CRISPR-CAS14A response photoelectrochemical sensing detection method and kit for detecting T2 toxin

A photoelectrochemical sensing detection method for detecting T2 toxin in a sample, comprising:

• (a) mixing magnetic beads with a T2 aptamer (T2-APT) to prepare a magnetic probe;
• (b) ultrasonically cleaning indium tin oxide (ITO) glass, preparing cadmium sulfide-gold (CdS—Au)/ITO electrode by electrochemical deposition;
• mixing and reducing sulfhydryl modified deoxyribonucleic acid-upconversion nanoparticles (DNA-UCNPs) and tris(2-carboxyethyl) phosphine hydrochloride (TCEP), followed by reacting with the CdS—Au/ITO electrode to obtain a CdS—Au-UCNPs/ITO electrode;
• (c) incubating the magnetic probe prepared in step (a) with a sample containing T2 toxin and adding complementary DNA (cDNA) of T2 aptamer and obtaining competitive cDNA through magnetic separation;
• (d) carrying out strand displacement amplification (SDA) isothermal amplification on the cDNA obtained in step (c) with different concentrations to obtain ssDNA corresponding to the competitive cDNA with different concentrations;
• (e) incubating Cas14a and sgRNA to form a Cas14a-sgRNA complex, adding the ssDNA obtained in step (d) with different concentrations of cDNA to obtain a mixed solution;
• soaking the CdS—Au-UCNPs/ITO electrode in the mixed solution or dropping the mixed solution onto a surface of the CdS—Au-UCNPs/ITO electrode, and then standing at 37 degree Celsius (° C.) for 1 hour (h);
• (f) adopting a three-electrode system, taking the CdS—Au-UCNPs/ITO electrode from step (e) as a working electrode, an Ag/AgCl electrode as a reference electrode, a Pt sheet electrode as an auxiliary electrode, and 980 nanometers (nm) as an excitation light source, and adopting a current-time curve method to carry out photoelectrochemical detection;
• (g) plotting a standard curve with an absorbance value measured by the photoelectrochemical detection as an ordinate and a concentration of the T2 toxin as an abscissa;
• and
• (h) detecting the T2 toxin in the sample by extracting the sample with a methanol/water
• solution, centrifuging and filtering to obtain a supernatant, diluting the sample with deionized
• water, subjecting the diluted sample to the competitive reaction of step c, performing steps d-f
• on the diluted sample, and determining the concentration of the T2 toxin in the sample using the
• standard curve of step g.