Magnetic mesoporous silica nanoparticle probe and its preparation method and application
A technology of mesoporous silica and nano-particles, which is applied in the direction of material analysis and instrumentation by chemical reaction of materials, material analysis by observing the influence of chemical indicators, etc., can solve the complex operation process, operation process error, High operation requirements, to achieve the effect of simple process, wide application field and good stability
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Embodiment 1
[0034] Example 1 Taking the detection probe of Newcastle disease virus as an example, constructing a magnetic mesoporous silica nanoparticle probe
[0035] 1. Characterization of M-MSN nanoparticles: Dilute M-MSN nanoparticles to 1μg / μL, then take 10μL spotting copper grid, and then observe the size and pore diameter of nanoparticles with transmission electron microscope and scanning electron microscope. The results are shown in figure 1 and figure 2 .
[0036]2. Loading of peroxidase (POD): the commercialized peroxidase and the diluted M-MSN nanoparticles in step 1 were in a mass ratio of 1:1 (i.e. 100 μg of M-MSN:100 μg of POD ) mixed, and the mixed solution was incubated on the mixture at 4°C for 8-12h; then the unbound peroxidase was separated and washed with a magnetic frame to obtain the supernatant and the precipitate, and the obtained precipitate was POD / M-MSN; the precipitate was dissolved in 100μL 20 μg of M-MSN nanoparticles were dissolved in 100 μL of PBS, and 2...
Embodiment 2
[0041] The detection of embodiment 2 peroxidase activity
[0042] In order to detect the activity of peroxidase in magnetic mesoporous silica, we chose to use TEM as the substrate to react with peroxidase in different states to develop color, and the specific grouping is pure peroxidase (POD ), magnetic mesoporous silica-coated peroxidase (POD / M-MSN), polyethyleneimine-coated magnetic mesoporous silica loaded with peroxidase (POD / M-MSN / PEI) and The completed nanoprobe (POD / M-MSN / PEI / Ab) was constructed, and its OD450 value was read using a microplate reader. The test results found that after the peroxidase was loaded into the magnetic mesoporous silica, there was no change in the enzymatic activity of the peroxidase in each state. The specific test results are as follows:
[0043] POD content OD450 POD0.2μg 3.9267 POD / M-MSN 0.2μg 3.9389 POD / M-MSN / PEI 0.2μg 3.9165 POD / M-MSN / PEI / Ab 0.2μg 3.7982
Embodiment 3
[0044] Example 3 Magnetic Mesoporous Silica Nanoparticle Probe Detection of Newcastle Disease Virus
[0045] 1. The magnetic mesoporous silica nanoparticle probe (POD / M-MSN / PEI / Ab) prepared in Example 1 is combined with Newcastle disease virus: the magnetic mesoporous silica nanoparticle probe is combined with Newcastle disease virus The ratio is 1:1, and react at room temperature for 1 hour. After 1 hour, use a magnetic stand to separate the supernatant and precipitate, and wash 3 times with 1×PBS. Set aside for precipitation.
[0046] 2. Extraction of viral RNA and reverse transcription: extract RNA from the precipitated product obtained in step 1 using an RNA extraction kit, and then use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA. The reverse transcription system is as follows:
[0047]
[0048] The reverse transcription process is 37°C, 15min, 85°C, 5s.
[0049] 3. Detection of binding efficiency: the reverse transcription product i...
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