Structure switching aptamer enhance sensitivity and specificity of photonic crystal-based sensors for RSV-G protein detection

Photonic crystal-based aptasensors for viral proteins detection offer the advantage of producing visible readouts. However, they usually suffer from limited sensitivity and high non-specific background noise. A significant contributing factor to these issues is the use of fixed-conformation aptamers in these sensors. To address this challenge, we have developed an approach to transforming aptamers into molecular switches that undergo conformational changes upon target binding. This strategy enhances the performance of colorimetric aptasensors by improving sensitivity and reducing background noise without the need for post-SELEX modifications. In this study, we present an efficient aptamer screening method designed to facilitate the development of target-responsive aptamer structure switches from pre-existing aptamers with stable conformations. A novel algorithm was developed that integrates binding motif information with secondary structure data to generate three ssDNA libraries. Through virtual screening, we identified an aptamer with structure-switching properties (MSA-Apt-16) (Kd = 7.6 nM), and its structure-switching property was further validated using molecular simulations and circular dichroism spectroscopy. Further, the binding kinetics and selectivity of both aptamers were evaluated using SPR. MSA-Apt-16 displayed a kon of 1.39 × 10⁵ Ms^-1 and a koff of 1.76 × 10^-4 s^{- 1}, signifying a high binding affinity and selectivity. In contrast, Apt-H8 showed a kon of 1.22 × 10^-4 Ms^-1 and a koff of 1.89 × 10^-3s^-1, showing a low binding affinity with RSV-G. Moreover, we demonstrated the enhanced sensitivity of the newly designed molecular switching aptamer (MSA-Apt-16) compared to the parent aptamer (Apt-H8) by incorporating them into the photonic crystal-based colorimetric sensing platform for the detection of RSV-G protein. The molecular switching aptasensor successfully detected RSV-G protein, exhibiting efficient color changes, significantly reduced background noise, and a limit of detection (LOD) of 1 pg/mL with a linear range of 1-500 pg/mL (signal-to-noise ratio = 3, R² = 0.9489), and an analytical recovery of 94.0 ± 1.5 % to 102.0 ± 1.5 % in artificial serum, saliva, and nasal swab samples. In contrast, the parent aptamer demonstrated a LOD of 1 ng/mL with a linear range of 1-500 ng/mL (R² = 0.9129). Our pipeline offers a robust and broadly applicable technique for developing aptamer switches personalized to specific targets, significantly reducing background noise and enhancing aptasensor efficiency, thus expanding the potential applications of aptasensors in various diagnostic fields.