Team members: Katherine Wang, Winnie He, Adonie Dong, Ginny Fan, Chloe Ou, Max Chen, Alex Wang, Roger Wang, Jacky Xu, Regina Ru, Evangeline Liu, Jerry Xie, Lambert Wang, Mandy Liang, and Alice Zhen.
Team PI: Dr. Yeqing Zong
Team Instructor: Dr. Yiming Dong
Advisors: Diana Tan, Uranium Chen, Alexis Zeng, Harry Jiang
Our project, ShroomSweeper, improves the current methods of mushroom poison detection by improving antibodies and our improved detection assay, ALFA - Aptamer Lateral Flow Assay, "sweeps" the minefield of mushrooms for the mushroom pickers. We aim to make an easy, cheap, rapid and portable mushroom toxin detector.
Before we started to design our product, we investigated the current methods of mushroom toxin detection and our stakeholders' expectations of our product through interviews, questionnaires and a visit to the Guangdong Institute of Microbiology.
LCMS, a current method to detect amanitin. It is expensive, unportable and difficult to operate.
The feedback we got from our stakeholders.
Also, the researchers from the Guangdong Institute of Microbiology suggested that we start from detecting a common mushroom toxin - amatoxin, which has lead to 90% of all mushroom poisoning death cases.
Combining all these opinions and suggestions from stakeholders, we decided to develop a rapid, easy, portable device for mushroom collectors, researchers and doctors to detect poisonous mushrooms, as well as the type of toxin they contain.
As one of the current methods for detecting amatoxins requires antibodies, and which has many limitations and disadvantages, we decided to first improve the antibodies used. scFv (single-chain fragment variable) is the variable fragment of an antibody connected by a linker, having the same or higher affinity towards antigens, and can be produced in bacteria instead of mammal cells or mammals.
Fig.1 The sketch map shows the basic structure of scFv.
scFv has a better binding affinity, higher stability, and lower cost as well as many other advantages compare with the traditional antibodies. We discovered an existing anti ɑ-amanitin scFv, which we have used as our detection molecule. And we have constructed a series of expression vectors to express the scFv in E.coli BL21(DE3).
Fig.2 The plasmid map of scFv expression vector.
We have optimized the production of scFv by linking a pelB tag to allow secretion to the periplasm, and used arginine extraction method to extract scFv. We also improved the culture conditions for scFv.
Fig.3 The SDS-PAGE shows different results of scFv expression in BL21(DE3) after arginine extraction.
We have tested the function of scFv by ic-ELISA(indirect competitive ELISA). The results showed that the scFv has a relatively low IC50(13.27ng/ml), meaning it has high affinity and sensitivity towards α-amanitin and has potential to test the presence of toxins in various kinds of samples, thus further developments like ELISA kit and colloidal gold test strips could be done to our scFv.
Fig.4 The result we collected from ic-ELISA, and we calculated the IC 50 of the scFv.
However, protein production in E.coli is not that cost-efficient and scFv still has some limitations; ic-ELISA and regular ELISA are both very time-consuming and requires laboratory apparatus, which are not what our project finally aims for. We need a better ligand, and a better method. Please click on our "Aptamer" part.
To make use of aptamers, we first verified the binding affinity of aptamers for α-amanitin from previous literatures, which we used for our hardware demonstration.
Fig.1 Structure of the aptamer that we found in previous literature
Fig.2 Result of testing the affinity of Best1 aptamer against α-amanitin.
Fig.3 The SELEX Procedure
Fig.4 The spectrum used to verify the conjugaton of amanitin and BSA
Apt1: 5'- CATGCTTCCCCAGGGAGATGTAGCGTCTGAAGCCGTTT
Apt2: 5'- CATGCTTCCCCAGGGAGATGGCCCGGGGTAACGTAGCC
Fig.5 The ELONA of our Apt1 and Apt2 toward β-amanitin.
The first model aims to determine the dissociation constant between amatoxin and its aptamer obtained from previous literature, as well as use this constant to determine the best aptamer concentration for competitive test on the test strip. We attempt to use the basic electrophoresis approach to determine the dissociation constant, in which the fluorescence of DNA bands could quantify the concentration of aptamer-target complex and free aptamer respectively.
Fig.1 Fitting curve for determining dissociation constant by varying aptamer concentration
Then, this value is used for the competitive test between immobilized toxin and free toxin, in which the aptamer concentration is covariate of total percentage of bound aptamer. At lower aptamer concentrationm, the dynamic range is more narrow so that it's more sensitive to toxin and allows a lower LOD.
A diffusion-convection-reaction partial differential equation was constructed with an initial condition and two boundary conditions. Upon solving with a set of hypothetical values for the parameters of the model, we obtained the following 3D plot for the concentration of the aptamer-toxin in sample conjugate at given positions on the test strip at different times:
We arrive at the conclusion that there exists an optimal test line position, where the aptamer-toxin equilibrium has completed to a certain extent, and the time is minimized to reduce the test time. We refer back to this conclusion when we are designing our test strip hardware, where an appropriate amount of space will be left between the starting position and the test line.
To meet the requirements of our stakeholders, we aim to develop hardware for amatoxin detection that meets their demands. Our original design is inspired by the traditional construction of LFIA (Lateral Flow ImmunoAssay), called ALFA(aptamer lateral flow assay). Different from the sandwich assay used in pregnancy test kit, we selected a competitive test principle since amatoxin is small in size for enough binding sites at upper and lower surface.
However, after contacting doctor Ying Yang, who deals with mushroom poisoning, we innovated another construction, incorporating a signal amplification method to amplify aptamer to lower the limit of detection as she suggested. Among all, we selected RPA(Recombinase Polymerase Amplification) for it can work under an isothermal environment. Through this methodology, we detect amplicon of toxin bound aptamer on test line instead. The limit of detection is also improved through competitive test, shown in the graph below.
We also create a manual in 4 languages to demonstrate the operation of hardware in wild circumstances as well as in clinical situation.
Our prototype of toolbox is tested by volunteer trial testers, who commented highly on our design. In the foreseeable future, we aim to industrialize our product and further decrease price, ensuring its popularity among our stakeholders.
In conclusion, we succeeded in designing an easy-to-operate, rapid (detection time within minutes), cheap (less than $15, much cheaper than current devices) and portable(small test strip) mushroom toxin detector. In the foreseeable future, we aim to industrialize our product and further decrease price, ensuring its popularity among our stakeholders.
In addition, we contacted professionals and experts in different fields and they provided us with useful suggestions throughout the project to help us improve. We also attended the Conference of China iGEMer Community and presented our project to the other attendees and judges, in which we won the two awards - Best Presentation and Best Poster as the only high school team among the winners.
Dr. Candace Bever provided us with suggestions about how to deal with amatoxins safely and helped us contact Professor Yanru Wang to create amanitin-BSA conjugates for our experiment.
Dr. Ciara O'Sullivan and Dr. Miriam Jauset-Rubio gave us precious suggestion on experiments, hardware design and models.
Dr. Wojciech Strzałka suggested that it will be worthy for us to select proper aptamers for beta-amanitin since most of the recent researches are based on alpha-amanitin.
Dr. Qinqin Han provided suggestions that helped us to improve our experimental method used in ELONA and Dot blot.
iGEM Team EPFL 2019 provided us with useful suggestions on the method of Recombinase Polymerase Amplification, hardware design and human practice activities.
Dr. Ying Yang from Shenzhen Hospital of Peking University provided detailed explanation of the current situation on treating the mushroom-poisoned patients.
Professor Wangqiu Deng offered us a visit to the Guangdong Institute of Microbiology, and provided suggestions for our hardware design as well as the organization of a trip to Baiyun Mountain for us to gain better understanding of the current situation.
Thanks to the mushroom collectors who was willing to chat with us, and filling out our questionnaires.
Thanks to all people who filled out our questionnaires on SynBio and mushroom poisoning.
Thanks to the sponsors and teachers in Shenzhen Bluepha Lab for guiding us through our project and providing laboratory for us to work in.
- Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M. “ScFv antibody: Principles and clinical application”. Clin Dev Immunol.
- Kiprijanov, Sergej. “Bacterial Expression, Purification, and Characterization of Single-Chain Antibodies”.
- He K, Mao Q, Zang X, Zhang Y, Li H, Zhang D. “Production of a broad-specificity monoclonal antibody and application as a receptor to detection amatoxins in mushroom”. Biologicals.
- Zhang X, He K, Zhao R, Feng T, Wei D. “Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors”. Food Anal Methods.
- Jauset-Rubio M, Svobodová M, Mairal T, et al. “Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers”. Anal Chem.
- Lobato IM, O’Sullivan CK. “Recombinase polymerase amplification: Basics, applications and recent advances”. TrAC - Trends Anal Chem.
- Li JJ, Fang X, Tan W. “Molecular aptamer beacons for real-time protein recognition”. Biochem Biophys Res Commun.
- Chen A, Yang S. “Replacing antibodies with aptamers in lateral flow immunoassay”. Biosens Bioelectron.
- Han Q, Xia X, Jing L, et al. “Selection and characterization of DNA aptamer specially targeting α-amanitin in wild mushrooms”. SDRP J Food Sci Technol.
- Hidding J. “A therapeutic battle: Antibodies vs. Aptamers”. Nanosci master Progr.
- Hu T, Wessels H, Fischer C, et al. “Aptamers Using Magnetic Separation and BEAMing”. Anal Chem.
- Muszyńska K, Ostrowska D, Bartnicki F, et al. “Selection and analysis of a DNA aptamer binding α-amanitin from Amanita pphalloides”. Acta Biochim Pol.
- Qian, S., & Bau, H. H. “A mathematical model of lateral flow bioreactions applied to sandwich assays”. Analytical Biochemistry.
- Ragavendar, M. S., & Anmol, C. M. “A mathematical model to predict the optimal test line location and sample volume for lateral flow immunoassays”. Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2012.
- Hoare, Sam R.J. “Receptor binding kinetics equations: Derivation using the Laplace transform method”. Journal of Pharmacological and Toxicological Methods.
- Levine HA, Nilsen-Hamilton M. “A mathematical analysis of SELEX”. Comput Biol Chem. Koussa MA, Halvorsen K, Ward A, Wong WP. “DNA nanoswitches: a quantitative platform for gel-based biomolecular interaction analysis”. Nat Methods.
- Koussa MA, Halvorsen K, Ward A, Wong WP. “DNA nanoswitches: a quantitative platform for gel-based biomolecular interaction analysis”. Nat Methods.
- Thevendran R, Navien TN, Meng X, Wen K, Lin Q, Sarah S, Tang TH, Citartan M. ”Mathematical approaches in estimating aptamer-target binding affinity”. Anal Biochem.
- Bever CS, Hnasko RM, Cheng LW, Stanker LH. “A rapid extraction method combined with a monoclonal antibody-based immunoassay for the detection of amatoxins”. Toxins (Basel).
- Bever CS, Swanson KD, Hamelin EI, et al. “Rapid, sensitive, and accurate point-of-care detection of lethal amatoxins in urine”. Toxins (Basel).
- Chen, Y., Yip, L., Corriden, R., Insel, P. A., & Junger, W. G. “Cell surface expression of A3 and A2A adenosine receptors defines the response of PMN to hypertonic saline”. Shock, 26(Supplement 1), 29.
- Yu Q, Zhao Q, Wang S, et al. “Development of a lateral flow aptamer assay strip for facile identification of theranostic exosomes isolated from human lung carcinoma cells”. Anal Biochem.
- Narongchai S, Narongchai P. “Deactivation Study of α-Amanitin Toxicity in Poisonous Amanita spp. Mushrooms by the Common Substances In Vitro”. J Forensic Res.