ShroomSweeper GreatBay_SCIE

Our project is based on the target-specific binding affinity of an oligonucleotide ligand (aptamer), selected by the iterative SELEX（Systematic Evolution of Ligands by EXponential enrichment) procedure from previous literature. We thus develop an ALFA (Aptamer Lateral-Flow Assay) test strip as a portable, rapid toolbox for amatoxin detection. The incorporation of RPA (Recombinase Polymerase Amplification) on the test strip could amplify the signal under room temperature and isothermally within 10 minutes. Finally, the presence of amatoxin above the limit of detection is demonstrated by a decolorized test line following the principle of a competitive assay, in which the red-blue color shift of the Au nanoparticle plays a vital role in reporting the results of the test.

Verification of Aptamer-Toxin Binding

Overall, the amatoxin we focused on is a cyclopeptide, which is much smaller than an 80bp aptamer which is used as a binding ligand. Ideally, the loop formed by the folding of the oligonucleotide sequence encloses and binds selectively with the aptamer. Hence, we attempt to verify this concept by examining an existing aptamer (named Best 1 and Best 2, selected by SELEX in previous literature[1]), specific to α-amanitin.

Fig.1 The secondary structures of the Best 1 and Best 2 aptamers

Based on the principle elaborated in "DNA Nano switches: a quantitative platform for gel-based biomolecular interaction analysis" [2], we planned an experiment to determine the association of the aptamer and the toxin by electrophoresis. To explain, it is predicted that the aptamer bound with toxin would be slightly lagged compared to the free aptamer, demonstrating 2 distinct bands on the agarose gel.

Fig.2 The two distinctive bands in the gel electrophoresis of free aptamers and bound aptamers

However, according to the experimental results, we found out the difference is insignificant due to the low resolution of the agarose gel, and the difference is yet to unveil even with the TBE-PAGE gel. It is probably owing to the size of amatoxin, which hardly leads to a conformational change of aptamer that significantly affects the speed of electrophoresis.

Thereafter, we searched for novel techniques enabling the verification of binding of the aptamer and the amanitins. We found methods based on the principles of ELISA, but are applicable with aptamers: ELONA (Enzyme-Linked OligoNucleotide Assay) and Dot Blot [3]. The toxin is initially immobilized on either a 96-well plate or a nitrocellulose membrane by electrostatic forces, then a biotin-labeled aptamer is added for the association. Any unbound aptamer is washed away. If the aptamer is bound to the immobilized toxins, then the subsequent addition of the streptavidin-HRP complex would further be immobilized onto the aptamer, and hence, onto the 96-well plate or the nitrocellulose membrane. Finally, HRP catalyzes the reaction of the TMB substrate added, to produce an observable color change.

After communicating with Dr. Han, the author of one of the previously-mentioned literature, we are instructed to follow the guidelines of ELONA and dot blot rigorously and thus plan an experiment verifying the aptamer-toxin association and comparing them with that using other polypeptides (β-amanitin and BSA) to demonstrate specificity.

The SELEX procedure

Apart from the verification and repetition of the state of art, we ambitiously intended to develop brand new aptamers for different subtypes of amanitins, given that different mushroom species under the Amanita genus may contain different subtypes of amanitins. Furthermore, this method can be easily adapted to other small molecules, so the test cassette could be adapted for a wide range of assays, aiming to detect various chemical substances.

According to the literature in which we obtained the sequence of α-amatoxin aptamer, we plan to conduct an iterative SELEX process for aptamer identification of β-amanitin. Firstly, a random pool of ssDNA is generated in the format "head-N40-tail", where the head and tail regions are designed for future amplification and the N(40) is randomized to a 4^40 chance of different sequences. The pool is incubated with the toxin and those who could specifically bind to β-amanitin are retained, amplified, and used as the pool for the next round.

Fig.3 The SELEX Procedure[4]

Fig.4 The BEAMing amplification procedure to amplify the selected ssDNA in each round of SELEX

Theoretically, we could replicate the exact chemical method mentioned in the literature, immobilizing the toxin on carboxylic magnetic beads [5], which is more suitable for small molecules compared to resin beads.

Fig.5 The chemical structure of amanitins

Fig.6 How the toxin is conjugated with the protein

Nevertheless, the alpha amatoxin used in the work was modified by a C4 spacer and NH2 group to form an amide bond with the carboxylic acid group. The original NH2 group lacking a primary amine group could not be utilized. Hence, to achieve the purpose of coupling toxin with magnetic beads, a conjugation of amatoxin to another protein is necessary. Based on the EDC/NHS carboxylic group activating principle[6], we plan to activate the carboxylic group presented only in β amanitin first, which is then conjugated with amine group in BSA, and finally coupled with magnetic beads.

Therefore, we attempt to have a 15-rounded SELEX with first 4 rounds of counterselection (to screen out the aptamers that bind specifically with BSA, in order to improve specificity towards toxin), monitoring the quality of the product by control PCR.

Preparation of Hardware

Our original design is referred to as ALFA (Aptamer Lateral Flow Assay), in which the aptamer specific to the amatoxins is conjugated with reporting Au nanoparticles. Inspired by the LFIA test strip construction, we primarily designed our test strip with four components: the sample pad for the entry of the mushroom extract containing amatoxin, the conjugate release pad for releasing the Au-nanoparticle-conjugated aptamers into the sample liquid, the main pad containing the test line and the control line, and finally the absorption pad to discard any excess reagents and sample liquids. As the sample fluid flows through, the toxin in the sample and the toxin immobilized on the test line compete for the aptamer binding. The more toxin is present in the sample, the less aptamer is left to bind with the immobilized toxin, which could be characterized by the absence of the red color caused by the aggregation of Au nanoparticles on the aptamers bound to the test line, and vice versa. The control line contains an ssDNA sequence which is complementary to that of the aptamer, allowing the aptamer and the Au particle to be able to bind to it at all times, verifying the functionality of the test strip.

Fig.7 ALFA test strip design 1

To further amplify the signal of detection, achieving a lower limit of detection to serve our stakeholders and potential users better, we incorporated RPA (Recombinase Polymerase Amplification) into our test and constructed an alternative design, involving a competitive assay. The aptamer is exposed simultaneously to both the sample liquid (which may contain free toxin molecules) and immobilized toxin molecules. After some time has elapsed for binding, the immobilized phase is removed, the aptamers are eluted from the free toxins, and are amplified through RPA. Through this competitive binding, elusion, and amplification, the presence of toxin in the sample could be sensitively indicated by the presence of amplified aptamers. Therefore, all we need to do next is to find a way to detect the presence of these aptamers in the amplification product.

Through specially-designed RPA primers, we could generate an extra sequence at the ends of the aptamer. At one end, we incorporate a poly-A strand, which could bind to the poly T strands on the test line, immobilizing the aptamers on the line. Also, we generate another sequence on the other end, which is complementary to a short ssDNA sequence connected to the Au nanoparticle by thiolate bonding. Hence, when the Au nanoparticle reporting probe flows through the test line, the gold particles would be captured by the immobilized aptamers, causing aggregation and hence revealing an observable red color [8]. The control line contains an ssDNA strand which is also complementary to the sequence conjugated to the Au reporting probe and should give a red color (through the same mechanism) as long as the reporting probe and the test strip is functional.

Fig.8 ALFA test strip design 2: Incorporating competitive assay and RPA

Before the construction of test strip components, the concept of RPA following a competitive assay should be verified, which is to carry out an aptamer-toxin binding test with a part of the toxin immobilized on the magnetic beads. The fluorescence of the band after RPA is recorded, and the ultimate aim should be finding the proper concentration of aptamer for the sensitive detection of toxin in various range. This part is specified in our model on the disassociation constant of aptamer-toxin binding.

scFv:

Another approach we have done is the production of scFv. The sequence comes from the scFv previously constructed by Dr.Dong[8]. There are various benefits of using scFv instead of antibodies. Firstly, scFvs are recombinant proteins and it is to express their sequences and produce them in E.coli , which can greatly increase the productivity and decrease the costs. Secondly, it is shown in Dr. Dong's work that the scFv they have constructed has a 10 fold greater sensitivity than the original antibody. Besides, the standard method of detecting molecules is by using antibodies, and the technology of producing antibodies is much maturer than the selection of aptamers, so it will be securer to make a working test strip with scFvs,even though it may have a relatively low sensitivity than our own ALFA test strip. We want to test the specificity, sensitivity and productivity of the antibody to construct our LFIA detecting α-amanitin. Our test strip design will be based on Dr. Bever's work[1], to which we will make some modifications [10] .

Fig.9 The cross reactivity and IC_50 data of the scFv[9]

Fig.10 The way we do the test[10]

Fig.11 The competitive test strip[1]

Go to results >

References

1. Muszyńska K, Ostrowska D, Bartnicki F, et al. Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides. Acta Biochim Pol. 2017;64(3):401-406. doi:10.18388/abp.2017_1615

2. Koussa MA, Halvorsen K, Ward A, Wong WP. DNA nanoswitches: A quantitative platform for gel-based biomolecular interaction analysis. Nat Methods. 2015;12(2):123-126. doi:10.1038/nmeth.3209

3. 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. 2018;3(6):497-508. doi:10.25177/JFST.3.6.2

4. https://www.creative-biogene.com/Services/Aptamers/Technology-Platforms.html

5. Hu T, Wessels H, Fischer C, et al. Aptamers Using Magnetic Separation and BEAMing. Anal Chem. 2014;86:10940-10947. doi:10.1021/ac503261b

6. Bever CS, Barnych B, Hnasko R, Cheng LW, Stanker LH. A new conjugation method used for the development of an immunoassay for the detection of amanitin, a deadly mushroom toxin. Toxins (Basel). 2018;10(7):11. doi:10.3390/toxins10070265

7. 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. 2016;88(21):10701-10709. doi:10.1021/acs.analchem.6b03256

8. Song Q, Qi X, Jia H, et al. Invader assisted enzyme-linked immunosorbent assay for colorimetric detection of disease biomarkers using oligonucleotide probe-modified gold nanoparticles. J Biomed Nanotechnol. 2016;12(4):831-839. doi:10.1166/jbn.2016.2257

9. 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. 2016. doi:10.1007/s12161-016-0509-3