Proof of concept
1. The results of aptamer based fluorescence method
We used aptamer fluorimetry separately to verify the specificity and affinity (binding capacity) of aptamer for the targets (3O-C12-HSL and C4-HSL) in the literature.
SYBR Green Ⅰ fluorescence method
We selected one aptamer each from the literature that specifically binds 3O-C12-HSL and C4-HSL. The excitation and quenching of SG fluorescence was used to verifying whether the aptamer could specifically bind small molecules. This method has been used several times as a label-free test and is based on the principle that SG acts as a DNA fluorescent dye that does not carry a fluorescent signal of its own (Abraham et al., 2018). When bound to the aptamer, it fluoresces because it is embedded in the secondary structure of the DNA. Thus when aptamer is bound to the SG dye, green fluorescence is produced when stimulated by excitation light（Excitation Wavelength 485 nm Emission Wavelength 535 nm）. However, when the target small molecule is added to the system, the specific binding of the small molecule to the aptamer results in competition with the SG for the binding site, and as a result, the fluorescence value of the system decreases.
In this project, the specificity of the aptamer to bind 3O-C12-HSL and C4-HSL, as screened in the literature, was first verified using this method. The total system was supplemented with 100 nM aptamer, 1 x SG and various concentrations of small molecules (0 nM, 100 nM, 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM and 2000 nM respectively). The fluorescence intensity of the buffers was also measured. The relative levels of fluorescence of the systems were observed based on this. (Two small molecules of aptamer, 3O-C12-HSL, and C4-HSL, were performed separately for this experiment).
The data we obtained showed a better indication of fluorescence quenching, indicating that both aptamers can bind small molecules better(Fig 1)
Fig 1. The fluorescence quenching effect by C4-HSL(left) and 3O-C12-HSL(right）
With the two experiments described above, we have successfully demonstrated the binding affinity and specificity of the chosen is the ligand.
FAM fluorescence quenching
TThis experiment uses fluorescein (FAM) modified at the 5'-end of the aptamer, along with a segment of ssDNA complementary to the aptamer with a Blackhole quencher (BHQ1) modified at the 3'-end. The complementary sequence is close to the 5' end of the aptamer, and the complementary sequence and the aptamer will bind together to form a duplex structure due to complementary pairing, which will result in the proximity of the FAM and BHQ, thus producing a loss of fluorescence. However, if there are small molecules in the environment that can bind specifically to the aptamer, the aptamer, due to the binding of the small molecules, changes its structure and is no longer in close proximity to the BHQ, producing a fluorescence relighting phenomenon.
However, as the length of the complementary single-stranded DNA sequence is something that needs to be explored, if it is fully complementary, the structure formed by the aptamer and the complementary sequence may be more stable, resulting in small molecules not being able to compete the anti-aptamer off the top of the aptamer. However, if it is too short, it may lead to poor quenching or other problems.
We performed FAM experiments using the redesigned aptamer and the results of the experiments showed good quenching and relighting as shown below (C4-HSL results on the left and 3O-C12-HSL results on the right.
Fig 2. The fluorescence qunenching and recovery of FAM modified aptamers caused by anti-aptamers and AHL>
2. Gold nanoparticle-aptamer coupling experiment
Nanogold is a very common marker with a wide range of applications in bioassays, as a chromogenic agent that binds well to the ligand and does not affect its properties. Based on this quality, nanogold is very widely used in qualitative or semi-quantitative rapid immunoassay methods. At a suitable concentration of sodium chloride solution, we have successfully produced colloidal gold-ligand couples for the next step in the preparation of test strips.
To verify whether the coupling of colloidal gold and aptamer was successful, we prepared 4 tubes of 200 μL of colloidal gold solution and added 4 μL of activated C4-HSL aptamer to tubes 3 as the experimental group, while tubes 1 served as the control group. We added 8.5 μL of sodium chloride to each tube in 17 separate doses. sodium, 0.5 μL at a time, making a final concentration of 80 mM. After shaking and mixing, the reaction was carried out at room temperature for 2 h and the solution was observed for agglomeration. If there is sufficient aptamer in the gold-labeled aptamer coupling solution to label the colloidal gold, the gold-labeled aptamer coupling solution will continue to remain red and stable after the addition of a high concentration of the salt solution, i.e. the aptamer is optimally labeled and the coupling is successful; whereas unlabelled colloidal gold solution or incompletely labeled colloidal gold solution will undergo agglomeration under the action of high salt ions and the colloidal gold particles will completely agglomerate to the bottom of the centrifuge tube.
Fig 3. The conjugation results of gold nanoparticles and aptamer
As can be clearly seen from the graph above, the experimental group remained stable in red, indicating that the optimal labeling amount of the aptamer was reached and the coupling was successful.
3.Aptamer-based lateral flow strip
The sequnces were:
- C4-HSL-apt: 5’-Thiol-CGCTGCCCCCTGTCCTGGGTTAGCTAGCGAGAGCG AAA AAA AAA AAA AAA AAA AA-3'
- Test probe: 5'-Biotin-（CH2)6-CGGTCACTAGTACACCGCACCAGAAGCGGGCCCCG-3'
- Control probe: 5'-Biotin-TTT TTT TTT TTT TTT TTT TT-3'
The principle of the designed aptamer-based strip method was based on the competitive reaction between the DNA probe 1 (test line) and the target C4-HSL molecule to combine with aptamers. If target C4-HSL molecule was in the detection solution, it would combine with aptamer–GNP probe, decreasing the aptamer–GNP that could hybridize to the DNA probe1 in the test line causing the red color intensity to become weaker. In other words, the more target C4-HSL molecule in the solution, the weaker intensity in the test line; Regardless of the presence of target C4-HSL molecule in the detection solution, the aptamer–GNP probe would definitely hybridize with DNA probe 2 in the control line, ensuring the validity of the detection. We have successfully verified that Probe 2 can successfully bind to the test paper.
If Probe 2 successfully binds to the test paper, the control line will appear red regardless of the presence or absence of the target small molecule. Based on this, we used pure water instead of a solution containing the target small molecule, with the following results.
Fig 4. The successfully construction of the control line
As can be seen in the above diagram, the control line successfully shows red, however, the test line should also show color under a negative result, but the test line failed to show color.
Next, we consulted with doc Zong, the instructor of the Greatbay_SCIE team, he suggested adding a C6 spacer between the biotin and the sequence to separate the modified group from the sequence and avoid affecting the function of the sequence, better able to unfold and combine with aptamer, so our modified sequence is 5'-Biotin-(CH2)6-CGGTCACTAGTACACCGCACCAGAAGCGGGCCCCG-3'. This allows us to finally successfully produce a test strip that shows color in both lines under negative conditions.
we also successfully verified that the test strips give a positive result when using a solution containing C4-HSL, as stated above, the more target C4-HSL molecule in the solution, the weaker intensity in the test line, so finally The test strip should give a colorless result for the test line and a red result for the control line. If there are no small C4-HSL molecules in the solution, the test line will appear red.
The results of the experiment are shown below.
Fig 5. The negative result demonstrated by the strip
Fig 6. The positive result demonstrated by the strip
Based on the results, it can be seen that both test strips successfully verified the previous theories and guesses, indicating that our test strips can successfully detect C4-HSL
4. Biosensor based on C4-HSL receiver cells
At the beginning of the project, GCGS planned to build a device to detect C4-HSL by starting with transcription factors and test strips. However, due to the time problem and the epidemic, GCGS finally succeeded in constructing test strips only, but during the preliminary research and experimental method design, GCGS saw in the literature that an author had successfully constructed a strain that used GFP as a reporter gene to detect C4-HSL(figure 7), so by contacting the author of the article, GCGS obtained the strain, tested it after obtaining the bacteria, and found that the bacteria successfully detected C4-HSL in the growth environment. After successful testing, GCGS shared the bacterium with NDNF, which combined the bacterium with NDNF's project Hidro to create a transcription factor-based live bacterium detection system that can be used in direct contact with food to detect food spoilage.
Fig 7.The genetic circuit structures of C4-HSL receiver cells (Du et al.,2020)
For more detail about the Hidro system, please visit NDNF_China 2021 and the results of the GCGS test (left) and the final test (right) with the Hidro system are shown in the figure 8. And you can also see our Partnership.
Fig 8. The testing of C4-HSL receiver cells(left) and the biosensor combined with hydro system(right)
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