title: Theophylline detection in serum using a self-assembling RNA aptamer-based gold nanoparticle sensor.
abstract: Recently, DNA aptamer-gold nanoparticle (AuNP) conjugates have emerged as novel biosensing tools. Although RNA aptamers are more advantageous than DNA aptamers, their vulnerable nature during the construction of these conjugates restricts the development of RNA aptasensors. In this study, we developed an RNA aptamer-based AuNP sensor for the detection of theophylline in serum, combining the high binding affinity and selectivity of a theophylline RNA aptamer and the fluorescence quenching ability of AuNPs. In order to prevent nuclease degradation during the experimental process, the single strand of the theophylline RNA aptamer (33-mer) was split at the end loop region into two shorter halves, which were able to reassemble to form the theophylline-binding pocket. One fragment was linked to a DNA sequence that included a 15 thymine (T15) spacer and a polyadenine (polyA, A12) tail. The chimeric RNA/DNA oligonucleotide was attached to AuNPs within a few minutes via adsorption of the polyA tail. The other fragment was labeled with a fluorophore (Cy3). The two individual fragments self-assembled in the presence of theophylline. Upon ligand binding, the fragments came into close proximity, resulting in fluorescence quenching. This sensor exhibited a low detection limit of 0.05 µM, with a linear dynamic range from 0.1 to 10 µM in serum. Moreover, the sensor did not recognize theophylline-related compounds (e.g., caffeine and theobromine), demonstrating its high selectivity. This strategy offers new possibilities for the application of RNA aptasensors in clinical settings.
authors.affiliation: Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Biomedical Material of Tianjin, Tianjin 300192, PR China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Biomedical Material of Tianjin, Tianjin 300192, PR China.
authors.forename: Kai
authors.initials: K
authors.lastname: Ling
authors.affiliation: Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Biomedical Material of Tianjin, Tianjin 300192, PR China.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Tao
authors.affiliation: Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Biomedical Material of Tianjin, Tianjin 300192, PR China. Electronic address: zhangqiqt@126.com.
authors.forename: Qiqing
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation*; D001993:Bronchodilator Agents / Q000097:blood; D003460:Crystallization / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*; D013806:Theophylline / Q000097:blood*
chemical_list: D052157:Aptamers, Nucleotide; D001993:Bronchodilator Agents; D006046:Gold; D013806:Theophylline
keywords: Aptasensor; Fluorescence quenching; Gold nanoparticle; RNA aptamer; Theophylline