A dual-mode aptasensor based on rolling circle amplification enriched G-quadruplex for highly sensitive IFN-γ detection.

Background: Aptasensors have been extensively utilized in target detection due to their advantages of high sensitivity and fast response. However, the reliability of the detection results of the single-mode aptasensor cannot be verified in time. Developing efficient detection methods with cross-validation capability is beneficial to achieving highly reliable detection. This study aims to design a colorimetric and fluorescent dual-mode aptasensor by skillfully engineering G-quadruplex assembly and rolling circle amplification for highly reliable IFN-γ detection.

Results: The complexes of anti-IFN-γ aptamers and complement sequences (cDNA) were modified on the magnetic beads. In the presence of IFN-γ, the preferential combination of aptamers with IFN-γ resulted in the release of cDNAs. The cDNAs were collected by magnetic separation and then used as primers to trigger rolling circle amplification reaction to generate enriched G-quadruplexes. The G-quadruplex could be utilized to combine with hemin to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine for colormetric mode or to couple with the fluorogenic dye Thioflavin T for fluorescent mode. The developed dual-mode aptasensor displayed a linear range of 1-10000 pM with a detection limit of 0.406 pM for the colormetric mode and a range of 0.1-10000 pM with a detection limit of 0.037 pM for the fluorescent mode. Further, the designed aptasensor was applied to IFN-γ detection in serum samples and achieved satisfactory recoveries.

Significance: This innovative dual-mode detection strategy skillfully leverages the effective target-binding ability of aptamer, dual-function of the G-quadruplex and the signal amplifying ability of rolling circle amplification. This approach not only provides a reliable testing tool for the detection of IFN-γ, but also promotes the development of multimode sensing platforms.