Immune PCR detection method based on functional graphene oxide and application of immune PCR detection method
A detection method and a functional technology, applied in the field of biochemistry, can solve problems such as the application of graphene oxide, and achieve the effects of safety and reliability, improved sensitivity, and high sensitivity
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Embodiment 1
[0047] Embodiment 1 prepares graphene oxide bioprobe
[0048] Take 50mgNaOH and 50mgClCH 2 Add COONa to 1mL, 1mg / mL GO water suspension, ultrasonic cracking for 1h, neutralize the mother liquor with dilute HCl, the obtained GO-COOH is repeatedly washed and centrifuged with ultrapure water until the product is evenly dispersed in ultrapure water , the GO-COOH suspension was dialyzed against ultrapure water for 48 h to remove all ions.
[0049] Mix the above GO-COOH suspension, 400mM EDC, 100mM NHS and 1mL, pH5.20 MES buffer, stir for 30min, centrifuge the mixture at 12000rpm for 5min, remove the supernatant, and then use MES buffer repeatedly Rinse to remove excess EDC and NHS, and disperse the obtained product in 1.0mL of 0.01M, pH7.40 PBS buffer solution and sonicate for 5min to obtain a uniform dispersion; then slowly add 50μL of biotin-labeled goat anti-rabbit IgG, 4°C Stir overnight, wash with 0.01M, pH7.40 PBS buffer, centrifuge at 4°C, 12000rpm for 5min, repeat 3 tim...
Embodiment 2
[0052] Example 2 GO-BA-iPCR detection system detects BDE-28 standard curve
[0053] Polypropylene PCR tubes were pretreated with 0.8% glutaraldehyde solution, 50 μL / well, incubated at 37°C for 6 h, and washed three times with ultrapure water, 200 μL / well. Dilute the original BDE-28 coating solution with 0.5M, pH9.60 CBS buffer, add it to the PCR tube treated with glutaraldehyde, 20μL / well, overnight at 4°C, pour off the coating solution the next day , repeatedly wash the tubes with PBST washing solution 3 times, 200 μL / well, add 200 μL 3% OVA to each tube to seal the PCR tube, incubate at 37°C for 1 hour, repeat washing the tubes 3 times with PBST washing solution, 200 μL / well, then add Put 10 μL of BDE-28 standard solution and 10 μL of appropriately diluted pAb-BDE-28 in a PCR tube, incubate at 37°C for 1 h, wash the tube three times with PBST washing solution, 200 μL / well; add 20 μL of biological reagent appropriately diluted with PBS to each tube Vein-goat anti-rabbit Ig...
Embodiment 3
[0057] Example 3 GO-BA-iPCR detection system detects BDE-28 in environmental samples
[0058] Utilize the GO-BA-iPCR detection system constructed by the present invention to detect PM 2.5 BDE-28 in which, PM 2.5 The samples were collected from the urban areas of Shanghai (S 1 -S 4 ), Suburbs (U 1 -U 4 ) and agricultural areas (C 1 -C 4 ), and the results are shown in Table 1. The actual sample of BDE-28 is also detected by GC-MS method for comparison, and the detection results of the two methods are compared, see the attached Figure 5 As shown, it can be seen that the detection value of BDE-28 has good consistency, and the correlation coefficient R 2 =0.9868, the GO-BA-iPCR detection system is accurate and reliable.
[0059] Table 1 Concentration of BDE-28 in different environmental samples
[0060]
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