Selection of Aptamers To Detect Lean Meat Agents: Ractopamine and Clenbuterol

In this work, a library-immobilization method was used to select DNA aptamers for ractopamine (RAC) and clenbuterol (CLB), two common "lean meat agents". Two RAC-binding aptamers, RAC-1 and RAC-5, exhibit dissociation constants (K_d) of 6.8 and 2.9 μM, respectively, based on isothermal titration calorimetry (ITC). Similar values were also obtained using a thioflavin T (ThT) fluorescence assay. A CLB aptamer named CLB-1 exhibited a K_d of 561 nM by the ThT assay and 885 nM by ITC. The RAC-5 aptamer showed a modest response to CLB, whereas CLB-1 exhibited strong specificity to CLB. Comparisons were made with previously published aptamers, and the aptamers reported here were shorter, had well-defined secondary structures, and were validated by rigorous binding assays. Furthermore, the ThT fluorescence responses of RAC-5 and CLB-1 were used to develop a label-free fluorescent sensor array to distinguish RAC and CLB from each other and from other structurally related molecules. These individual sensors had limits of detection of 1.2 μM for RAC and 0.16 μM for CLB. Finally, the label-free aptamer sensor was applied to spiked cat food and pork samples. These results demonstrate the potential of these aptamers for practical applications in food additive monitoring and safety assessment.