▎ 摘　　要

(CN212404103-U) NOVELTY - The utility model belongs to the technical field of nucleic acid detection, relating to a double-layer microfluidic chip for nucleic acid detection. A double-layer micro-fluidic chip, comprising a valve control layer of the upper layer, a flow channel layer and a functional substrate in the middle, the flow channel layer is provided with a micro-cavity, micro-flow channel connected with the micro-cavity, and a sample inlet and a sample outlet; the valve control layer comprises a control valve, a valve inlet connected with the control valve; the functionalized substrate is divided into a detection area and a reaction area; the detection area is corresponding to the micro-cavity; the reaction area is corresponding to the micro-flow channel. The double-layer micro-fluidic chip of the utility model has simple structure, easy preparation, short time for detecting miRNA, simple steps, high sensitivity, small detection sample demand, capable of realizing fast and efficient detection of miRNA. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are also included for: (1) breast cancer miRNA detection kit, comprising double-layer microfluidic chip, breast cancer miRNA standards, and fluorescently labeled miRNA probes; (2) preparing breast cancer miRNA detection kit, comprising preparing double-layer microfluidic chip, synthesizing breast cancer miRNA standard products, and synthesizing fluorescent probes; (3) preparing the double-layer microfluidic chip, (1) using two reagents, RTV 615A and RTV 615B to prepare valve control layer and flow channel layer respectively, (2) preparing functionalized substrate, (3) combining the valve control layer and the flow channel layer, and then bonding with the functionalized substrate, and (4) laying fluorescent probes in the reaction zone of the functionalized substrate; and (4) detecting breast cancer miRNA, comprising (i) diluting the breast cancer miRNA standard into a gradient concentration, injecting the miRNA standard solution of each concentration into the sample inlet of the double-layer microfluidic chip with fluorescent probes, and using nitrogen to blow it into the micro flow channel from the injection port, (ii) incubating in the micro-channel for 20-30 minutes, using nitrogen to blow the liquid in the micro-channel into the micro-cavity from the injection port, and rinsing the micro-channel with a flushing reagent to flush it into the micro-cavity, (iii) incubating the liquid in the microcavity for 10-15 minutes, (iv) soaking the entire chip in 3% bovine serum albumin, removing the double-layer PDMS on the substrate, and soaking the substrate for 10-15 minutes for sealing, (v) soaking the sealed substrate in 100% phosphate buffered saline, 50% phosphate buffered saline, and ultrapure water, and drying for fluorescence detection, (vi) performing linear fitting on the logarithm of different concentrations and fluorescence values to make a standard curve, and (vii) performing fluorescence detection of miRNA in the sample to be tested, and bringing in the measured fluorescence value according to the standard curve to obtain the miRNA content in the sample to be tested.