Highly stable nano-gold probe functional complex and preparation method thereof
A nano-gold probe and complex technology, applied in the field of molecular biotechnology and biological detection, can solve the problems of increasing the risk of cross-contamination of enzymatic amplification reaction products, limited sensitivity of target nucleic acid, and inability to meet real samples, and achieve a short time High-sensitivity closed-tube nucleic acid detection, low cost, and high stability
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[0052] This embodiment also relates to the preparation method of the highly stable nano-gold probe functional complex, comprising the following steps:
[0053] (S1) first mix the gold nanoparticles and the functional nucleic acid probe at a ratio of 1:300-1000;
[0054] (S2) Add fluorosurfactant, then add buffer and sodium chloride solution to mix evenly, hybridize and incubate at room temperature for 2-10 hours, centrifuge and wash and resuspend in buffer; wherein, the fluorosurfactant is FS-10, At least one of FS-30, FS-31, FS-3100, FS-50, FSN-100; the buffer can be conventional phosphate buffer, Tris salt buffer, MOPS salt buffer;
[0055] (S3) Add a stabilizer, hybridize and incubate at room temperature for 20-30 hours, and then centrifuge, wash and resuspend in deionized water.
[0056] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the technical field of th...
Embodiment 1
[0060] Firstly, the gold nanoparticles and the thiol-containing probe (probe 1 sequence: SH-(CH 2 ) 6 - AGC TGA AAT AAT GATTAT CAA TAT T-FAM, SEQ ID NO.1) mixed in a certain ratio (such as at 1:400), adding a thiol-containing surfactant such as FSN-100, and adding a potassium phosphate buffer at pH 6.0 Mix well with sodium chloride solution, hybridize and incubate at room temperature for 3 hours, centrifuge and wash and resuspend in potassium phosphate buffer, add a mercapto-containing stabilizer such as mercaptomethoxysilane, and incubate for 24 hours at room temperature, then centrifuge and wash and resuspend in ion-free Reserve in water. The prepared nano-gold probe complexes were incubated under Tris buffer conditions at 60°C, 80°C, 90°C, and 95°C for 40 minutes, and the percentage of nucleic acid remaining on the surface of the gold nanoparticles was calculated (gold nanoparticles The total number of surface nucleic acids was calculated using DTT elution and was 100%). ...
Embodiment 2
[0063] Firstly, the gold nanoparticles and the thiol-containing probe (probe 1 sequence: SH-(CH 2 ) 6 -AGC TGA AAT AAT GATTAT CAA TAT T-FAM, SEQ ID NO.1) mixed in a certain ratio (such as 1:400), adding surfactants such as FSN-100, surfactants were selected FS-30, FS- 31. FS-3100, FS-50, and FSN-100 were compared, and potassium phosphate buffer and sodium chloride solution were added to mix well, hybridized and incubated at room temperature for 3 hours, centrifuged and washed, resuspended in potassium phosphate buffer, and thiol-containing stabilized Agents such as mercaptomethoxysilane, hybridization incubation at room temperature for 24 hours, centrifuged after washing and resuspended in deionized water for later use. The prepared nano-gold probe complexes were treated with DTT to calculate the number of modified probes.
[0064] image 3 It is the result of surfactant selection in the preparation of nano-gold probe functional complexes. The results show that the number o...
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