Transketolases (TKs) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the nonoxidative branch of the pentose phosphate pathway. They are considered as interesting therapeutic targets in numerous diseases and infections (e.g., cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming, and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, the enzyme loading, electrochemical protocol, and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 min, which allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identified to inhibit TK from E. coli with an IC of 63 μM and an inhibition constant ( K) of 3.4 μM. The electrochemical assay was also used to propose an inhibition mechanism.

Analysis of a large number of samples by electrochemical methods for rapid optimization of amperometric biosensors or screening applications is still a challenge. To overcome this limitation, a system with 96 screen‐printed electrodes was developed to assay H2O2 produced by free and immobilized galactose oxidase as a model for oxidases. Detection was based on two electrochemical methods: intermittent pulse amperometry (IPA) and electrochemiluminescence (ECL). This 96‐well electrochemical device allowed rapid optimization of the parameters in half a day, regardless of the method used (i.e., IPA or ECL). We demonstrate that ECL is more sensitive for the detection of H2O2 (limit of detection of 1 μm for ECL vs. 10 μm for IPA) with lower interelectrode variability (1 vs. 19 %). Rapid determination of sensitivities, linear ranges, and limits of detection of galactose oxidase substrates (e.g., dihydroxyacetone and l‐erythrulose) by using free or immobilized enzyme was conducted in only 50 min by the mean of 5 different sets of 96 screen‐printed electrodes.