Overview

MIKROSKIN aims to be an easy to use rapid test for the detection of dysbiosis in the skin microbiota. To develop our rapid test, we divided the project into smaller parts, to make the development easier and faster, as we were able to work on several parts simultaneously. Our project is divided into three different parts.

The first part is the aptamer selection, where we use SELEX to select aptamers against the common skin bacteria C.Acnes and one aptamer against all gram-positive skin bacteria. The second part of the project is the analysis of an already discovered aptamer against S.aureus. The third part is the development of the detection method, where we used the colorimetric properties of the polymer PCDA to induce a color change upon aptamer-target binding.

Our goal for our project was to develop a prototype of our rapid-test, which we could use in presenting our product to potential stakeholders, for the establishment of our business model.

Aptamer Selection

The aptamer selection was done by using cell-SELEX for the selection of an C.acnes aptamer and in a bead based-SELEX for selection of a Protein A aptamer. Both protocols needed optimization to make them work properly. We obtained results for the first SELEX round of both targets, showing that some aptamers of the initial library bind our target. However, due to the shortage of time, we did not manage to show an increase in affinity over multiple SELEX rounds.

In theory, an increased binding affinity should be shown by using FACS analysis. The fluorescent tag of the aptamer enables to show target binding by measuring fluorescence intensity, as done by Van Simaeys et al. and others.

Aptamer Testing

To develop a functional and highly sensitive rapid test, it is important to have sensitive aptamers. We analysed the binding affinity with three different approaches: affinity chromatography, bead based purification and fluorescent detection.

Chromatography is a widely used technique to test aptamer target binding. However, in our experiment no binding of the Protein A aptamer to the HiTrap Mabselect protein A affinity chromatography column was detectable.

An alternative approach to the chromatography is magnetic-bead based affinity analysis. The aptamer affinity was tested using magnetic beads conjugated with Protein A. Still, no binding of the protein A aptamer was detected.

We decided to test the affinity of the aptamers additionally by using an affinity assay based on fluorescence detection. The affinity of the aptamers against the immobilized target were measured by detecting emitted fluorescence. This approach is highly sensitive in detecting target binding, because low aptamer concentration in the pico molar range could potentially be detected, according to the calibration curve.

Therefore, the protein A aptamer was tagged with a fluorophore on the 5’end.

The affinity of purchased protein-A aptamer was tested as described. Unfortunately, the aptamer affinity to bind protein-A was calculated to be around XXX%.

In conclusion, the purchased protein A aptamer was not able to bind its target.

Detection Method

Our rapid test is based on a colorimetric change induced by the aptamer binding its target. Therefore, the aptamer is coupled to PCDA vesicles. Target binding induces mechanical stress to the PCDA vesicles, which induces a conformational change, which shifts the PCDA color from blue to red.

We successfully synthesized PCDA vesicles and showed their color changing ability due to external stress, in the form of heat and UV-light, as seen in Figure 1.

Figure 1: Our example of the colour-changing ability

This colorimetric properties of PCDA vesicles are utilized in several developed biosensors. It could be shown by XXX et al. that PCDA vesicles conjugated to aptemers change color upon target binding.

We conjugated the PCDA vesicles with the Protein A aptamer. However, no color change was detectable. This is expected, because as previously mentioned, the binding affinity of the aptamer was very low.

Functional Testing

The final step in the project is the functional testing of the PCDA-Aptamer conjugates on facial swabs. This step is necessary to show that the detection method is working under application conditions.

Conclusion

In our project we aimed to develop an aptamer-based rapid test to detect skin bacteria in a semi-quantitative manner. We were able to develop an experimental procedure for the development of the rapid test. The developed protocols need further optimization to succeed in producing a functional test, however, we succeeded in obtaining promising results for some parts of the process.

After the successful optimization of the experimental procedures, we will develop a customizable rapid-test with a wide range of aptamers. You can read more about our implementation strategy here

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