An ultrasensitive and specific fluorescence split-aptasensor for VEGF165 detection based on nicking enzyme-assisted 3D DNA walker coupling with CRISPR-Cas12a.

The overexpression of vascular endothelial growth factor 165 (VEGF165) in cancer cells plays a pivotal role in promoting tumor metastasis by facilitating their excessively rapid proliferation and division. Hence, the development of analytical methods possessing high sensitivity and resistance to interference is imperative for the detection of VEGF165. Various types of aptasensors have been devised for VEGF165 detection; however, the performance of these biosensors can be influenced by non-target signals caused by conformational changes in unbound aptamers. The paper shows the creation of a precise and sensitive fluorescence biosensor designed to detect VEGF165 by using a VEGF165-specific split aptamer. Additionally, this biosensor employs nicking enzyme-assisted DNA walker coupling with CRISPR-Cas12a to achieve dual-signal amplification. The VEGF165 calibration curve shows a detection limit of 268 fM and has a broad linear range from 5 to 4000 nM. The fluorometric biosensor was utilized to detect VEGF165 in human serum and cellular homogenate samples, yielding good outcomes. The innovative design serves as proof of concept and demonstrates significant potential in detecting various targets.