CN115786266A - Preparation method of lectin microarray chip and method for capturing cell exosomes - Google Patents
Preparation method of lectin microarray chip and method for capturing cell exosomes Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a preparation method of a lectin microarray chip and a method for capturing a cell exosome. The preparation method comprises the following steps: (1) Boiling the agarose solution until the agarose solution is completely dissolved, covering 1-5ml of the agarose solution on the surface of a substrate, and drying to obtain an agarose gel membrane substrate; (2) Activating the agarose gel membrane substrate in sodium periodate solution for 10-120min; (3) And (3) carrying out point sampling on the activated agarose gel membrane substrate by the prepared point sampling solution, and reacting to obtain the lectin modified agarose gel membrane microarray chip. The agarose gel membrane substrate is modified by agglutinin, and the obtained microarray chip can be seen through an AFM scanning picture, and the surface of the microarray chip presents a concave-convex structure, so that the specific surface area is large, and the capture of exosomes is facilitated.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a preparation method of a lectin microarray chip and a method for capturing cell exosomes.
Background
Exosomes (exosomes) are small vesicles of about 30-150 nm in diameter secreted by living cells, with a typical lipid bilayer structure. Exosomes carry important information such as various proteins, lipids and RNA, play an important role in the transmission of substances and information between cells, are expected to become early diagnosis markers of various diseases, and become one of the main targets of liquid biopsy. Currently, many techniques and devices based on physical (size, density) or biological (antibody antigen, aptamer) properties of exosomes have been developed for efficient enrichment and detection of exosomes. The current major methods for exosome extraction are: the differential centrifugation method has the defects that the requirement on equipment is high, time is consumed, the high-strength centrifugal force can cause mechanical damage to exosomes, and aggregation of lipoprotein and the like is easily caused; 2. density gradient centrifugation has the disadvantages of low sample processing capacity, high equipment requirement, time consumption, and mechanical damage to exosomes caused by high-strength centrifugal force; 3. the ultrafiltration membrane centrifugal separation method has the defects of low purity and easy membrane blockage;
the microarray chip is an important tool for the research of genomics, proteomics and glycomics at present, and thousands of different probe molecular lattices are constructed on the surface of a solid phase carrier with the magnitude of square centimeter mainly by a mechanical arm spotting technology or a microelectronic photoetching technology so as to realize the accurate, rapid and large-information-quantity detection of nucleic acid, protein, saccharide and other biological components, and the microarray chip has the advantages of high flux, automation, small sample consumption and the like; therefore, in order to solve the problems of obtaining exosomes, a method for preparing a lectin microarray chip and a method for capturing cell exosomes have been developed.
Disclosure of Invention
The first object of the present invention is to: aiming at the defects in the prior art, the preparation method of the lectin microarray chip is provided.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
a preparation method of lectin microarray chip comprises fixing lectin on agarose gel membrane substrate by Schiff base reaction to obtain lectin microarray chip; the preparation method comprises the following steps:
(1) Boiling 0.5-2wt% agarose solution to dissolve completely, covering 1-5ml agarose gel film substrate with thickness of 50-100nm on the surface of the agarose gel film substrate, and drying;
(2) Putting the agarose gel membrane substrate in the step (1) into 1-15mmol/L sodium periodate solution for activation for 10-120min;
(3) Spotting the activated agarose gel membrane substrate by the prepared spotting solution, reacting for 1-18h, and then blocking unreacted aldehyde groups by a PB buffer solution containing 0.1-1wt% of bovine serum albumin, 1-5 v/v of ethanolamine, 0.05-0.3mol/L of sodium chloride, pH7.5 and 0.01-0.2mol/L to obtain the lectin-modified agarose gel membrane microarray chip; wherein the sample solution comprises 0.01-0.2mol/L HEPES buffer solution with the pH value of 8.0 and agglutinin, and the concentration of the agglutinin in the sample solution is 0.05-4mg/mL; the HEPES buffer solution contains 0.001-0.01wt% of bovine serum albumin, 10-50% of glycerol, 0.01-0.05% of Tween-20 and 0.05-0.3mol/L of sodium chloride.
As an improved technical scheme, the boiling time in the step (1) is controlled to be 1-3min.
As an improved technical scheme, in the step (3), the activated agarose gel membrane substrate is spotted by using a spotting solution, and the reaction is carried out under the conditions that the temperature is 5-37 ℃ and the relative humidity is 10-60%.
As an improved technical scheme, the agglutinin in step (3) is lentil agglutinin (LCA), vitexin I (UEA-I), phaseolin (PHA) or ricin agglutinin (RCA).
A second object of the present invention is to: aiming at the defects in the prior art, the method for capturing exosomes in cell culture supernatant by the lectin microarray chip is provided.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
a method for capturing exosomes in cell culture supernatant by a lectin microarray chip comprises the following steps:
(1) Centrifuging the cell culture solution to be detected for the first time, continuously centrifuging the collected supernatant for the second time, continuously centrifuging the collected supernatant for the third time, filtering the collected supernatant, and performing ultrafiltration concentration on the collected filtrate to obtain a concentrated solution, namely a spare cell culture supernatant sample;
(2) And taking a spare cell culture supernatant sample, co-incubating the cell culture supernatant sample with the lectin-modified agarose gel membrane microarray chip, centrifuging and removing the supernatant to obtain the microarray chip with the exosomes captured.
As an improved technical scheme, the incubation temperature in the step (2) is 18-37 ℃, and the incubation time is 1-12h.
By adopting the technical scheme, compared with the prior art, the invention has the following advantages:
the invention coats a layer of agarose membrane on the substrate (glass sheet) to form the microarray carrier, the three-dimensional gel modified microarray carrier not only has simple manufacture, low cost and low fluorescence background signal, but also obviously improves the fixed amount of protein compared with the two-dimensional surface modified microarray carrier, and keeps the natural conformation and activity to a greater extent. The lectin is a non-enzymatic and non-antibody carbohydrate-binding protein, has the capacity of specifically binding specific glycosyl, and is used for modifying an agarose gel membrane substrate, so that the obtained microarray chip can be seen through an AFM scanning image, and the surface of the microarray chip presents a concave-convex structure, has a large specific surface area and is more beneficial to trapping exosomes; in addition, the exosomes secreted by tumor cells and the exosomes secreted by normal cells usually have the difference of surface glycosyl compound expression, so the lectin-modified microarray chip can well capture the exosomes of different subtypes, and further can be used for downstream analysis to further explain the heterogeneity among the exosomes.
Drawings
FIG. 1 is a SEM photograph of a substrate of a lectin microarray chip of example 2 of the present invention;
FIG. 2 is an AFM scan of the substrate in the lectin microarray chip of example 2 of the present invention;
FIG. 3 is a graph showing fluorescence signals of a lectin microarray in example 5 of the present invention;
FIG. 4 is a fluorescent amplification graph of example 5 of the present invention.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A lectin microarray chip is prepared by immobilizing lectin (lentil lectin) on an agarose gel membrane substrate by Schiff base reaction.
The preparation method comprises the following steps:
(1) Boiling 0.5wt% agarose solution (prepared from agarose and water) for 1min until completely dissolved, covering 1ml agarose gel film substrate with agarose coating thickness of 50nm on the substrate surface, and drying;
(2) Activating the agarose gel membrane substrate in the step (1) in 1mmol/L sodium periodate solution for 10min;
(3) Spotting the activated agarose gel membrane substrate by the prepared spotting solution under the conditions that the temperature is 5 ℃ and the relative humidity is 10 percent, after reacting for 1h, blocking unreacted aldehyde groups by a PB buffer solution containing 0.1wt percent of bovine serum albumin, 1 v/v percent of ethanolamine, 0.05mol/L of sodium chloride, pH7.5 and 0.01mol/L to obtain the lectin modified agarose gel membrane microarray chip; wherein the spotting solution comprises 0.01mol/L HEPES buffer solution with pH8.0 and lectin (lentil lectin), and the concentration of the lectin in the spotting solution is 0.05mg/mL; HEPES buffer solution contains 0.001wt% of bovine serum albumin, 10% v/v of glycerol, 0.01% v/v of Tween-20 and 0.05mol/L of sodium chloride.
The method for capturing exosomes in a human colon cancer cell SW480 culture supernatant sample by the lectin microarray chip comprises the following steps of:
(1) Collecting 20mL of cell culture solution of human colon cancer cells SW480, and centrifuging for 10min at 4 ℃ under the condition of 500g to remove dead cells in the culture solution; centrifuging the collected supernatant at 4 deg.C and 2000 Xg for 20min to remove cell debris; continuously centrifuging the collected supernatant for 30min at 4 ℃ and 20000 Xg to remove microvesicles, filtering the collected supernatant through a 0.22um filter membrane, and carrying out ultrafiltration concentration on the collected filtrate by using an ultrafiltration membrane with the aperture of 100Kd (centrifuging for 60min at 4 ℃ and 5000 Xg), wherein the collected concentrated solution is a cell culture supernatant sample to be detected;
(2) Taking a spare 2ml cell culture supernatant sample, co-incubating with the agglutinin modified agarose gel membrane microarray chip (incubation temperature is 25 ℃, incubation time is 12 h), centrifuging and removing the supernatant to obtain the microarray chip with the captured exosomes.
Example 2
A lectin microarray chip is prepared by immobilizing lectin (vitex negundo lectin I) on an agarose gel membrane substrate by Schiff base reaction.
The preparation method comprises the following steps:
(1) Boiling 1wt% agarose solution (prepared from agarose and water) for 1.5min until completely dissolved, covering 2.5ml agarose gel film substrate with agarose coating thickness of 65nm on the substrate surface, and drying;
(2) Putting the agarose gel membrane substrate in the step (1) into 5mmol/L sodium periodate solution for activation for 10-120min;
(3) Spotting the activated agarose gel membrane substrate by the prepared spotting solution under the conditions that the temperature is 15 ℃ and the relative humidity is 40 percent, and after 8 hours of reaction, blocking unreacted aldehyde groups by PB buffer solution containing 0.4wt percent of bovine serum albumin, 2.5 v/v percent of ethanolamine, 0.15mol/L of sodium chloride, pH7.5 and 0.05mol/L to obtain the lectin-modified agarose gel membrane microarray chip; wherein the spotting solution comprises 0.05mol/L HEPES buffer solution with pH of 8.0 and lectin (vitex bean agglutinin I), and the concentration of lectin in the spotting solution is 1.5mg/mL; HEPES buffer solution contains 0.005wt% bovine serum albumin, 25% v/v glycerol, 0.02% v/v Tween-20 and 0.15mol/L sodium chloride.
The method for capturing exosomes in the human colon cancer cell SW480 culture supernatant sample by the lectin microarray chip comprises the following steps:
(1) Collecting 20mL of cell culture solution of human colon cancer cells SW480, and centrifuging for 10min at 4 ℃ under the condition of 500 Xg to remove dead cells in the culture solution; centrifuging the collected supernatant at 4 deg.C and 2000 Xg for 20min to remove cell debris; continuously centrifuging the collected supernatant for 30min at the temperature of 4 ℃ and the temperature of 20000 Xg to remove the microvesicles, filtering the collected supernatant through a filter membrane of 0.22um, centrifuging the collected filtrate for 60min at the temperature of 4 ℃ and the temperature of 5000 Xg to perform ultrafiltration concentration on the collected filtrate by using an ultrafiltration membrane with the aperture of 100Kd, wherein the collected concentrated solution is a cell culture supernatant sample to be detected;
(2) Taking a spare 2ml cell culture supernatant sample, incubating the sample with the agglutinin modified agarose gel membrane microarray chip (incubation temperature is 30 ℃, incubation time is 8 h), centrifuging and removing the supernatant to obtain the microarray chip with the captured exosomes.
Example 3
A lectin microarray chip is prepared by immobilizing lectin (phaseolus vulgaris lectin) on an agarose gel membrane substrate by Schiff base reaction.
The preparation method comprises the following steps:
(1) Boiling 1.5wt% agarose solution (prepared from agarose and water) for 2min until completely dissolved, covering 3.5ml agarose gel film substrate with agarose coating thickness of 80nm on the substrate surface, and drying;
(2) Activating the agarose gel membrane substrate in the step (1) in a 10mmol/L sodium periodate solution for 80min;
(3) Spotting the activated agarose gel membrane substrate by prepared spotting solution under the conditions that the temperature is 37 ℃ and the relative humidity is 60 percent, after 12 hours of reaction, blocking unreacted aldehyde groups by PB buffer solution containing 0.8wt percent of bovine serum albumin, 3.5 v/v of ethanolamine, 0.2mol/L of sodium chloride, pH7.5 and 0.15mol/L to obtain the lectin modified agarose gel membrane microarray chip; wherein the spotting solution comprises 0.15mol/L HEPES buffer solution with pH8.0 and lectin (phaseolus vulgaris agglutinin), and the concentration of the lectin in the spotting solution is 2.5mg/mL; HEPES buffer solution contains 0.08wt% bovine serum albumin, 35% v/v glycerol, 0.03% v/v Tween-20 and 0.2mol/L sodium chloride.
The method for capturing exosomes in the human colon cancer cell HCT116 cell culture supernatant sample by the lectin microarray chip comprises the following steps:
(1) Collecting 20mL of cell culture solution of human colon cancer HCT116 cells, and centrifuging for 10min at 4 ℃ and 500 Xg to remove dead cells in the culture solution; centrifuging the collected supernatant at 4 deg.C and 2000 × g for 20min to remove cell debris; centrifuging the collected supernatant for 30min to remove the microvesicles under the conditions of 4 ℃ and 20000 Xg, filtering the collected supernatant through a filter membrane of 0.22um, and performing ultrafiltration concentration on the collected filtrate by using an ultrafiltration membrane with the aperture of 100Kd (centrifuging for 60min under the conditions of 4 ℃ and 5000 Xg), wherein the collected concentrated solution is a cell culture supernatant sample to be detected;
(2) Taking a spare 2ml cell culture supernatant sample, incubating the sample with the agglutinin modified agarose gel membrane microarray chip (incubation temperature is 18 ℃, incubation time is 1 h), centrifuging and removing the supernatant to obtain the microarray chip with the captured exosomes.
Example 4
A lectin microarray chip is prepared by immobilizing lectin (ricinus communis lectin) on an agarose gel membrane substrate by Schiff base reaction.
The preparation method comprises the following steps:
(1) Boiling 2wt% agarose solution (prepared from agarose and water) for 3min until the agarose solution is completely dissolved, covering 5ml agarose solution on the surface of the substrate, and drying to obtain agarose gel film substrate with agarose coating thickness of 100 nm;
(2) Activating the agarose gel membrane substrate in the step (1) in 15mmol/L sodium periodate solution for 120min;
(3) Spotting the activated sepharose gel membrane substrate by the prepared spotting solution under the conditions that the temperature is 25 ℃ and the relative humidity is 20 percent, after reacting for 18 hours, blocking unreacted aldehyde groups by a PB buffer solution containing 1wt percent of bovine serum albumin, 5 percent of ethanolamine by volume and 0.3mol/L of sodium chloride, the pH value of which is 7.5 and the pH value of which is 0.2mol/L, so as to obtain the lectin modified sepharose gel membrane microarray chip; wherein the spotting solution comprises 0.2mol/L HEPES buffer solution with pH8.0 and lectin (ricin lectin) at a concentration of 4mg/mL in the spotting solution; HEPES buffer solution contains 0.01wt% bovine serum albumin, 50% v/v glycerol, 0.05% v/v Tween-20 and 0.3mol/L sodium chloride.
The method for capturing exosomes in the culture supernatant sample of the human colon cancer cells SW620 by the lectin microarray chip comprises the following steps:
(1) Collecting 20mL of cell culture solution of human colon cancer cells SW620, and centrifuging for 10min at 4 ℃ and 500 Xg to remove dead cells in the culture solution; centrifuging the collected supernatant at 4 deg.C and 2000 Xg for 20min to remove cell debris; continuously centrifuging the collected supernatant for 30min at 4 ℃ and 20000 Xg to remove microvesicles, filtering the collected supernatant through a 0.22um filter membrane, performing ultrafiltration concentration on the collected filtrate by adopting an ultrafiltration membrane with the aperture of 100Kd (centrifuging for 60min at 4 ℃ and 5000 Xg), and collecting the concentrated solution which is a cell culture supernatant sample to be detected;
(2) Taking a spare 2ml cell culture supernatant sample, co-incubating with the agglutinin modified agarose gel membrane microarray chip (incubation temperature is 37 ℃, incubation time is 6 h), centrifuging and removing the supernatant to obtain the microarray chip with the captured exosomes.
Example 5
The microarray chip with exosomes captured in example 2 was used for downstream miRNA analysis, comprising the following steps:
(1) Extracting miRNA of exosome captured on the surface of a microarray by using a commercial miRNA extraction kit (sparkjade AC 1501), and the specific steps are as follows:
1) On the surface of the microarray chip, 1mL of lysine/Binding buffer was added, carefully blown, thoroughly lysed and mixed.
2) The mixture was left at room temperature for 5min to fully isolate the nucleic acid protein complex, and the lysate was transferred to a centrifuge tube.
3) 200 μ L of chloroform was added thereto, followed by vigorous shaking for 15 seconds, standing at room temperature for 2 to 3min, and centrifugation at 12,500 rpm (14,500 Xg) for 10 min.
4) Transferring the supernatant into a new centrifuge tube, adding 1.5 times of anhydrous ethanol, and mixing by vortex.
5) The mixture was applied to an adsorption column RA (adsorption column placed in collection tube), centrifuged at 12,000rpm (13,400 Xg) for 60 s and the waste liquid discarded.
6) Add 700. Mu.L Washsolution 1, 12,000rpm (13,400 Xg) and centrifuge for 30s, discard waste.
7) Add 500. Mu.L Washsolution 2/3, centrifuge at 12,000rpm (13,400 Xg) for 30s, discard waste and repeat the procedure.
8) The adsorption column RA was returned to the empty collection tube and centrifuged at 12,500 rpm (14,500 Xg) for 2 min.
9) Taking out the adsorption column RA, placing into an RNaseFreeFree centrifuge tube, and adding 30 μ L RNaseFree H in the middle part of the adsorption membrane 2 O。
10 Placed at room temperature for 1min,12,000 rpm (13,400 Xg), and centrifuged for 1 min.
(2) Real-time fluorescence quantification of mir-221-3p in exosomes was performed using stem-loop method RT-qPCR using a commercial kit (sparkjade AG0502, AH 0501), and the fluorescence amplification curve is shown in fig. 4, and the specific steps are as follows:
1) Thawing template RNA on ice, RNaseFree H 2 O was thawed at room temperature (15-25 deg.C), quickly placed on ice after thawing, and all components were placed on ice until ready for use.
2) Preparing the following mixed solution, lightly blowing and uniformly mixing by using a pipettor: total RNA 4. Mu.L, 5 XgDNAwipermix 2. Mu.L, RNaseFree H 2 O 4μL。
3) At 42 ℃ for 2min, and after the reaction is finished, the mixture is placed on ice.
4) Continuously and directly adding the following components into the same tube, and lightly blowing and mixing by using a pipettorHomogenizing: 10. Mu.L of the mixture of the previous step, RNaseFree H 2 O5. Mu.L, stem-loop primer 1. Mu.L, 10 × RTmix 2. Mu.L, SPARKSCIPTIIIenzymemx 2. Mu.L.
5) The first strand cDNA synthesis reaction was performed under the following conditions: 25 ℃ for 5min,50 ℃ for 15min,85 ℃ for 5min.
6) Preparing a PCR reaction solution: 2 × miRNA SYBR qPCR Master Mix 5 μ L, reaction solution 4 μ L of the previous step, forward primer 0.2 μ L, reverse primer 0.2 μ L, RNaseFree H 2 O 0.6μL。
7) qPCR amplification was performed under the following conditions: the fluorescence signal was collected at 97 ℃ for 2min for 40 cycles (94 ℃ for 5s,60 ℃ for 34 s).
The present patent is not limited to the above-mentioned embodiments, and those skilled in the art can make various changes without creative efforts from the above-mentioned conception, and fall within the protection scope of the present patent.
Claims (6)
1. A preparation method of a lectin microarray chip is characterized in that the lectin microarray chip is obtained by fixing lectin on an agarose gel membrane substrate by Schiff base reaction; the preparation method comprises the following steps:
(1) Boiling 0.5-2wt% agarose solution until it is completely dissolved, covering 1-5ml agarose gel film substrate with agarose coating thickness of 50-100nm on the surface of the substrate, and drying;
(2) Putting the agarose gel membrane substrate in the step (1) into 1-15mmol/L sodium periodate solution for activation for 10-120min;
(3) Spotting the activated agarose gel membrane substrate by the prepared spotting solution, reacting for 1-18h, and then blocking unreacted aldehyde groups by a PB buffer solution containing 0.1-1wt% of bovine serum albumin, 1-5 v/v of ethanolamine, 0.05-0.3mol/L of sodium chloride, pH7.5 and 0.01-0.2mol/L to obtain the lectin-modified agarose gel membrane microarray chip; wherein the sample solution comprises 0.01-0.2mol/L HEPES buffer solution with the pH value of 8.0 and agglutinin, and the concentration of the agglutinin in the sample solution is 0.05-4mg/mL; the HEPES buffer solution contains 0.001-0.01wt% of bovine serum albumin, 10-50% of glycerol, 0.01-0.05% of Tween-20 and 0.05-0.3mol/L of sodium chloride.
2. The method of preparing a lectin microarray chip as claimed in claim 1, wherein the boiling time in step (1) is controlled to 1-3min.
3. The method of claim 1, wherein the activated Sepharose membrane substrate is spotted using a spotting solution in the step (3) and the reaction is carried out at a temperature of 5 to 37 ℃ and a relative humidity of 10 to 60%.
4. The method of claim 1, wherein the lectin used in step (3) is selected from the group consisting of lentil lectin, vitellus agglutinin I, phaseolus vulgaris agglutinin, and ricin agglutinin.
5. A method for capturing extracellular secretion by using the lectin microarray chip obtained by the production method as claimed in claim 1, comprising the steps of:
(1) Centrifuging the cell culture solution to be detected for the first time, continuously centrifuging the collected supernatant for the second time, continuously centrifuging the collected supernatant for the third time, filtering the collected supernatant, and performing ultrafiltration concentration on the collected filtrate to obtain a concentrated solution, namely a spare cell culture supernatant sample;
(2) And taking a spare cell culture supernatant sample, co-incubating the cell culture supernatant sample with the lectin-modified agarose gel membrane microarray chip, centrifuging and removing the supernatant to obtain the microarray chip with the exosomes captured.
6. The method for capturing extracellular secretion using a lectin microarray chip as claimed in claim 5, wherein the incubation temperature in step (2) is 18 ℃ -37 ℃ for 1-12h.
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