Multi-protein simultaneous online enrichment detection method based on multiple nucleic acid aptamers
A nucleic acid aptamer and protein technology, which is applied in the field of high-efficiency, high-selectivity enrichment and online sensitive detection, can solve problems such as simultaneous detection of multiple proteins, and achieve improved selective recognition ability, good mechanical strength, and small non-specific adsorption. Effect
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Embodiment 1
[0023] The capillary hydrophilic monolithic column modified with thrombin nucleic acid aptamer and histone nucleic acid aptamer is prepared according to the following procedure:
[0024] The sulfhydryl-modified thrombin nucleic acid aptamer and histone nucleic acid aptamer (the sulfhydryl group and the nucleic acid aptamer are connected by spacer arms, that is, 6 methylene groups, purchased from Bao Biological Engineering (Dalian) Co., Ltd.) in equimolar ratios (1:1) dissolved in nucleic acid aptamer buffer PBS, in which the final concentration of each nucleic acid aptamer was 25 μM, and continuously passed into a nano-gold doped capillary hydrophilic monolithic column with an inner diameter of 200 μm and a length of 10 cm, at room temperature Reaction 4h.
[0025] The preparation method of nano-gold doped GMA-PEGDA capillary hydrophilic monolithic column: the GMA-PEGDA capillary monolithic column was continuously fed with 1M cysteamine solution (dissolved in 1M NaOH solution)...
Embodiment 2
[0026] The capillary hydrophilic monolithic column modified with thrombin nucleic acid aptamer and cytochrome C nucleic acid aptamer is prepared according to the following procedure:
[0027] The aldehyde-modified thrombin nucleic acid aptamer and cytochrome C nucleic acid aptamer (the modified aldehyde group and the nucleic acid aptamer are connected by spacer arms, that is, 18 methylene groups, purchased from Bao Biological Engineering (Dalian) Co., Ltd. company) was dissolved in the aptamer buffer PBS at a molar ratio (1:4), wherein the concentration of the thrombin aptamer was 5 μM, and the concentration of the cytochrome C aptamer was 20 μM, and the aptamer with amino groups on the surface was continuously introduced GMA-PEGDA capillary hydrophilic monolithic material with an inner diameter of 100 μm and a length of 6 cm (the preparation method of the hydrophilic monolithic column is basically the same as in Example 1, the difference is that 10 mg / mL of PEI (Mn=600) is add...
Embodiment 3
[0029] Using the capillary hydrophilic monolithic column modified with thrombin nucleic acid aptamer and histone nucleic acid aptamer prepared in Example 1 as a liquid chromatography column, thrombin (T), histone extract (H) and thrombin and The mixture (T+H) of histone extracts is detected separately, and the specific determination steps are:
[0030]1) On the liquid chromatography system, aptamer buffer PBS was used as the loading buffer, and the flow rate was 2.4 μL / min. Load 10 ng / μL of thrombin (T), histone extract (H) and 20 μL of the mixture of thrombin and histone extract (T+H) in sequence, and wash with 2M NaClO 4 Gradient elution was carried out for 10 minutes as the elution solution; within 10 minutes, the percentage of the elution phase to the total volume of the elution phase and the loading buffer was changed from 0% to 100%. Adopt ultraviolet detector to detect, detection wavelength is 280nm, the result is as follows figure 1 shown. figure 1 Among them, when ...
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