Simultaneous and sensitive detection of two pathogenic genes of thrombotic diseases using SPRi sensor with one-step fixation probe by a poly-adenine oligonucleotide approach.

Single-base mutations of Factor V Leiden G1691A and Prothrombin gene G20210A are the main genetic risk factors for inherited thrombotic tendency. The establishment for rapid and efficient detection method is of great significance to the prevention of venous thrombosis. In this work, a multiplexed, highly sensitive and regenerable surface plasmon resonance imaging (SPRi) sensor is described to identify and detect the two pathogenic genes by fixing probes in one-step. The probes are fixed by ployA, which is a simpler, faster and lower cost modification method compared with traditional thiol (-SH). PolyA-DNA-AuNPs is used to amplify the signal to improve sensitivity. The detection limit of the sensor is 8 pM, and it has a wide dynamic range between 8 pM and 100 nM and a good linear relationship between 8 pM to 50 pM. The equilibrium dissociation constant (KD) of 3.0 (± 0.3) pM indicates a high binding capacity. Based on the advantages of high-throughput detection, the SPRi chip can simultaneously identify and detect two genes related to thrombotic Diseases. In addition, more than 90% signal intensity can still be obtained on the surface of the chip after being regenerated of 25 times, indicating that this SPRi sensor has good stability and reproducibility. The established SPRi sensor has the advantages of high-throughput, high-sensitivity, label-free and no need for amplification, which is expected to become an effective technical means for real-time online detection of gene point mutations, and can be extended to detect and quantify a wider range of DNA mutation diseases.