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

Active Publication Date: 2018-10-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although graphene oxide has been used in many immunoassay detection methods, according to literature research, there is no application of graphene oxide in BA-iPCR method.

Method used

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  • Immune PCR detection method based on functional graphene oxide and application of immune PCR detection method
  • Immune PCR detection method based on functional graphene oxide and application of immune PCR detection method
  • Immune PCR detection method based on functional graphene oxide and application of immune PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses an immune PCR detection method based on functional graphene oxide and application of the immune PCR detection method. The method comprises the following steps: labeling biotinylated goat anti rabbit onto a graphene oxide carrier according to the traditional BA-iPCR technical principle, firstly preparing a functional graphene oxide probe, and establishing a BA-iPCR detectionsystem (GO-BA-iPCR) for the functional graphene oxide on the basis of the functional graphene oxide probe. The method has the advantages that second antibody is replaced with biotinylated second antibody modified graphene oxide, meanwhile a biotin-avidin combined system is utilized, so that detection signals are double magnified, the amount of biotinylated DNA on a solid phase carrier in a reaction system can be increased, the sensitivity of the detection method is further improved, and the method is applied to detection of trace pollutants in an environment medium.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to the preparation of functionalized graphene oxide probes, in particular to an immuno-PCR detection method based on functionalized graphene oxide and its application, which is used for trace amounts of 2,4,4'-trimonium oxide in environmental media Detection of brominated diphenyl ethers (BDE-28). Background technique [0002] Real-time fluorescence quantitative immuno-PCR (rt-iPCR) has many advantages such as strong specificity, ultra-high sensitivity, simple and fast operation, simple sample pretreatment steps, and low test cost, and is often used in biomedicine, food safety and other fields to trace Quantitative detection of target analytes. Biotin-avidin amplified immuno-PCR method (BA-iPCR) is a PCR-amplified fluorescent substance labeled template DNA as a reaction signal, and then uses a biotin-avidin binding system to connect the antibody to the template double-stranded DNA, Real-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804
CPCC12Q1/6804C12Q2531/113C12Q2563/131
Inventor 庄惠生马振
Owner SHANGHAI JIAO TONG UNIV
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