Firstly, the use of a buffer as a model ignores the complex conditions and various solutes in saliva or
serum. Therefore, based on literature, our use of steady-state fluorescence measurements may not work in
complex biological fluids due to the background fluorescence. A solution would be to replace the dyes with
luminophores that rely on mechanisms such as Bioluminescent Resonance Energy Transfer to create an effect
similar to FRET, but without the need for excitation and background fluorescence. A proof-of-concept has
been created by Wickramaratne et al using Europium-doped molecules. Another option would be to use
time-resolved fluorescence, similar to commercially available DELFIA assays.
Despite our achievements, we are cognizant of the fact that currently no satisfactory biomarkers exist for
the early detection of breast cancer. Even cutting edge tests that make use of multiple biomarkers, such as
the CancerSEEK assay pioneered by researchers at Johns Hopkins University, showed relatively poor
sensitivity when detecting breast cancer (meaning that the risk of an incorrect diagnosis in a clinical
setting is disturbingly high). Therefore, although our experiments have successfully demonstrated the
feasibility of using aptamer probes as a potentially noninvasive and cost-effective diagnostic tool, current
research indicates that our kit is still not specific and accurate enough to be used by patients.
However, current research indicates that effective biomarkers highly specific to breast cancer, such as
miRNAs, could be used in early diagnosis. Although current methods of detecting miRNAs are complicated and
expensive, we believe that our aptamer-based system will offer a viable alternative to current methods,
provided that suitable aptamers for said miRNAs are found in the near future.
Furthermore, the proof-of-concepts demonstrated here require the use of a well plate reader, which may not
be cost-effective or practical in some clinical settings. Therefore, we believe that further work must be
done on making this technology more accessible if our project is to be implemented.
Firstly, we believe that the design of an accessible machine with rudimentary well plate reading and
fluorescence quantification functionality is crucial for this kit to be successfully used in a clinical
setting.
Furthermore, we also believe that our kit should be self-contained and as compact as possible, so that even
clinics in underserved areas are able to afford them. Also, according to our human practices research, we
have found that the kit should be able to test multiple samples at once.
In our assay, the main component is a buffer solution with the aptasensor suspended in it. The patient will
have to collect a sample of serum of 1 mL, then mix approximately 1000µL of concentrated aptamer solution
into the sample and allow it to incubate for 30 minutes. The resulting solution is then read with a well
plate reader, and the results may be compared with the standard curve, using software to generate a
semi-quantitative result for the concentration of biomarkers in the sample.
The upside to this test is that it offers the sensitivity and specificity of traditional immunoassays (such
as ELISA) without requiring long periods of incubation or cumbersome washing steps, thus making it more
realistic to implement in a clinical setting.
As our kit is still in its early stages, we believe that it should first be administered in a clinical
setting, as the risk of contamination and false positives is very high if administered at home, by those who
are not certified healthcare professionals.
Secondly, the kit described above makes use of biomarkers that have not yet been approved for the early
detection of breast cancer (as no such marker currently exists). Therefore, future teams must first be able
to find a suitable and highly specific biomarker that successfully indicates the presence of breast cancer
with high sensitivity before applying our work in designing an aptasensor that will successfully detect said
biomarker. That being said, it is also important to note that such tests are not guaranteed to be comparable
to mammograms and other invasive methods in terms of accuracy, and thus should not be used as the sole
method in diagnosing a patient. Instead, the results may be used to corroborate data gleaned from
traditional methods.
According to the FDA
Diagnostic Device Development Process, we are currently attempting to develop a
prototype for preclinical studies. As of now, we have already shown that this device is workable.
The first step in the process would be to expand the production of high-quality dual-labeled ssDNA for
aptasensors, which may involve working with a pharmaceutical company and mass production lines. The first
step will also involve seeking out reliable biomarkers with high diagnostic value for breast cancer.
The second step would be to test the device using biological samples spiked with predetermined amounts of
biomarker and evaluating the accuracy and specificity of the device. We will also need to see whether
long-term exposure to the device has adverse effects on health.
The third step would be to go through premarket approval and the 510(k) process. If the device is not found
to be substantially similar to one already on the market, then it must go through a PMA process to evaluate
its risks. As our product is not life-supporting but still requires the use of human samples, we foresee
that it will be placed in the Class II category.
The fourth step is the FDA review, whereby the Food and Drug Administration makes a decision on the validity
of the product. As our product is most likely in the Class II category, we must prove that it is
substantially similar to other products already being marketed. In our case, multiple groups have already
designed liquid biopsies for breast cancer in the lab, showing the feasibility of such an endeavor. In
particular, CancerSeek, a technology very similar to our own in that it detects multiple biomarkers
associated with breast cancer in blood, was approved by the FDA in 2019. Other commercially available tests
similar to ours in the sense that they rely on the detection of specific molecules include the AdnaTest,
which targets circulating tumor cells, and Videssa Breast, a protein-based test that searches for biomarkers
in the blood. Therefore, we believe that our product is substantially similar to other products already
being marketed, and thus has the possibility of being approved by the FDA.
The final step is post-market surveillance and evaluation by the FDA for possible risks.
As shown on our results page, aptasensor solutions are relatively stable and can tolerate being stored in the refrigerator for at least two weeks at a time. Furthermore, our results show that very little aptamer solution is required for each test. Therefore, we believe that our aptamer solutions will be easy to transport and store for clinics, even those in far-flung or underserved areas.
Following recommendations from Dr. Yuza, we propose the use of multiple bodily fluids in our tests to prevent false positives. We believe that we could collect saliva and blood as Mucin 1 is expressed in both bodily fluids, and only a small amount of samples are required for our assay to make this feasible.
The next steps in our project will be as follows:
