A ratiometric pH sensor based on a tetrahedral framework nucleic acid dual CRISPR system, its preparation method and application

Abstract

This invention discloses a target-gated ratiometric pH sensor based on a dual CRISPR system of tetrahedral framework nucleic acids, belonging to the fields of biomedicine and nucleic acid nanotechnology. The sensor uses tetrahedral framework nucleic acids as a rigid scaffold, precisely assembling Cas12a, Cas13a, and their corresponding fluorescent reporter molecules in a 1:1 stoichiometric ratio. Their orthogonal pH responses form the basis of the ratiometric sensing. Target recognition-pH sensing logic gating is achieved through target-specific crRNA design. Cholesterol modification allows for stable anchoring on the lipid membrane surface. This invention also discloses the sensor's fabrication, membrane anchoring, pH monitoring, and target-gated function implementation methods. This sensor possesses advantages such as high-precision ratio measurement, accurate spatial arrangement, high specificity, stable membrane anchoring, and a wide pH response range, and can be used for precise monitoring of pH dynamics at biomembrane interfaces and during extracellular vesicle fusion.

Images