Development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification and CRISPR/Cas12a sensing system

Code: 241E07CS

The detection of gene and cell doping in athletes is crucial to maintain fair competition and prevent the use of performance-enhancing substances that can be harmful to an athlete's health. In this project, we propose the development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a system with on-chip target gene amplification and detection functions. This chip will facilitate the sequential execution of multiple isothermal nucleic acid amplification and CRISPR/Cas12a sensing reactions directly on small blood samples, eliminating the need for separate pre-processing steps. Our proposed all in one gene doping chip paper features three key components: a sample pad, four conjugation pads, and a test membrane. The sample pad, preloaded with dried reaction buffer, allows for simple heat treatment of whole blood, releasing transgene DNA directly onto the chip without pre-preparation step. Each conjugation pad, linked to a specific target gene, combines on-chip RPA with a Cas12a reaction. In just 15 minutes at 37°C, RPA amplifies the target gene using specific primers. Next, Cas12a cleaves a reporter molecule (biotin-FAM) in the presence of the target exogenous DNA, but leaves it intact in its absence. Finally, the test membrane, pre-coated with streptavidin and anti-anti-FAM lines, captures the reporter depending on the target presence.