A novel fluorescent biosensor based on affinity-enhanced aptamer-peptide conjugate for sensitive detection of...

Lead contamination is a major concern in food safety and, as such, many lead detection methods have been developed, especially aptamer-based biosensors. However, the sensitivity and environmental tolerance of these sensors require improvement. A combination of different types of recognition elements is an effective way to improve the detection sensitivity and environmental tolerance of biosensors. Here, we provide a novel recognition element, an aptamer-peptide conjugate (APC), to achieve enhanced affinity of Pb²⁺. The APC was synthesized from Pb²⁺ aptamers and peptides through clicking chemistry. The binding performance and environmental tolerance of APC with Pb²⁺ was studied through isothermal titration calorimetry (ITC); the binding constant (K_a) was 1.76*10⁶ M^-1, indicating that the APC's affinity was increased by 62.96% and 802.56% compared with the aptamers and peptides, respectively. Besides, APC demonstrated better anti-interference (K⁺) than aptamer and peptide. Through the molecular dynamics (MD) simulation, we found that more binding sites and stronger binding energy between APC with Pb²⁺are the reasons for higher affinity between APC with Pb²⁺. Finally, a carboxyfluorescein (FAM)-labeled APC fluorescent probe was synthesized and a fluorescent detection method for Pb²⁺ was established. The limit of detection of the FAM-APC probe was calculated to be 12.45 nM. This detection method was also applied to the swimming crab and showed great potential in real food matrix detection.