CRISPR-CAS14A response photoelectrochemical sensing detection method and kit for detecting T2 toxin
Disclosed are a CRISPR-Cas14a responsive photoelectrochemical sensing detection method and a kit for detecting T2 toxin, relating to the technical field of small molecule toxin detection; the detection method includes: (1) preparation of a magnetic probe; (2) electrode modification; (3) identification of magnetic beads; (4) SDA isothermal amplification; (5) cutting; (6) photoelectrochemical detection; and (7) standard curve plotting. According to the present application, using SDA to enlarge and amplify the signal isothermally, target is detected by Cas14a trans cleavage property and photoelectric signal with good stability, high specificity of reaction, high sensitivity.
1 . 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.
2 . The photoelectrochemical sensing detection method for detecting T2 toxin according to claim 1 , wherein a preparation method of the magnetic probe in the step (a) comprises: taking 100 μL of the magnetic beads, magnetically separating and washing, adding 500 μL of the T2-APT, and mixing at a room temperature for 30 minutes (min) to obtain the magnetic probe.
3 . The photoelectrochemical sensing detection method for detecting T2 toxin according to claim 1 , wherein a preparation method of the CdS—Au/ITO electrode in the step (b) comprises: ultrasonically cleaning the ITO glass, using CdS and Na 2 S 2 O 3 solutions as an electrolyte, depositing CdS nanoparticles on a surface of the ITO glass by cyclic voltammetry to obtain a CdS/ITO electrode, followed by placing the CdS/ITO electrode into an Au/ITO preparation solution to produce the CdS—Au/ITO electrode.
4 . The photoelectrochemical sensing detection method for detecting T2 toxin according to claim 1 , wherein the incubating in the step (e) is carried out under a reaction condition of 37° C. for 10 min.