In this research, a screening procedure was applied for designing new aptamers with the enhanced binding affinity for tetracycline detection through a common theoretical and experimental study. In the first step, among the specific aptamers introduced from the experimental studies, the 8-mer aptamer was chosen toward tetracycline with a low limit of detection. The different mutants were designed by changing the nucleotides of the native sequence and, their MD simulations were performed in the presence of tetracycline. The conformational factor (Pi) was computed as a parameter that indicates the affinity of each aptamer nucleotides to target. To calculate the DGbind as a suitable factor for the specificity of aptamer toward the target, the umbrella sampling simulation was applied. Theoretical results proved that some of the mutants, such as the M-5T and M-5C mutants possess enhanced binding affinity toward tetracycline compared to the native aptamer. In the next step, the enhanced selective detection of designed mutants toward tetracycline was investigated by utilizing an easy colorimetric test relying on the gold nanoparticles (AuNPs). Based on the experimental outcomes, the limit of detection values of 159, 1.2 and, 0.1 pM were determined for the native, M-5T and, M-5C aptamers, respectively. The results of colorimetric showed that the designed mutants have good selectivity to tetracycline compared to other antibiotics. Besides, the designed mutants were successfully applied to detect tetracycline in milk samples. The agreement among the theoretical and experimental results proves the ability of theoretical approaches for designing the new aptamers with enhanced binding affinity to identify different targets.

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