CN109929832A - A kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material - Google Patents
A kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material Download PDFInfo
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- 229910052681 coesite Inorganic materials 0.000 claims description 7
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 6
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- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical group NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims description 5
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- 238000002360 preparation method Methods 0.000 claims description 4
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Abstract
The present invention provides a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material, belongs to field of biotechnology.The present invention uses dispersive solid-phase extraction mode, the specific steps are as follows: 1) first balances DNA enrichment material using sample solution, which is mixed with biological sample with mass ratio 100-1000:1, hatch 0.5-720min, filtering or centrifuge separation, abandon upper mixed layer clear liquid, collect precipitating；2) it is mixed, is hatched -60 minutes 0.5 minute with precipitating using 1-6 cleaning solution, filtering or centrifuge separation abandon supernatant liquor, collect precipitating；3) it is mixed, is hatched -60 minutes 0.5 minute with precipitating using pH 8-13 eluent, supernatant is collected in filtering or centrifuge separation, obtains ctDNA, and above-mentioned whole process is carried out at 10-60 DEG C, and the DNA enrichment material is bi-component polymeric.Method of the invention shows the features such as highly selective and high-throughput in terms of extracting Circulating tumor DNA, and efficiently separating and being enriched with for Circulating tumor DNA may be implemented.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of enrichment of bi-component polymeric functionalization material to extract circulation
The method of Tumour DNA.
Background technique
Circulating tumor DNA, i.e. ctDNA (circulating tumor DNA), refer to tumour cell by Apoptosis or
The DNA fragmentation that person's necrosis generates discharges into the circulatory system.CtDNA is present in the body fluid such as blood, synovial fluid and cerebrospinal fluid.By
It is 1 hour or so in ctDNA half-life period, can accurately reflect tumour present case, so ctDNA can be used as the non-of tissue biopsy
Invasive alternative solution.In addition, ctDNA plays a role in early diagnosis of cancer, prognosis and measurement drug therapy reaction.
The ctDNA mutation detection techniques of mainstream include based on PCR amplification technique and based on DNA sequence dna detection technique two at present
Class.But whether which kind of sequencing technologies, requires all to be enriched with DNA and extracted, to reduce to amplification or subsequent sequencing
Interference.
The main extracting method of ctDNA is mainly based upon silicon ball, the solid phase extraction method of magnetic ball and based on phenol-chlorine
Imitative liquid-liquid extraction method.Main problem is extraction efficiency and reproducibility.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of side of bi-component polymeric functionalization material enrichment cycles Tumour DNA
Method.
A kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material, using dispersive solid-phase extraction
Mode, the specific steps are as follows:
1) DNA enrichment material is first balanced using sample solution, by the DNA enrichment material and biological sample with mass ratio 100-
1000:1 mixing hatches 0.5-720min, filtering or centrifuge separation, abandons supernatant liquor, collects precipitating；
2) it is mixed, is hatched -60 minutes 0.5 minute with precipitating using 1-6 cleaning solution, upper layer is abandoned in filtering or centrifuge separation
Clear liquid collects precipitating；
3) it is mixed, is hatched -60 minutes 0.5 minute with precipitating using pH 8-13 eluent, filtering or centrifuge separation are received
Collect supernatant, obtain ctDNA, above-mentioned whole process is carried out at 10-60 DEG C；
The DNA enrichment material is bi-component polymeric functionalization material.
The bi-component polymeric, structure are as follows:
Wherein R is carboxyl, nitro, methyl, group-4 ethyl formate or hydrogen；X=0.01-0.5.
The material of its substrate is Si, Cu, Ag, Au, Pt, CuO, Fe3O4, porous SiO2, porous Al2O3, porous TiO2Or it is more
Hole ZrO2In any one or two or more arbitrary proportions mixing.
The bi-component polymeric functionalization material the preparation method comprises the following steps: utilizing atom transition free radical polymerization reaction machine
System, bi-component functional copolymer is grafted on inorganic semiconductor, metal or metal oxide surface, it is made to obtain intelligent response
Property polymers function surface；The inorganic semiconductor is Si or SiO2In in any proportion mixed of any one or two kinds
It closes.The metal is the mixing of any one or two or more arbitrary proportions in Au, Ag or Pt, the metal oxidation
Object is CuO, Al2O3、TiO2、ZrO2Or Fe3O4In any one or two or more arbitrary proportions mixing.
The preparation method of the bi-component polymeric functionalization material specifically: sequentially added in the flask of 25mL
0.4mmol N-isopropylacrylamide, the thiocarbamide function monomer of 0.1mmol, the two molar ratio is 4:1, while 6mL H is added2O with
And 6mL DMF makees solvent；It is passed through nitrogen under stiring, after sufficiently dissolving to monomer, catalyst is added under nitrogen protection
CuBr 0.032g and PMDETA or bipyridine ligand 0.16mL, following reaction system vacuumize-inflated with nitrogen, remove reactant
Remaining oxygen in system；By the processed Si of bromination, SiO2、Au、Ag、Pt、CuO、Al2O3、TiO2、ZrO2Or Fe3O4In appoint
The mixture for one or two kinds of any of the above ratio of anticipating immerses configured reaction solution；The temperature of flask is controlled at 60 DEG C
Stand reaction 4-6 hours；DMF, H are used after reaction2O successively washing copolymer grafted surface, obtains bi-component copolymer,
The functionalized surface of this bi-component copolymer with a thickness of 10-50nm, be dried with nitrogen surface be placed on it is spare in vacuum desiccator.
The thiocarbamide function monomer structural formula are as follows:
The partial size of the bi-component polymeric functionalization material is 0.2-50 μm, and aperture is
The biological sample is blood, synovial fluid, cerebrospinal fluid or urine.
The sample solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile, methanol or ethyl alcohol, is had
Machine acid is formic acid, acetic acid or trifluoroacetic acid, and organic solvent volume concentration is 50-90%, pH=1-6.
The cleaning solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile, methanol or ethyl alcohol, is had
Machine acid is formic acid, acetic acid or trifluoroacetic acid, and organic solvent volume concentration is 50-90%, pH=1-6.
The eluent is the mixed liquor of organic solvent, organic base and water, and organic solvent is acetonitrile, methanol or ethyl alcohol, is had
Machine acid is ammonium hydroxide or triethylamine, and organic solvent volume concentration is 10-50%, pH=8-13.
The invention has the benefit that invention method shown in terms of extracting Circulating tumor DNA it is highly selective with
Efficiently separating and being enriched with for Circulating tumor DNA may be implemented in the features such as high-throughput.
Specific embodiment
Embodiment 1
Bi-component polymeric functionalization material structure is as follows:
Wherein R is carboxyl, nitro, methyl, group-4 ethyl formate or hydrogen；X=0.01-0.5.
In the present invention, base material is inorganic semiconductor, metal or metal oxide.The inorganic semiconductor be Si or
SiO2In any one or two kinds of mixing in any proportion.The metal be Au, Ag or Pt in any one or
The mixing of the two or more arbitrary proportions of person, the metal oxide are CuO, Al2O3、TiO2、ZrO2Or Fe3O4In it is any one
The mixing of kind or two or more arbitrary proportions.
By taking X=0.2 as an example, 0.4mmol N-isopropylacrylamide is sequentially added in 25ml three-necked flask, 0.1mmol's
Thiocarbamide function monomer, the two molar ratio is 4:1, while 6mL deionized water is added and 6mLDMF makees solvent；It is passed through under stiring
After sufficiently dissolving to monomer, catalyst CuBr 0.032g and bipyridyliums ligand is added in nitrogen under nitrogen protection
0.16mL, following reaction system vacuumize-inflated with nitrogen, remove oxygen remaining in reaction system；By the processed substrate of bromination
Immerse configured reaction solution；The temperature control of flask is reacted 4-24 hours in 60-80 DEG C of standing；It uses after reaction
DMF and deionized water successively washing copolymer grafted surface, obtain Circulating tumor DNA enrichment material, the enrichment of this Circulating tumor DNA
Material surface bi-component copolymer layer with a thickness of 10-80nm, be dried with nitrogen surface be placed on it is spare in vacuum desiccator.
In the present embodiment, the base material of selection is silica gel, structural formula are as follows:
Embodiment 2
A kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material, is prepared using embodiment 1
Bi-component polymeric functionalization material；Using dispersive solid-phase extraction mode, the specific steps are as follows:
1) DNA enrichment material is first balanced using sample solution, the DNA enrichment material and blood is mixed with mass ratio for 100:1
It closes, hatches 12 hours, filtering or centrifuge separation abandon upper mixed layer clear liquid, collect precipitating；
2) it is mixed, is hatched 60 minutes with precipitating using pH 1-6 cleaning solution, supernatant liquor is abandoned in filtering or centrifuge separation,
Collect precipitating；
3) it being mixed, is hatched 60 minutes with precipitating using pH 8-13 eluent, supernatant is collected in filtering or centrifuge separation,
CtDNA is obtained, above-mentioned whole process is carried out at 30 DEG C,
The sample solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile, and organic acid is formic acid,
Organic solvent volume concentration is 50-90%, pH=1-6.
The cleaning solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile, and organic acid is formic acid,
Organic solvent volume concentration is 50-90%, pH=1-6.
The eluent is the mixed liquor of organic solvent, organic base and water, and organic solvent is acetonitrile, and organic acid is ammonium hydroxide,
Organic solvent volume concentration is 10-50%, pH=8-13.
It is evaluated by standard items, after the extraction of DNA enrichment material, the extraction recovery of DNA is 84%, and coverage is
86%.
Embodiment 3
A kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material, is prepared using embodiment 1
Bi-component polymeric functionalization material；Using dispersive solid-phase extraction mode, the specific steps are as follows:
1) DNA enrichment material is first balanced using sample solution, with mass ratio is 500:1 by the DNA enrichment material and cerebrospinal fluid
Mixing is hatched 6 hours, and filtering or centrifuge separation abandon upper mixed layer clear liquid, collect precipitating；
2) it is mixed, is hatched 30 minutes with precipitating using pH 1-6 cleaning solution, supernatant liquor is abandoned in filtering or centrifuge separation,
Collect precipitating；
3) it being mixed, is hatched 30 minutes with precipitating using pH 8-13 eluent, supernatant is collected in filtering or centrifuge separation,
CtDNA is obtained, above-mentioned whole process is carried out at 10 DEG C,
The sample solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is ethyl alcohol, and organic acid is trifluoro second
Acid, organic solvent volume concentration are 50-90%, pH=1-6.
The cleaning solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is ethyl alcohol, and organic acid is trifluoro second
Acid, organic solvent volume concentration are 50-90%, pH=1-6.
The eluent is the mixed liquor of organic solvent, organic base and water, and organic solvent is ethyl alcohol, and organic acid is three second
Amine, organic solvent volume concentration are 10-50%, pH=8-13.
It is evaluated by standard items, after the extraction of DNA enrichment material, the extraction recovery of DNA is 83%, and coverage is
85%.
Embodiment 4
A kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material, is prepared using embodiment 1
Bi-component polymeric functionalization material；Using dispersive solid-phase extraction mode, the specific steps are as follows:
1) DNA enrichment material is first balanced using sample solution, with mass ratio is 1000:1 by the DNA enrichment material and synovial fluid
Mixing, hatches 0.5 minute hour, and filtering or centrifuge separation abandon upper mixed layer clear liquid, collect precipitating；
2) it is mixed using pH 1-6 cleaning solution with precipitating, hatches 0.5 minute minute, upper layer is abandoned in filtering or centrifuge separation
Clear liquid collects precipitating；
3) it is mixed, is hatched 0.5 minute minute with precipitating using pH 8-13 eluent, filtered or be centrifugated, in collection
Clear liquid obtains ctDNA, and above-mentioned whole process is carried out at 60 DEG C,
The sample solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is methanol, and organic acid is acetic acid,
Organic solvent volume concentration is 50-90%, pH=1-6.
The cleaning solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is methanol, and organic acid is acetic acid,
Organic solvent volume concentration is 50-90%, pH=1-6.
The eluent is the mixed liquor of organic solvent, organic base and water, and organic solvent is methanol, and organic acid is three second
Amine, organic solvent volume concentration are 10-50%, pH=8-13.
It is evaluated by standard items, after the extraction of DNA enrichment material, the extraction recovery of DNA is 87%, and coverage is
89%.
Claims (10)
1. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material, it is characterised in that using dispersion
Solid Phase Extraction mode, the specific steps are as follows:
1) DNA enrichment material is first balanced using sample solution, by the DNA enrichment material and biological sample with mass ratio 100-1000:1
Mixing hatches 0.5-720min, filtering or centrifuge separation, abandons upper mixed layer clear liquid, collects precipitating；
2) it being mixed, is hatched -60 minutes 0.5 minute with precipitating using 1-6 cleaning solution, supernatant liquor is abandoned in filtering or centrifuge separation,
Collect precipitating；
3) it is mixed, is hatched -60 minutes 0.5 minute with precipitating using pH 8-13 eluent, filtered or be centrifugated, in collection
Clear liquid obtains ctDNA,
Above-mentioned whole process is carried out at 10-60 DEG C,
The DNA enrichment material is bi-component polymeric functionalization material.
2. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 1,
It is characterized in that the bi-component polymeric functionalization material, structure are as follows:
Wherein R is carboxyl, nitro, methyl, group-4 ethyl formate or hydrogen；X=0.01-0.5；
The material of the substrate of the bi-component polymeric functionalization material is Si, Cu, Ag, Au, Pt, CuO, Fe3O4, porous SiO2、
Porous Al2O3, porous TiO2Or porous ZrO2In any one or two or more arbitrary proportions mixing.
3. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 1,
It is characterized in that the bi-component polymeric functionalization material the preparation method comprises the following steps: utilizing atom transition free radical polymerization reaction machine
System, bi-component functional copolymer is grafted on inorganic semiconductor, metal or metal oxide surface, it is made to obtain intelligent response
Property polymers function surface；The inorganic semiconductor is Si or SiO2In in any proportion mixed of any one or two kinds
It closes；The metal is the mixing of any one or two or more arbitrary proportions in Au, Ag or Pt；The metal oxidation
Object is CuO, Al2O3、TiO2、ZrO2Or Fe3O4In any one or two or more arbitrary proportions mixing.
4. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 3,
It is characterized in that, the preparation method of the bi-component polymeric functionalization material specifically: sequentially added in the flask of 25mL
0.4mmol N-isopropylacrylamide, the thiocarbamide function monomer of 0.1mmol, the two molar ratio is 4:1, while 6mL H is added2O with
And 6mL DMF makees solvent；It is passed through nitrogen under stiring, after sufficiently dissolving to monomer, catalyst is added under nitrogen protection
CuBr 0.032g and PMDETA or bipyridine ligand 0.16mL, following reaction system vacuumize-inflated with nitrogen, remove reactant
Remaining oxygen in system；By the processed Si of bromination, SiO2、Au、Ag、Pt、CuO、Al2O3、TiO2、ZrO2Or Fe3O4In appoint
The mixture for one or two kinds of any of the above ratio of anticipating immerses configured reaction solution；The temperature of flask is controlled at 60 DEG C
Stand reaction 4-6 hours；DMF, H are used after reaction2O successively washing copolymer grafted surface, obtains bi-component copolymer；This
The functionalized surface of bi-component copolymer with a thickness of 10-50nm, be dried with nitrogen surface be placed on it is spare in vacuum desiccator.
5. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 4,
It is characterized in that the thiocarbamide function monomer structural formula are as follows:
6. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 1,
It is characterized in that the partial size of the bi-component polymeric functionalization material is 0.2-50 μm, aperture is
7. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 1,
It is characterized in that the biological sample is blood, synovial fluid, cerebrospinal fluid or urine.
8. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 1,
It is characterized in that the sample solution be organic solvent, organic acid and water mixed liquor, organic solvent be acetonitrile, methanol or ethyl alcohol,
Organic acid is formic acid, acetic acid or trifluoroacetic acid, and organic solvent volume concentration is 50-90%, pH=1-6.
9. a kind of method that Circulating tumor DNA is extracted in the enrichment of bi-component polymeric functionalization material according to claim 1,
It is characterized in that the cleaning solution be organic solvent, organic acid and water mixed liquor, organic solvent be acetonitrile, methanol or ethyl alcohol,
Organic acid is formic acid, acetic acid or trifluoroacetic acid, and organic solvent volume concentration is 50-90%, pH=1-6.
10. the side that Circulating tumor DNA is extracted in a kind of bi-component polymeric functionalization material enrichment according to claim 1
Method, it is characterised in that the eluent is the mixed liquor of organic solvent, organic base and water, and organic solvent is acetonitrile, methanol or second
Alcohol, organic acid are ammonium hydroxide or triethylamine, and organic solvent volume concentration is 10-50%, pH=8-13.
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