A Method Based on Enzymatic Click Reaction Signal Amplification Magnetic Relaxation Time Immunosensor for Detection of Veterinary Drug Residues
A click reaction and veterinary drug residue technology, applied to the detection of veterinary drug residues, based on enzymatic click reaction signal amplification, magnetic relaxation time immunosensor detection of veterinary drug residues, can solve the problem of unsuitable detection of small molecular substances, low sensitivity, and lack of signal amplification System and other issues
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Embodiment 1a
[0048] Response of embodiment 1aMRS and nMRS sensors to divalent copper ions
[0049] Azide-MNP 30 Conjugates and Alkynyl-MNPs 30 Preparation of conjugates: Take two 200μL 5mg / mL MNP 30 , add 20 μL of 10 mg / mL EDC and 10 μL of 10 mg / mL sulfo-NHS respectively, and shake the mixed solution at room temperature for 30 minutes to activate. After activation of the magnetic beads, add 500 μL of PBS solution (pH=7.4, 10 mM) to the mixed solution, and then add 20 μL of 10 mg / mL alkyne-PEG 4 -NH 2 and azide-PEG 4 -NH 2 , the mixed solution was reacted with mild shaking at room temperature for 1 hour for coupling. After coupling, magnetically separate three times to remove excess alkyne-PEG 4 -NH 2 and azide-PEG 4 -NH 2 . Azide-MNP 30 Conjugates and Alkynyl-MNPs 30 The conjugates were resuspended in 200 μL PBS solution and stored at 4°C for later use. We conjugated alkyne-PEG 4 -NH 2 and azide-PEG 4 -NH 2 MNP before and after 30 Infrared spectroscopy was carried out, a...
Embodiment 2
[0053] Example 2 Optimization of nMRS Immunosensor Reaction Conditions
[0054] Dilute alkaline phosphatase to different concentrations (100, 80, 10, 5, 2, 1 U / L), add 50 μL of 20 mM AAP solution to 50 μL of alkaline phosphatase solutions of different concentrations, and mix the solution at 37 ° C Gently vortex the reaction for 30 minutes. After the reaction is over, add 50 μL of 1 mM Cu to the mixed solution sequentially. 2+ solution, 50 μL 400 μg / mL alkynyl-MNP 1000 Conjugate and 50 μL of different concentrations (20, 40, 80 μg / mL) of azide-MNP 30 After the conjugate solution, shake and react at room temperature for 10 minutes, magnetically separate after the reaction, and take the MNP containing MNP that cannot be magnetically separated. 30 The solution measured T 2 value to optimize the azide-MNP 30 The concentration of the conjugate, the results are as follows Figure 7 shown.
[0055] Dilute alkaline phosphatase to different concentrations (50, 10, 5, 2, 1, 0.5, 0....
Embodiment 3
[0058] The response of embodiment 3nMRS sensor to alkaline phosphatase
[0059] First, dilute the alkaline phosphatase to different concentrations (1000, 500, 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0 U / L), in 50 μL of alkaline phosphatase solutions with different concentrations Add 50μL of 20mM AAP solution respectively, and the mixed solution was gently vortexed at 37°C for 30 minutes. After the reaction, 50μL of 1mM Cu was added to the mixed solution in turn. 2+ solution, 50 μL 40 μg / mL azide-MNP 30 Conjugate solution and 50 μL of 400 μg / mL alkynyl-MNP 1000 After the conjugate solution, shake and react at room temperature for 10 minutes, magnetically separate after the reaction, and take the MNP containing MNP that cannot be magnetically separated. 30 The solution measured T 2 value. Experimental results such as Figure 10 Shown, T 2 The variation of the value gradually increases with the concentration of ALP, indicating that T 2 There is a good response rela...
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