Triple signal amplification magnetic relaxation sensing immunoassay method
A technology of immune analysis and signal amplification, which is applied in the field of detection and analysis, can solve the problems of low sensitivity and poor stability, and achieve the effect of high sensitivity, strong stability and accurate detection results
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Embodiment 1
[0061] The characterization results before and after the coupling of gold nanoparticles in embodiment 1
[0062] Take 2 mL of AuNPs solution with pH 8.0 (concentration 0.5 mg / mL), add chlorpyrifos monoclonal antibody (200 μL, 0.15 mg / mL) and react at 37 °C for 60 min; then add iDNA (1 μM, 500 μL) at 4 °C React for 16h; then add 3mL Tris-HCl buffer (pH=8.2) to the above mixture and continue to react for 24h at 4°C; finally centrifuge the above mixture (9500r / min, 40min, 4°C) and wash with PBST 3 times. The gold nanoparticles were characterized by TEM, particle size and potential. The results are as follows: Figure 1 to Figure 3 As shown, the absorption spectrum and particle size of gold nanoparticles changed after coupling, indicating that Ab-AuNPs-iDNA was successfully prepared.
[0063] Under this condition, the amount of Ab coupled to gold nanoparticles is 30 μg, and the amount of iDNA is 0.5 nmol. By adding different amounts of monoclonal antibodies and iDNA, gold nanopa...
Embodiment 2 3
[0064] Example 2 Triple Signal Amplification Magnetic Relaxation Sensor for Fe 3+ change of state
[0065] Take the prepared Ab-AuNPs-iDNA 200μL (concentration 0.5mg / mL) and mix with bio-H1, bio-H2 (50μL, 0.5μM), and react at 37℃ for 90min; then add 100μL of HRP with different dilution ratios -SA (dilution ratio 1:1000, 1:10000) continues to react at 37°C for different time (0, 2, 5, 10, 20, 30min); then add 100μL of 25mM dopamine hydrochloride solution to the above mixture, 37°C Under reaction 10min. The result is as Figure 4 As shown, the more HRP-SA, the faster the conversion rate of dopamine hydrochloride into polydopamine. And when HRP-SA is replaced by streptavidin-labeled alkaline phosphatase (ALP-SA), it cannot promote the rate of dopamine hydrochloride conversion into polydopamine, so Fe 3+ status did not change, indicating that it has high specificity ( Figure 5 ). Gel electrophoresis also proves that iDNA triggers the HCR reaction, the first three lanes are ...
Embodiment 3
[0067] The optimization of embodiment 3 detection conditions
[0068]The chlorpyrifos complete antigen was coated on the microtiter plate and blocked, and then 100 μL of different concentrations of chlorpyrifos standard solution and the same volume of Ab-AuNPs-iDNA solution were added (the amount of Ab coupled to gold nanoparticles was 10, 20, 30 , 40 μg, and the amount of iDNA is 0.5 nmol), react at 37°C for 20 minutes; after washing three times, add 50 μL of 0.5 μM bio-H1 and bio-H2 and continue to react at 37°C for 90 minutes; after washing three times, add diluted 100 μL of HRP-SA with a multiple of 1:1000 continued to react at 37°C for 10 min; after washing five times, add 100 μL of 25 mM dopamine hydrochloride solution and continue to react at 37°C for 10 min; after washing five times, add 200 μL of 0.2 mM Fe 3+ The solution continued to react at 37°C for 10 min; the supernatant was taken for T 2 Value detection to optimize Ab dosage. The result is as Figure 8 As sh...
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