ELONA Assay and Experimental Determination of Dissociation Constant

We followed established protocols for ELONA, an ELISA assay modified to accommodate aptamers, in order to determine the dissociation constant. We used a protocol modified from Sang et al to determine the Kd value using the Hill equation, a Scatchard plot, and non-linear regression. However, the results were inconclusive so we decided to find our Kd value through the 3D modeling software, UNAFold.

Aptamer Resuspension and Characterization

We followed standard aptamer resuspension protocols provided by the manufacturer, and were able to successfully resuspend our aptamers in the buffers used in the original papers. More information on this is provided in our lab notebook.

We were able to conduct a shelf life test on the aptamers by constructing a standard curve (results shown in next section), leaving the aptamer solution in the refrigerator for two weeks before testing the concentration of various standardized biomarker samples with known concentrations using the aptamer probes without resuspending or reheating them. The results below exemplify that the aptamer probes still successfully fluoresce 2 weeks after resuspension with fluorescence values close to their relative biomarker concentrations.

The results show that the aptamers have reasonable shelf life, even when they are not resuspended, if handled properly and kept in the dark using tinfoil. Therefore, we believe that this highlights practicality of our design in a clinical setting, where the time needed to prepare this assay is drastically shorter than traditional immunoassays. Furthermore, we have also demonstrated the superior durability of aptamers over antibodies, thus also making a strong case for the feasibility of this technology.

We were also able to test the specificity of our aptamer probes to our biomarkers using a solution with BSA. The results are shown below. For more information, please refer to our lab notebook.

Overall, we have shown that our probe is extremely sensitive and able to function well, even in complex environments.

Fluorescence of ssDNA Mucin 1 Aptasensor

We suspended the Mucin 1 aptasensors in the presence and absence of Mucin 1, and used a 96 well plate reader to excite the probe and monitor its fluorescence. Mucin aptamer S2.2 was used. The resulting data is shown below. As one can see, the presence of Mucin 1 greatly increased the fluorescence of the DNA probe solution, thus indicating that our design was successful.

Fluorescence in Standard Solutions

We added aptamer solutions to standard solutions of their respective biomarkers (Mucin 1) in a buffer solution, then excited them using a 96 well-plate reader to find the fluorescence of the aptamer probes in different concentrations of biomarker. Using this data, we were able to create standardized fluorescence vs. concentration curves for the aptamer probes. Then, using mathematical manipulation (see the Modeling page for more information), we were able to devise a simple method to calculate the total concentration of biomarkers in the original solution from the fluorescence of the solution by devising a computer algorithm based on the Hill equation.

We were able to determine the limit-of-detection and sensitivity of our aptasensors by measuring the fluorescence in decreasing concentrations of their respective biomarkers and assessing the range at which the decrease in fluorescence became negligible. The results are shown below. Furthermore, the error between the measured concentrations (using the protocols detailed in the lab notebook and using the program described on the Modeling page) and actual concentrations of standard solutions are shown below, demonstrating that our aptasensor can assess the concentrations of biomarkers in solution with relative accuracy.