extraction, extraction buffer (water) in bottles, syringe to add extract to the sample pad, 2 spare EP tube, and a disposable test kit. This device portable and the instruction manual mentioned below would be printed and provided to users, too, especially the safety precaution:
Amatoxin Detection
1. Tear open the plastic film wrapped outside the test strip. The head of the test strip is the end with the red logo and at the tail of the strip is a hole for adding samples. For new test strips, two red lines should be present in the results window.
2. Cut off a small part of the mushroom with a small blade and place it into the vial containing liquid solution. Shake the bottle gently and mix well.
3. After mixing, add drops of the liquid with a syringe into the hole of the test strip and let stand for about 5 minutes.
4. After 5 minutes, pull the chip on the side of the test strip and slide it to the left and right once each. Continue to let stand for about 10 minutes.
5. After 10 minutes, if only one red line is shown in the results window (the color of the other red line fades), the mushroom is toxic and should be destroyed and discarded immediately. If the lines do not change color, then the mushroom does not contain amanitin.
Disposal after use
1. The test strip, vial, and syringe should be tightly sealed in the plastic bag within the package box. Please do not carelessly discard any items in this test kit.
2. If garbage sorting services are available, please sort the remainder of this product into the "Harmful Garbage" container. If no such conditions apply, please dispose of it as ordinary plastic garbage.
Caution
- This product is for single use. Do not use the test strip multiple times.
- This product contains small parts and potentially harmful chemicals. Do not let children approach this product to avoid ingestion/suffocation/poisoning.
Multilingual versions of our user manual.
Click the corresponding links to view the multilingual versions of the user's manual: EN_USZH_CNFR_FRJA_JP
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A diagram demonstrating how the sliding chip could be slided left and right.
Pictures of our test kit.
We also invited a volunteer tester to try out our product.
A volunteer trying to use ShroomSweeper.
In the foreseeable future, our ALFA product could be industrialized and produced at an extremely low cost, since the components of the test strip would be purchased in bulk and produced by ourselves without extra cost due to intermediary retailers. This is essential for mushroomers according to our previous interview (their expected cost is around 100 RMB, approximately equal to 13 dollars) since they take the importance of the balance between the market value of edible mushrooms and the cost of detection. All in all, the predicted cost of our hardware now is even much lower than state of art methods, with merely 13.59 USD per kit thus edged in the market competition and has the potential to be popular.
Overall, the feasibility of ALFA is proved by convergent evidence, and feedback from different stakeholders as well as voluntary testers. Shroomsweeper ultimately solves the problem of amatoxin detection using synthetic biology, however, we didn't develop a quantitative system to test the toxin in a standardized way. In the near future, we could develop software to quantify the color intensity in the test line, and thus use a calibrated curve to find toxin concentration in the sample. Moreover, the design of our hardware might inspire future research in multiple disciplines to detect amatoxin in different contexts(e.g blood or urine sample) and detect brand new molecules sharing similarities, like phalloidin, ricin, etc.
Sources:
[1] Shanghai Simp Bio-Science Co.，Ltd.