Development of an Activity-Based Ratiometric Electrochemical Switch for Direct, Real-Time Sensing of Pantetheinase in Live Cells, Blood, and Urine Samples

Pantetheinase is a key biomarker for the diagnosis of acute kidney injury and the monitoring of malaria progression. Currently, existing methods for sensing pantetheinase, also known as Vanin-1, show considerable potential but come with certain limitations, including their inability to directly sense analytes in turbid biofluid samples without tedious sample pretreatment. Here, we describe the first activity-based electrochemical probe, termed VaninLP, for convenient and specific direct targeting of pantetheinase activity in turbid liquid biopsy samples. The probe was designed such that cleavage of the pantetheinase amide linkage, triggered by a self-immolative reaction, simultaneously ejects an amino ferrocene reporter. Among the distinctive properties of the VaninLP probe for sensing pantetheinase are its high selectivity, sensitivity, and enzyme affinity, a wide linear concentration range (8-300 ng/mL), and low limit of detection (2.47 ng/mL). The designed probe precisely targeted pantetheinase and was free of interference by other electroactive biological species. We further successfully applied the VaninLP probe to monitor and quantify the activity of pantetheinase on the surfaces of HepG2 tumor cells, blood, and urine samples. Collectively, our findings indicate that VaninLP holds significant promise as a point-of-care tool for diagnosing early-stage kidney injury, as well as monitoring the progression of malaria.