Huang, Po-Jung JimmyLiu, Juewen2025-09-172025-09-172022-02-10https://doi.org/10.1021/acs.analchem.1c0434910.1021/acs.analchem.1c04349https://hdl.handle.net/10012/22452This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.1c04349With the growing consumption of caffeine-containing beverages, detection of caffeine has become an important biomedical, bioanalytical, and environmental topic. We herein isolated four high-quality aptamers for caffeine with dissociation constants ranging from 2.2 to 14.6 μM as characterized using isothermal titration calorimetry. Different binding patterns were obtained for the three single demethylated analogues: theobromine, theophylline, and paraxanthine, highlighting the effect of the molecular symmetry of the arrangement of the three methyl groups in caffeine. A structure-switching fluorescent sensor was designed showing a detection limit of 1.2 μM caffeine, which reflected the labeled caffeine concentration within 6.1% difference for eight commercial beverages. In 20% human serum, a detection limit of 4.0 μM caffeine was achieved. With the four aptamer sensors forming an array, caffeine and the three analogues were well separated from nine other closely related molecules.enAttribution-NonCommercial-ShareAlike 2.5 Canadahttp://creativecommons.org/licenses/by-nc-sa/2.5/ca/Article