[Research advances in non-immobilized aptamer screening techniques for small-molecule targets]

Aptamers obtained through systematic evolution of ligands by exponential enrichment (SELEX) techniques are single stranded deoxyribonucleic acid (ssDNA) or RNA molecules capable of specifically recognizing target molecules. Such aptamers are easily chemically synthesized and modified, highly thermally stable, and are low toxicity and low immunogenicity. Aptamers that target small molecules have broad applications prospects for the development of new drugs, treating tumors, diagnosing diseases, monitoring environmental pollution, detecting drugs, and in ultrafast and sensitive detection applications. However, the simple structures and low molecular masses of small molecules, along with the limited number of binding groups available for interacting with nucleic acids lead to unstable aptamer-small molecule binding, which poses significant challenges for aptamer screening and sensor development. Efficient screening techniques are crucial for identifying aptamers with excellent performance characteristics. At present, the aptamer screening techniques suitable for small-molecule targets are mainly divided into three categories: target-immobilized-based screening technique, nucleic acid library-immobilized-based screening technique, and target-non-immobilized screening technique. Among them, target-non-immobilized screening technique require fewer screening rounds and result in aptamers with superior (typically nmol/L level) affinities. This paper summarized non-immobilized aptamer screening techniques for small-molecule targets, including principle, advantages, disadvantages and application progress associated with graphene oxide (GO)-SELEX, capillary electrophoresis (CE)-SELEX, and gold nanoparticle-assisted (GNP)-SELEX techniques. In addition, strategies for selecting control targets in aptamer-specific evaluation were summarized.