Development of an activity-based ratiometric electrochemical substrate for measuring circulating dipeptidyl peptidase-IV/CD26 in whole blood samples

Dipeptidyl peptidase-IV (DPP-IV) is a circulating blood biomarker that diagnose pancreatic and thyroid cancers, as well as type 2 diabetes. Although current DPP-IV detection methods show promise, real-time detection in whole blood is limited, as blood samples require tedious pre-treatment. To overcome these limitations, a DPP-IV targeted electrochemical substrate, DPPLPOH (DiPeptidyl Peptidase Latent Probe-OH (with Hydroxyl group)), was designed. When coupled with an electrochemical analytical method, this substrate enabled direct and convenient detection of DPP-IV in complex biofluids, including whole blood samples. In these assays, DPP-IV selectively hydrolyzed DPPLPOH, which underwent a self-immolative reaction to generate a masked electrochemically sensitive amino ferrocene reporter (AFOH). This electrochemical analytical tool demonstrated excellent sensing performance, characterized by exceptional enzyme binding properties. DPPLPOH showed excellent sensitivity and selectivity, with a detection limit of 0.021 ng/mL and a broad linear detection range of 0.1–100 ng/mL. The probe was specific to DPP-IV without interference from other electroactive species, enzymes, or hydrolases. Furthermore, DPPLPOH enabled real-time monitoring of DPP-IV activity on tumor cell surfaces and direct tracking of DPP-IV concentration in whole blood without a tedious separation process. This method may be a valuable tool in the early detection of pancreatic and thyroid cancers and in post-treatment surveillance.