Aptamer-enhanced particle aggregation inhibition assay for simple homogeneous protein detection using DNA aptamer and thermo-responsive magnetic nanoparticles

A simple and sensitive homogeneous protein detection system is required for the early detection of biomarkers. Thermo-responsive magnetic particles (TM) have already been developed to achieve easy bound/free separation at the homogeneous protein detection system, but they are still limited owing to the requirement of secondary antibodies and negatively charged polymers, and it is challenging to control the TM aggregation behavior because of the size of the TM. Therefore, at new method to control TM aggregation behavior that is simple, easy, and highly sensitive is required. In this study, we developed a DNA aptamer-based TM assay as a simple protein detection system without additional secondary molecular recognition elements or negatively charged polymer. In the first attempt, a DNA aptamer was modified on the TM surface, and its aggregation behavior was monitored depending on the target molecule concentration. The TM aggregation rate during the heating process decreased depending on the amount of the DNA aptamer and increased depending on the target protein level. This suggests that the DNA aptamer prevented TM aggregation owing to its negative charge and achieved target protein detection owing to the cancellation of repulsion. Capturable aptamers were used in the TM assay to improve the sensitivity and limit of detection. The designed Capture DNA was modified on the TM surface, and the aptamer was captured in the presence of the target protein through a conformational change. Eventually, Capturable aptamer-based TM assay achieved a sub-nanomolar limit of detection and higher sensitivity than that of our initial investigation. Through this study and the ease of the DNA aptamer design, it was shown that the DNA aptamer-modified TM assay enabled the development of a simple and sensitive homogeneous protein detection system.