Set of sulfamethazine specifically-bound aptamers and screening method and applications thereof
A technology of sulfamethazine and nucleic acid aptamer, which is applied in the field of nucleic acid aptamer that specifically binds to sulfamethazine and its screening, can solve the problems of limited use of antibodies, difficulty in reproducible production and the like, and achieves simple and convenient methods, Stable properties and small molecular weight
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Embodiment 1
[0058] Example 1. Screening of nucleic acid aptamers specifically binding to sulfamethazine
[0059] In this embodiment, SELEX technology is used for in vitro screening of nucleic acid aptamers, and sulfamethazine (SM 2 ) and Sulfamethazine (Sulfamerazine, SMR) are coupled with magnetic beads (Magneticbeads, MBs) to obtain SM 2 -MBs and SMR-MBs; and with SM 2 -MBs are positive screening targets, SMR-MBs are reverse screening targets, and a magnetic bead-assisted-exponential amplification ligand phylogenetic evolution screening system (Mag-SELEX) for sulfamethazine nucleic acid aptamer screening was established , the nucleic acid aptamer specifically binding to sulfamethazine was obtained from a random oligonucleotide library synthesized in vitro through 9 rounds of screening. Specific steps are as follows:
[0060] 1. Coupling of sulfamethazine, sulfamethazine and carboxyl magnetic beads
[0061] (1) Take 100 μL of carboxylated magnetic beads (about 0.6 μmol) into a 2 mL c...
Embodiment 2
[0087] Embodiment 2, the dissociation constant of nucleic acid aptamer is measured by ultrafiltration
[0088] The reaction equilibrium process was simulated by ultrafiltration, and the dissociation constant (Kd) of each nucleic acid aptamer SA04, SA06, SA07 and SA41 obtained in Example 1 was determined. Specific steps are as follows:
[0089] 1. Dilute the aptamers SA04, SA06, SA07 and SA41 with binding buffer to concentrations of 3 μM, 1.6 μM, 0.8 μM, 0.4 μM, 0.2 μM, 0.1 μM and 0.05 μM, respectively.
[0090] 2. Denature at 95°C for 6 minutes, then quickly place on ice for 10 minutes, and then let stand at room temperature for 5 minutes.
[0091] 3. Take 100 μL of the above-mentioned nucleic acid aptamers at each concentration and add to an equal volume of 20 μM sulfamethazine solution (the solvent is binding buffer), and incubate at 25° C. for 30 minutes.
[0092] 4. Add the entire amount of the reaction solution to Ultra-0.5 Centrifugalfilter (10KD), and centrifuge at 12...
Embodiment 3
[0098] Embodiment 3, competition method determines the specificity of nucleic acid aptamer
[0099] 1. Take sulfamethazine (SM 2 ), sulfamethazine (SMR) and sulfamethazine (SDZ) standard stock solutions were diluted with binding buffer to a final concentration of 4 μM.
[0100] 2. Dissolve 5 μg of nucleic acid aptamers SA07 and SA41 in 100 μL of drug-free binding buffer, denature at 95°C for 6 minutes, quickly place on ice for 10 minutes, and then let stand at room temperature for 5 minutes.
[0101] 3. Add 100 μL of sulfamethazine, sulfamethazine and sulfamethazine prepared in step 1 to the nucleic acid aptamer solution prepared in step 2 to obtain incubation solutions respectively, and incubate at 25°C for 30 minutes. The incubation solution contains unbound nucleic acid aptamers, unbound drugs and drug-nucleic acid aptamer complexes.
[0102] 4. Add the full amount of incubation solution to 7×10 7 Sulfamethazine-coated magnetic beads were incubated at 25°C for 30 min. F...
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