Theophylline detection in serum using a self-assembling RNA aptamer-based gold nanoparticle sensor

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 0.05µM, with a linear dynamic range from 0.1 to 10µM 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. •An RNA aptamer-based AuNP sensor was first developed for the detection of serum theophylline.•The RNA aptamer of theophylline was first splitted into two shorter fragments, which self-assembled an complex in the presence of the ligand.•Chimeric RNA/DNA oligonucleotide was first attached to AuNP within a few minutes via adsorption of a polyA tail.•In this aptasensor system, AuNPs not only act as quenchers but also enhance the biostability of the attached RNA fragments.•This sensor design can prevent the degradation of RNA molecule during the experimental process, which will expand the bioanalytical applications of RNA aptamers in clinical settings.