SELEX strategy

The isolation of aptamers was carried out via a parallel-and-serial selection strategy. The whole aptamer isolation process consisted of five (for ethylone and butylone) or nine (for alpha-pyrrolidinopentiophenone, α-PVP) rounds of parallel selection and two cycles of serial selection. Detailed information regarding the conditions for each round of selection are listed in Supplementary Table S1. Since each sequence has a low copy number at the beginning of SELEX, high target concentrations were used during the first few rounds to ensure retention of all possible target binders. Moreover, for counter-SELEX, counter-target concentrations were initially kept low to remove high-affinity interferent binders while avoiding loss of target binders. In later rounds of SELEX, selection stringency was increased by decreasing the target concentration and increasing the counter-target concentration; this was done to obtain aptamers with high target affinity and specificity. The counter-SELEX protocol was rationally designed to encompass known and commonly observed interferents found in seized drug samples, including cutting agents (e.g. caffeine and acetaminophen), adulterants (e.g. procaine, lidocaine and promazine), other illicit drugs (e.g. cocaine and methamphetamine) or structurally related non-cathinone molecules (e.g. amphetamine, pseudoephedrine and ephedrine) to ensure that the isolated aptamer did not bind to them.