Lian Xia,Jia Zheng,Jian Su,Yue Tang,Zhen Wu,Xiaojuan Niu,Yuangen Wu
Amygdalin, a cyanogenic glycoside abundant in apricot kernels, poses a food safety risk, requiring rapid detection that conventional chromatographic methods cannot fulfill for on-site application. Thus, this study aims to construct a novel colorimetric sensing platform and improve detection performance through a multivalent aptamer strategy. This pioneering work introduces a novel bivalent aptamer strategy for ultra-sensitive on-site amygdalin detection. The aptamers specific to amygdalin were screened using a five-segment single-stranded DNA library and Capture-SELEX, with truncated variants optimized to obtain a high-affinity core sequence. A key innovation involves the first design and synthesis of a bivalent aptamer that exploits synergistic binding to enhance target affinity. The bivalent aptamer (Bapt 4-4) exhibited doubled affinity for amygdalin compared to its monovalent truncated counterpart (Amyg 4–26), achieving a dissociation constant ( K d ) of (2.19 ± 1.03) nM. By integrating the bivalent aptamer with the laccase-like activity of silver oxide nanoparticles (Ag 2 O NPs), an aptasensor with high sensitivity and specificity was developed, yielding a detection limit of 0.008 μg mL −1 , which is lower than that of the monovalent aptamer-based sensor (0.0137 μg mL −1 ). This multivalent aptamer strategy provides an effective approach for highly sensitive amygdalin detection. Furthermore, the constructed bivalent aptasensor provides a robust technical tool for the on-site monitoring of plant-derived cyanogenic glycoside toxins, with broad application prospects in the domains of food safety and traditional Chinese medicine safety.
Spermine contamination ranks as one of the food safety issues, it will cause some adverse reactions if the intake of spermine is excessive in human body. So it is of great significance to establish fast and efficient analysis method to detect spermine in foods. In this study, the spermine aptamers with high affinity and specificity were obtained by the capture systematic evolution of ligands by exponential enrichment (Capture-SELEX) technique. Forty-one aptamer sequences were obtained by cloning and sequencing, and were divided into eight families based on homology and secondary structure analysis. The affinity and specificity of candidate aptamers was analyzed by isothermal titration calorimetry (ITC) and fluorescence assay . The aptamers named APJ-6 was picked out as the optimal aptamer that recognizes spermine specifically with the Kd value of 9.648 ± 0.896 nM. In order to verify the practicability of the selected aptamers, the sensitive aptamer-based fluorescene assay was designed. Under optimized conditions, this aptasensor exhibited a low detection limit of 0.052 nM, as well as a linear within the range of 0.1–20 nM. Besides, it has been further applied for the determination of spermine in pork samples and the recoveries ranged from 86.45% to 98.15%, showing its great potential for sensitive analysis in food safety control.
Yuzhe Ding,Qingyu Chen,Yicheng Heng,Emily Hoi Pui Chao,Yunus A. Kaiyum,Philip E. Johnson,Juewen Liu
Kanamycin A, or simply referred to as kanamycin, is an aminoglycoside antibiotic with a narrow therapeutic window. Aptamers are useful recognition molecules for their detection and continuous monitoring. However, a short and high-affinity kanamycin aptamer that works under physiological conditions is still lacking. In this work, we revisited a previous aptamer selection done at pH 8, which had been abandoned due to poor sequence enrichment. Its top sequence, named KAN8–1, shows a Kd of 51 nM at pH 7.5 for kanamycin as measured by isothermal titration calorimetry, and its affinities to kanamycin A and B are similar. Using NMR spectroscopy methods, the KAN8–1 aptamer undergoes ligand-induced folding and likely has a better-defined structure compared to the KAN6–1 aptamer, which was highly enriched in the pH 6 selection. Using the KAN8–1 aptamer, a strand-displacement biosensor was developed, and it has a limit of detection of 0.9 μM with excellent selectivity. This sensor also has a similar performance in serum. The reason for the poor enrichment of KAN8–1 was attributed to its low hybridization efficiency and poor hybridization stability to the capture strand as demonstrated by a fluorescence titration assay and melting analysis, which indicated a limitation of the capture-SELEX method.
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