Rapid and one-pot detection of nucleic acids within mRNA vaccines based on a dual amplification strategy with PER-CSD.

[BACKGROUND] mRNA vaccines have emerged as a promising and innovative approach in cancer immunotherapy, offering high selectivity, rapid development, and the potential for personalized treatment. Analyzing the concentration of mRNA molecules in serum and urine samples enables researchers to assess stability, delivery efficiency, and potential degradation, all of which impact therapeutic efficacy and safety. Traditional detection methods are associated with challenges, including operational complexity and time consumption. Therefore, it is of great significance to establish a rapid, reliable, and easy-to-operate detection strategy.

[RESULTS] In this research, we have established a novel analytical platform based on primer exchange reaction (PER) and catalytic strand displacement (CSD), enabling one-pot and rapid detection of mRNA vaccines for non-small cell lung cancer (NSCLC). Specifically, the target can activate the upstream PER, which features a fully enclosed dumbbell-shaped hairpin structure, bringing about the generation of substantial trigger molecules. Thereafter, the trigger binds to the exposed toehold domain of the probe, which is modified with fluorescent groups, initiating the CSD process and producing a prominent fluorescence signal. In this system, triggers can be recycled to enable a secondary stage of signal amplification. In addition, this strategy performs dual signal amplification for sensitive detection in an isothermal, rapid, and one-pot way.

[SIGNIFICANCE] Ultimately, this research innovatively established a fluorescence biosensor based on primer exchange reaction and catalytic strand displacement, achieving dual signal amplification for rapid, one-pot, and isothermal detection. The fully enclosed dumbbell-shaped hairpin structure facilitates enhanced stability and reduced background noise. These advancements have created a convenient and user-friendly platform for the in vitro detection of nucleic acids within mRNA vaccines and may inspire scalable detection platforms for other mRNA treatments.