Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904-0495, Japan.
Center for Microbiome and Human Health, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 44195, USA.
Sci Rep. 2019 Nov 13;9(1):16659. doi: 10.1038/s41598-019-52876-1.
Histamine produced by bacteria through decarboxylation of histidine in spoiled foods such as fish is known to cause food poisoning. Therefore, accurate and facile measurement of histamine is of practical importance. Using the recently discovered RNA aptamer that specifically recognizes histamine (A1-949 aptamer), we developed an aptasensor based on the structure-switching mechanism. Specifically, the aptamer A1-949 was fluorescently labeled at the 5' end and hybridized with a short quencher DNA strand that is partially complementary to the aptamer. The quencher strand was modified with a fluorescence quencher at its 3' terminus. Displacement of the quencher strand upon histamine binding results in an increased fluorescence. After optimizing the assay condition, the enantiomeric version of the aptasensor (L-RNA and L-DNA) was synthesized which could detect the achiral analyte with identical sensitivity and improved biochemical stability. The aptasensor performance was validated by measuring fish samples spiked with known concentrations of histamine. Finally, histamine content in spoiled fish samples was measured, and the results were compared with the measurements using a commercial enzymatic assay kit.
通过细菌对变质食物（如鱼）中组氨酸的脱羧作用产生的组胺已知会引起食物中毒。因此，准确、简便地测量组胺具有实际意义。我们使用最近发现的专门识别组胺的 RNA 适体（A1-949 适体），基于结构切换机制开发了一种适体传感器。具体来说，将适体 A1-949 的 5' 端进行荧光标记，并与短的淬灭 DNA 链杂交，该短链部分与适体互补。淬灭链在其 3' 末端修饰有荧光淬灭剂。组胺结合导致淬灭链置换，从而增加荧光。在优化了测定条件后，合成了对映体版本的适体传感器（L-RNA 和 L-DNA），它可以检测非手性分析物，具有相同的灵敏度和改善的生化稳定性。通过测量已知浓度组胺添加的鱼样本来验证适体传感器的性能。最后，测量了变质鱼样中的组胺含量，并将结果与使用商业酶法测定试剂盒的测量结果进行比较。
