Aptamer sensor for self-assembly of DNA hydrogel based on rolling circle amplification product and application of aptamer sensor
A technique of aptamer sensor and rolling circle amplification, which is applied in the determination/inspection of microorganisms, instruments, biological tests, etc., and can solve the problems of inconvenient visual detection of DNA hydrogel
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Embodiment 1
[0058] Embodiment 1 Preparation of SH-PEG functionalized gold nanoparticles:
[0059] (1) 1g HAuCl 4 4H 2 Dissolve O in 100 mL of pure water to make 1% HAuCl 4 4H 2 O in water; take 0.5 mL of 1% HAuCl 4 4H 2 O aqueous solution was added into 50mL pure water, heated to boiling under stirring;
[0060] (2) Add 0.4mL 5% aqueous sodium citrate solution therein, keep boiling for 30min, cool the wine red solution prepared to room temperature, and store it in the dark at 4°C for subsequent use; the ultraviolet-visible absorption spectrum and transmission electron microscope of the gained nano gold Figure respectively as figure 2 As shown in A and 2B;
[0061] (3) Take 5mL of the wine red solution prepared in step (2), add 0.25mL of the solution with a concentration of 5×10 -5 M SH-PEG, incubated at 37°C for 12h, then centrifuged at 10,000rpm for 10min, discarded the supernatant, and obtained a precipitate that was SH-PEG functionalized gold nanoparticles, and dispersed the p...
Embodiment 2
[0064] The preparation of embodiment 2 DNA hydrogel:
[0065] (1) 20μL 10 -5 M OTA aptamer and 20 μL 10 -5 The M primers were mixed and annealed at 95°C for 10 min, and then hybridized by the gradient cooling method to obtain the aptamer-primer hybridization complex; the hybridization condition of the gradient cooling method was 95°C for 10 min, and then naturally cooled to 25°C.
[0066] (2) Add 5 μL of exonuclease I to enzymolyze the unbound DNA single strand to prepare the purified aptamer-primer hybrid complex; React for 15 minutes.
[0067] (3) Take 10.5 μL of the purified aptamer-primer hybridization complex obtained in step (2), add OTA with a concentration of 10 ng / mL, and add 10.5 μL of 10 -5 M padlock probe and 1.0 μL T4 DNA ligase were mixed evenly and incubated at 37°C for 45 min; the enzyme activity of T4 DNA ligase was 5.0 U / mL;
[0068] (4) Then add 6.4 μL 10mM dNTPs and 2.0 μL 10u / mL Phi29 DNA polymerase to it, mix well and incubate at 37°C for 2.5h, then i...
Embodiment 3
[0072] A preparation method of said aptasensor, said preparation method comprising the following steps:
[0073] Add 5 μL SH-PEG functionalized gold nanoparticles prepared in Example 1 to 0.03 μL DNA hydrogel (10 ng / mL) prepared in Example 2 to obtain AuNPs / DNA hydrogel; incubate at 90°C for 30min, then cool to room temperature;
[0074]The AuNPs / DNA hydrogel was centrifuged at 2000 rpm for 10 min, and its absorbance at 530 nm was measured with a UV-1800 ultraviolet spectrophotometer.
[0075] The transmission electron microscope images and photos of AuNPs / DNA hydrogel are as follows image 3 D, shown in 3E. Depend on image 3 D It can be seen that the shape of AuNPs / DNA hydrogel is similar to that of DNA hydrogel, which is flower-shaped and the size is also micron-scale. It can be seen that the gold nanoparticles are wrapped inside the DNA hydrogel. Photos of DNA hydrogels and AuNPs / DNA hydrogels as image 3 As shown in E, it can be seen that the AuNPs / DNA hydrogel is re...
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