Generation of cross-reactive DNA aptamers to construct the fluorescent sensing array for identifying the origin of...

Toad venom, a traditional Chinese medicine, has long been used to treat various challenging ailments. Its effectiveness and toxicity can vary depending on the types and concentrations of bufadienolides, which vary from region to region. However, identifying the origin of toad venom is challenging due to the absence of distinct visual characteristics of the original animals. Therefore, developing a scientific and practical method for origin identification is crucial to ensure the safety and efficacy of toad venom. Integrating a fluorescent sensing array with cross-reactive aptamers provides a promising solution to this issue. We isolated cross-reactive aptamers using a combination of complex target-directed SELEX and convergent selection strategies. During the selection process, we used an immobilized stem-loop library to select aptamers and evaluated the enrichment rate and pool affinity using gel elution assays. After high-throughput sequencing, we selected three cross-reactive aptamers designated N4.8, N2.4, and S1 that exhibit distinct binding profiles for bufadienolides as the biorecognition elements for the fluorescent sensing array. This sensor is capable of distinguishing toad venom from different origins with high accuracy (98.7 %), offering convenient operation, and providing a new method for detecting the origin of toad venom.