▎ 摘　　要

NOVELTY - Preparing self-calibrating dual-signal biosensor involves layering graphene oxide which is self-assembled layer on a hydroxylated Si/SiO2 substrate through a silane coupling agent, annealing and reducing at 220-250 degrees C for 8-10 hours to obtain an reduced graphen oxide (RGO) film. The RGO film is patterned by depositing an aluminum film on the RGO film using a copper mesh template, the RGO film that is not protected by the aluminum film is removed by oxygen plasma, and the obtained RGO array is used as the source/drain electrode of the field-effect transistors (FET), continue to self-assemble graphene oxide layer by layer on the source/drain electrode of the FET and the silicon (Si)/silicon oxide (SiO2) substrate around the source/drain electrode, annealed and reduced at 150-180 degrees C for 6-8h, as the semiconductor layer of the FET and a fully covalently bonded graphene field effect transistor is constructed. USE - Method for preparing self-calibrating dual-signal biosensor used in preparing a device for detecting miRNA (claimed). ADVANTAGE - The method enables to prepare self-calibrating dual-signal biosensor, which fixes the DNA modified by the fluorescent group on the fully covalently bonded graphene field effect transistor as a sensitive probe for detecting miRNA, utilizes characteristics of the covalent bond connection between the layer-to-layer structure, realizes stability of the device in the solution phase for multiple subsequent processing, realizes simultaneous detection of dual signals of miRNA, meets the detection requirements of stability, reliability, high sensitivity, high selectivity, fast and simple detection, and provides unique ideas for miRNA detection. DETAILED DESCRIPTION - Preparing self-calibrating dual-signal biosensor involves layering graphene oxide which is self-assembled layer on a hydroxylated Si/SiO2 substrate through a silane coupling agent, annealing and reducing at 220-250 degrees C for 8-10 hours to obtain an reduced graphen oxide (RGO) film. The RGO film is patterned by depositing an aluminum film on the RGO film using a copper mesh template, the RGO film that is not protected by the aluminum film is removed by oxygen plasma, and the obtained RGO array is used as the source/drain electrode of the field-effect transistors (FET), continue to self-assemble graphene oxide layer by layer on the source/drain electrode of the FET and the Si/SiO2 substrate around the source/drain electrode, annealed and reduced at 150-180 degrees C for 6-8h, as the semiconductor layer of the FET and a fully covalently bonded graphene field effect transistor is constructed. The fully covalently bonded graphene field-effect transistor is soaked in a fluorescein-modified DNA solution for 30-60 minutes, rinsed after incubation, and soaked in a blocking solution for 1-2 hours, washed and dried, and self-calibrating dual-signal biosensor is obtained. An INDEPENDENT CLAIM is included for a method for detecting miRNA using the self-calibrated dual-signal biosensor, which involves: (A) testing the transfer curve of the self-calibrating dual-signal biosensor and obtaining the Dirac point grid voltage value D1; (B) taking series of miRNA solutions with a concentration gradient, and testing the fluorescence intensity I1 of the miRNA solution respectively; (C) immersing the self-calibrated dual-signal biosensor in a series of concentration gradient miRNA solutions and incubate for 15-60 minutes; (D) testing transfer curve of the self-calibrated dual-signal biosensor after immersion and obtain the Dirac point grid voltage value D2; (E) calculating Delta VCNP=D2-D1, using as an electrical signal sensor, and establishing linear equation I between the Dirac point grid pressure difference Delta VCNP and miRNA concentration; (F) testing fluorescence intensity I2 of the miRNA solution after soaking, calculating Delta I=I2-I1, using as a fluorescent signal sensor, and establishing a linear equation II between the difference in fluorescence intensity Delta I and the miRNA concentration; and (G) immersing self-calibrated dual-signal biosensor in the solution to be tested and incubate for 15-60 minutes, testing the Dirac point grid pressure difference Delta VCNP of the self-calibrated dual-signal biosensor before and after immersion and the difference I of the fluorescence intensity of the solution to be tested before and after immersion, substituting into linear equation I and linear equation II respectively, the electrical signal sensing detection result and the fluorescent signal sensing detection result of the miRNA in the solution to be tested are obtained.