Rational Design and Evaluation of a Novel Pepper Aptamer-Based Biosensor for Human Carboxylesterase 2 and Its Application in the Plasma Detection of Obese Children

Human carboxylesterase 2 (hCES2) has emerged as a protein of significant interest in obesity research. However, whether its activity is increased or decreased in the plasma of children with obesity remains a subject of debate. This study aimed to develop a novel Pepper-based biosensor for the in situ detection and imaging of hCES2 activity and to investigate the relationship between hCES2 activity and adipocyte differentiation. Leveraging the specific catalytic properties of hCES2, we designed and synthesized four probes based on the high-performance Pepper aptamer. Among these, BBC-HBC₅₃₀ demonstrated superior selectivity and sensitivity for hCES2 compared to the other three candidates. Molecular docking studies revealed the molecular interactions between the four synthetic probes and hCES2/Pepper and explained the reasons for the high selectivity and high sensitivity of BBC-HBC530 for hCES2 detection. Subsequent experiments also confirmed that BBC-HBC₅₃₀ could be applied to effectively quantify and visualize endogenous hCES2 in living cells. Furthermore, analysis of clinical samples using BBC-HBC₅₃₀ revealed that the activity of hCES2 in the plasma of obese children was significantly increased. In conclusion, the aptamer-based biosensor BBC-HBC₅₃₀ represents a promising molecular tool for investigating the biological functions of endogenous hCES2 in living systems and provides an important reference for the development of biosensors for other hydrolases.