The article presents a rational approach to optimizing structure-switching aptamers (SSAs) for biosensing applications, focusing on improving binding affinity and introducing target-dependent conformational switching. Key steps include: 1. **Structural Characterization**: Using NMR and computational modeling to identify critical sequence motifs and conformational changes in the presence of the target. 2. **Microarray-Based Mutation Analysis**: Conducting large-scale mutational analysis to map regions permissive to mutation and identify combinations with stronger binding affinity. 3. **Optimization in Biofluids**: Ensuring the optimized aptamer performs well in relevant biofluids to facilitate transition to biosensing platforms. The approach is demonstrated using a cortisol binding aptamer, which is crucial for monitoring stress levels in real-time. The optimized sequence shows a 3-fold improvement in binding affinity and selectivity compared to the original aptamer, and performs well in filtered human serum, a surrogate for interstitial fluid (ISF). The method is believed to be broadly applicable for optimizing SSAs for various targets and accelerating the development of rapid, continuous, and wearable biosensing applications.The article presents a rational approach to optimizing structure-switching aptamers (SSAs) for biosensing applications, focusing on improving binding affinity and introducing target-dependent conformational switching. Key steps include: 1. **Structural Characterization**: Using NMR and computational modeling to identify critical sequence motifs and conformational changes in the presence of the target. 2. **Microarray-Based Mutation Analysis**: Conducting large-scale mutational analysis to map regions permissive to mutation and identify combinations with stronger binding affinity. 3. **Optimization in Biofluids**: Ensuring the optimized aptamer performs well in relevant biofluids to facilitate transition to biosensing platforms. The approach is demonstrated using a cortisol binding aptamer, which is crucial for monitoring stress levels in real-time. The optimized sequence shows a 3-fold improvement in binding affinity and selectivity compared to the original aptamer, and performs well in filtered human serum, a surrogate for interstitial fluid (ISF). The method is believed to be broadly applicable for optimizing SSAs for various targets and accelerating the development of rapid, continuous, and wearable biosensing applications.