Deoxynivalenol aptamer affinity column as well as preparation method and application thereof

A technology of aptamers and affinity columns, applied in chemical instruments and methods, separation methods, preparation of test samples, etc., can solve problems such as poor linearity of standard curves, large coefficient of variation, matrix interference, etc., to reduce Cross-reactivity, high purification efficiency, and high-affinity effects

Inactive Publication Date: 2017-10-20

BEIJING MEIZHENG BIOLOGICAL TECH

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AI-Extracted Technical Summary

Problems solved by technology

However, there are the following problems in GC chromatography: poor linearity of the standard curve, response drift, retention and memory...

Abstract

The invention relates to a deoxynivalenol aptamer affinity column as well as a preparation method and an application thereof. The affinity purification column adopts a chemical modified solid-phase carrier, a nucleic acid aptamer of deoxynivalenol and the carrier are subjected to covalent coupling, and then the affinity column is filled. The aptamer affinity column is mainly used for purifying deoxynivalenol in food samples, feed samples, milk samples, blood samples and other various samples, so that following HPLC (high performance liquid chromatography) detection and fluorescence detection of deoxynivalenol in the samples can be facilitated.

Application Domain

Other chemical processesPreparing sample for investigation +1

Technology Topic

Solid phasesMilk sample +8

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Examples

Experimental program(6)

Example Embodiment

[0040] Example 1: Preparation of affinity column using amino-modified deoxynivalenol aptamer

Example Embodiment

[0041] A preferred embodiment of the present invention for preparing the deoxynivalenol aptamer affinity column is as follows:

[0042] 1. Carrier activation

[0043] Select N-hydroxysuccinimide (NHS) modified agarose carrier sepharose4B for activation

[0044] Take 1g of N-hydroxysuccinimide-modified agarose, add 50ml of 1mM HCl, swell for 30min, wash the gel 6 times with 100ml of 1mM HCl, and then wash with 100ml of ultrapure water

[0045] 2. The activated agarose gel sepharose4B with coupling buffer (0.1M NaHCO 3 , 0.8M NaCl, pH8.2) washed 3 times. Add 20mol/L of amino-modified deoxynivalenol aptamer, and couple at room temperature for 8 hours

[0046] 3. Wash the coupled deoxynivalenol aptamer-agarose carrier with 20 mM phosphate buffered saline PBS at pH 7.4 for 3 times

[0047] 4. Close

[0048] Add 100mmol/L Tris.ClPH8.0 to the coupled deoxynivalenol aptamer-agarose carrier, and react at room temperature for 2 hours

[0049] 5. Wash the blocked deoxynivalenol aptamer-agarose carrier with 20mM, PH7.4 phosphate buffered saline PBS three times

[0050] 6. Load the deoxynivalenol-agarose carrier into the chromatography column as required, and prepare deoxynivalenol affinity purification columns of different capacities as required

[0051] 7. Pack the column

[0052] The cross-linked deoxynivalenol-agarose gel was resuspended in 10 ml of 20 mM PBS pH 7.4, and then loaded into an empty affinity purification cartridge

[0053] 1) Take the empty cylinder, add the lower sieve plate, add 1ml PBS, and let it dry naturally

[0054] 2) Add the plug of the sample outlet below, add 1 ml of the above-treated deoxynivalenol aptamer-agarose carrier to the column, stand still for 5 minutes, and allow the carrier to settle naturally

[0055] 3) Add the upper sieve plate and press the sieve plate to make it reach the top of the carrier

[0056] 4) Add the piston adapter, the adapter has a pressing effect, so that the upper sieve plate is close to the carrier

[0057] 5) Put the stabilizing cuff on the column from below so that it is close to the injection port

[0058] 6) Pull out the plug of the sample outlet, use a syringe to take 5ml of PBS, connect the syringe to the sample inlet, slowly inject PBS into the affinity column, and keep the liquid output rate at 1-2 drops/second. Until all the liquid is injected into the affinity column

[0059] 7) Put on the sample outlet plug, put on the sample inlet plug, and store the prepared deoxynivalenol aptamer affinity column at 4°C.

Example Embodiment

[0060] Example 2: Preparation of affinity column using biotin-modified deoxynivalenol aptamer

[0061] A preferred embodiment of the present invention for preparing the deoxynivalenol aptamer affinity column is as follows:

[0062] 1. Take 1ml of streptavidin (SA) modified agarose gel sepharose4B, wash 3 times with 10ml of ultrapure water

[0063] 2. The (SA)-modified Sepharose 4B was washed 3 times with coupling buffer (0.02M in PBS, 0.8M NaCl, pH 7.4). Add 20mol/L biotin-modified deoxynivalenol aptamer, and couple at room temperature for 4 hours

[0064] 3. Wash the coupled deoxynivalenol aptamer-agarose carrier with 20 mM phosphate buffered saline PBS at pH 7.4 for 3 times

[0065] 4. Packing the column

[0066] The deoxynivalenol-agarose carrier is loaded into the chromatography column as required, and different capacity deoxynivalenol affinity purification columns can be prepared as required.

[0067] 1) Take the empty cylinder, add the lower sieve plate, add 1ml PBS, and let it dry naturally

[0068] 2) Add the plug of the sample outlet below, add 1 ml of the above-treated deoxynivalenol aptamer-agarose carrier to the column, stand still for 5 minutes, and allow the carrier to settle naturally

[0069] 3) Add the upper sieve plate and press the sieve plate to make it reach the top of the carrier

[0070] 4) Add the piston adapter, the adapter has a pressing effect, so that the upper sieve plate is close to the carrier

[0071] 5) Put the stabilizing cuff on the column from below so that it is close to the injection port

[0072] 6) Pull out the plug of the sample outlet, use a syringe to take 5ml of PBS, connect the syringe to the sample inlet, slowly inject PBS into the affinity column, and keep the liquid output rate at 1-2 drops/second. Until all the liquid is injected into the affinity column

[0073] 7) Put on the sample outlet plug, put on the sample inlet plug, and store the prepared deoxynivalenol aptamer affinity column at 4°C.

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Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

Simple and efficient operation
Improve efficiency

Deoxynivalenol aptamer affinity column as well as preparation method and application thereof

AI-Extracted Technical Summary

Problems solved by technology

Abstract

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Examples

Example Embodiment

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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