| CPC C12Q 1/6825 (2013.01) [G01N 27/3278 (2013.01); G01N 27/48 (2013.01); C12Q 2600/178 (2013.01)] | 7 Claims |
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1. A homogeneous detection method for continuous detection of a miRNA by a metal organic framework (MOF)-based nanozyme, comprising:
step 1: conducting a duplex-specific nuclease (DSN)-assisted signal amplification strategy in a homogeneous solution;
step 2: conducting continuous detection of the miRNA by a flow injection-batch method, comprising: in a flow injection mode, enriching single-stranded DNAs by a single-stranded DNA adsorption capacity of an indium tin oxide (ITO) electrode, wherein the ITO electrode is modified by an MOF-based nanozyme as an MOF-based nanozyme-modified ITO electrode; and in a batch mode, further amplifying the signal by using a peroxidase-mimicking activity of the MOF-based nanozyme for detection of the miRNA; and
step 3: in a flow mode, competitively eluting DNA on the MOF-based nanozyme-modified ITO electrode using a complementary DNA (cDNA) as a mobile phase; and repeating the steps 1 to 3 to conduct a next round of the miRNA detection;
wherein in the step 1, the DSN-assisted signal amplification strategy comprises: heating a methylene blue-labeled hairpin DNA sequence to 95° C. for 5 min, and cooling to a room temperature to form a hairpin structure; adding a DSN at a final concentration of 0.15 U to 0.5 U and the hairpin DNA at a final concentration of 0.8 μM to 1.2 μM to a nucleic acid amplification buffer, adding a miRNA sample to be tested, and conducting incubation at 45° C. to 60° C. for 50 min to 70 min; and adding a DSN stop solution to an obtained reaction solution to terminate the signal amplification; and
in the step 2, the flow injection-batch method comprises: in the flow injection mode, injecting a solution obtained in the step 1 into an electrochemical analysis cell by a flow injection, and conducting incubation in a batch mode for 30 min to 60 min; injecting a phosphate-buffered saline (PBS) containing hydrogen peroxide into the electrochemical analysis cell by flow injection; conducting detection by differential pulse voltammetry (DPV) or linear voltammetry or an impedance method in the batch mode using a three-electrode system to obtain an electrochemical signal, and obtaining a concentration of the miRNA sample to be tested according to a relationship between the electrochemical signal and a miRNA concentration, wherein recycling of the MOF-based nanozyme modified ITO electrode and continuous detection of the miRNA are realized.
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