See our experiments page for the details of how we obtained these results and where we got the protocols. See measurement for a more detailed additional perspective on which findings are novel and their significance.
Quencher and Target binding Test (PFOA)
Results
We measured the KD of the PFOA
Aptamer PFOA_JYP_21 in
replication of an experiment cited in the literature.
Graphs:
We found the KD to be approximately 2 ⋅ 10−4M. A pdf printout of a notebook containing our raw readings and code for analysis (fitting Hill curves, etc) is available here.
Reflection
The change in fluorescence with increasing concentration of the target molecule indicates that the DNA binds its target. This shows that we can detect aptamer-target binding in our hands.
- The experiment went remarkably well for the first try. We observed expected trends in the fluorescence data.
- Our KD
was somewhat at variance with the originally reported 5.5 ⋅ 10−6M
- Our computed Kd value was one to two orders of magnitude higher than reported, which may have improved if we took time to optimize the experiment.
- PFOA was difficult to dissolve- required heat and shaking over a couple hours
- Next time we would perform the whole experiment in the lab with the plate reader, so we don’t have to move spaces and introduce fluctuations in temperature and light.
- Applications of this information
- Offers a simple platform for measuring binding affinity of an aptamer using fluorescence. Does require ordering a modified aptamer and optimizing a quencher strand, though they are inexpensive
- Aptamers can be used in a variety of ways in therapeutic, diagnostic, and basic research applications.
- Next steps: look into other methods for validating aptamer binding affinity.
Isothermal Titration Calorimetry (ITC) Experiment (progesterone):
Results
We measured the KD of the
progesterone aptamer P4G032 with ITC to be 50 nM.
Our results, as reported by the Nanoanalyze software:
| Measurement | Units | Value |
|---|---|---|
| Kd | M | 5.000 ⋅ 10−8 |
| ΔH | kJ/mol | −50 |
| Ka | M−1 | 2.000 ⋅ 107 |
| −TΔS | kJ/mol | 8.326 |
| ΔG | kJ/mol | −41.67 |
| ΔS | J/(mol ⋅ K) | −27.92 |
This differs slightly from the only value reported in literature to
our knowledge, of 9.63 ± 3.12 nM, however
both values are in the tens of nanomolar range. This guaruntees a
certain level of sensitivity of the bare P4G03 aptamer to
progesterone.
Reflection
- These results will allow us to have confidence that an aptamer sensor using this specific aptamer sensor can sensitively be able to detect concentrations of progesterone of greater than 50 nM.
- The measurement difference between our KD value and the single value we could find in literature could in part be due to differences in methodology, we measured KD using ITC, while in the literature the reported measurement comes through fluoresence.
- The main point that could be improved from this experiment would be the development of an aptamer that has a lower dissociation constant (Kd) than 50 nM.
- We learned from these experiences how important it is to be detail focused when determining specificity. This is because the buffers used in each ITC sample must be equivalent in concentration to be compared against one another. This means one must be as exact as possible to determine that equivalent buffers are used in all of the samples. To verify the KD, one could use the Electrophoretic Mobility Shift Assays (EMSA).
- The information collected using the ITC allows other IGEM teams to immediately have a dissociation constant for this progesterone aptamer. This will give them the ability to immediately manipulate the aptamer without having to determine the KD to then manipulate the aptamer. This information allows our stakeholders to have confidence that we will be able to build an aptamer sensor with specificity to concentrations in the nanomolar range. The next steps are to build a sensor and use experimentally determined KD to be able to calculate concentration based on the electrical signal. We need to test this device to receive cosistent measurements of concentrations of the aptamer binding.
Circular Dichroism (CD) experiments (progesterone):
Introduction
Download all CD data from Experiment 1 as a .csv
here.
Download all CD data from Experiment 1 as a .csv
here.
Download all CD data from Experiment 1 as a .csv
here.
Experiment 1 – May 14: Finding Working Concentrations
Rationale
Before collecting real data, we needed to make sure the aptamer and progesterone concentrations produced readable signals without baseline drift or background noise.
Results
We collected clean, usable spectra across ratios ranging from 1:2 to 2:1 progesterone to aptamer. The data showed consistent ellipticity between 240 and 280 nm, confirming that the setup and concentrations were in the right range. One of the best outcomes of this test was that it helped us confirm that our experimental design worked well and set the stage for later runs.
Figure 1. CD spectra showing aptamer response to progesterone across ratios from 1:2 to 2:1.
Experiment 2 – June 6: Confirming Conformational Change
Rationale
Once we had the basic setup figured out, our goal was to confirm that the P4G03 aptamer actually changes shape when it binds to progesterone and to see how that change depends on concentration.
Results
We tested 0.2:1, 1:1, 5:1, 10:1, 20:1, and 40:1 progesterone: aptamer ratios. The signal got stronger as the concentration of progesterone increased (view graph below). At 40:1, the signal dropped below that of 20:1, likely because the solution started to become oversaturated (could use acetone in the future to keep progesterone in solution). is run gave us our first clear evidence of Progesterone induced folding of the aptamer.
Figure 2. CD spectra showing Progesterone induced folding of P4G03 across ratios up to 40:1. Ellipticity increased until 20:1, then decreased at 40:1.
Experiment 3 – September 17: Testing Specificity
Rationale
After confirming that P4G03 folds when it binds progesterone, we wanted to know if that response was unique to progesterone or if similar hormones could trigger it too. We obtained P4G03 as it claimed to only bind to progesterone but we wanted to be sure.
Results
We compared progesterone, DHT, and cholesterol at 0.2:1, 5:1, and 20:1 ratios. Only progesterone caused a noticeable change in the CD spectrum. DHT showed little to no difference, and cholesterol began to come out of solution at the 20:1 ratio (could use acetone in the future to keep cholesterol in solution). These results showed that the folding pattern we saw was unique to progesterone.
Figure 3. CD spectra comparing P4G03 binding specificity to progesterone, DHT, and cholesterol at ratios up to 20:1. Only progesterone caused a clear conformational shift, while DHT and cholesterol showed no significant spectral changes.
Overall Findings
- Experiment 1 helped us find a concentration range that produced consistent, high-quality data (1:2 to 2:1).
- Experiment 2 confirmed that P4G03 undergoes a measurable conformational change when it binds to progesterone, with the largest shift at the 20:1 ratio compared to 10:1.
- Experiment 3 showed that this structural change only happens with progesterone and not with DHT or cholesterol.
- Taken together, these results show that P4G03 folds specifically in response to progesterone under body-like conditions, making it a strong candidate for use in a hormone-monitoring biosensor.
Future Experiments
- We’re planning a competitive binding assay at body temperature to measure how progesterone interacts with both albumin and our aptamer. By tracking how quickly it binds and unbinds, we’ll calculate the key constants (kₐ, k_d, and K_d) and see how well the aptamer holds up in realistic biological conditions, where natural competition could affect detection accuracy.
Broader Impact
Future iGEM Teams
This work gives a simple, repeatable method for measuring how aptamers fold when they bind their targets. Future teams can use this same approach to validate new aptamers, especially when developing biosensors or diagnostic tools.
Synthetic Biology
Our results show how aptamer folding can be directly observed and validated using accessible CD spectroscopy. This connects the molecular-level understanding of aptamer behavior with the practical side of sensor design—something that’s essential for building reliable biological devices.
Stakeholders (Researchers, Women Experiencing Miscarriage, Clinicians)
By confirming progesterone-specific folding, this work supports the long-term goal of creating a biosensor that can continuously track hormone levels during pregnancy. A tool like this could help detect early warning signs of complications and collect data that can lead to further research. Eventuly we might be able to offering more proactive care for women and giving clinicians a better way to monitor hormonal changes that influence pregnancy outcomes.
AC Voltammetry Experiments
Results
Raw quantitative data – see graphs
- Experiment 1:
- Our first AC Voltammetry experiment taught us that 6-mercaptohexanol is necessary for the strengthening of the aptamer monolayer. We only used 2-mercaptohexanol in our first experiment and it resulted in the Aptamers being wiped from the surface of the electrode.
- Experiment 2:
We ran AC Voltammetry experiments over time and found a gradual drift downward in data over time. We didn’t see a big difference in peak levels from our low concentration of Progesterone solution and our PBS Buffer (with no progesterone). At minute 125, we decided to try a higher level of progesterone and found a dramatic increase in the peak current that continued to rise as we took measurements. This indicated that the progesterone had bound. When we returned the electrode to PBS buffer, we saw the return to a level that followed the previous trend.
Experiment 3
- In our next experiment, we ensured the Progesterone was evenly distributed throughout the mixture before it interacted with the gold electrode. These results were inconclusive however because it had been a week after the formation of our aptamer monolayer so we saw extreme degrading of our sensor. This is visible in the drop in current each time a new substance flowed over the surface of the electrode. And including some human-fault data due to bumping the electrodes (seen in the random increase in current)
Reflection
- One theory of why we were not able to visually see differences in the PBS vs the low concentration of Progesterone in Experiment 2 is that Progesterone molecules clump together, being non-polar, thus none would be free to bind to the aptamers.
- Our inconclusive results mean we need to do more research before achieving a minimum viable product. However, the fact that we were able to see some change in the peak current with changing levels of progesterone gives us hope that some additional optimization and troubleshooting of our current sensor model will be worth the effort.
- This influences stakeholders because the greatest technical risk in our product is getting the aptamer impedance based sensing to work sufficiently well. Once we achieve this in conditions sufficiently close to a living person, we will have a technology with novel capabilities which will improve women’s health. Our mixed results mean that we haven’t achieved this result yet, but provide us with some guidance in what we need to work on next.
References
Park, J., Yang, K.-A., Choi, Y. & Choe, J. K. Novel ssDNA aptamer-based fluorescence sensor for perfluorooctanoic acid detection in water. Environ. Int. 158, 107000 (2022).
Jiménez, G. C. et al. Aptamer-Based Label-Free Impedimetric Biosensor for Detection of Progesterone. ACS Publications https://pubs.acs.org/doi/pdf/10.1021/ac503639s (2015) doi:10.1021/ac503639s.
Xiao, Y., Lai, R. Y. & Plaxco, K. W. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat Protoc 2, 2875–2880 (2007).