CN110836965A - Sealing liquid for liquid chip, sealing method and application - Google Patents
Sealing liquid for liquid chip, sealing method and application Download PDFInfo
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- CN110836965A CN110836965A CN201911154930.1A CN201911154930A CN110836965A CN 110836965 A CN110836965 A CN 110836965A CN 201911154930 A CN201911154930 A CN 201911154930A CN 110836965 A CN110836965 A CN 110836965A
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- liquid
- preservative
- magnetic beads
- sealing
- phosphate buffer
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- 239000007788 liquid Substances 0.000 title claims abstract description 53
- 238000007789 sealing Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims description 17
- 239000011324 bead Substances 0.000 claims abstract description 53
- 239000003755 preservative agent Substances 0.000 claims abstract description 24
- 230000002335 preservative effect Effects 0.000 claims abstract description 24
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 150000001413 amino acids Chemical class 0.000 claims abstract description 16
- 239000004094 surface-active agent Substances 0.000 claims abstract description 14
- 238000000576 coating method Methods 0.000 claims abstract description 12
- 230000000903 blocking effect Effects 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 27
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 18
- 235000018102 proteins Nutrition 0.000 claims description 16
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 15
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 15
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 15
- 235000001014 amino acid Nutrition 0.000 claims description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 11
- 229940098773 bovine serum albumin Drugs 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 10
- 239000004471 Glycine Substances 0.000 claims description 8
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 7
- 239000004472 Lysine Substances 0.000 claims description 7
- 239000005018 casein Substances 0.000 claims description 7
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 7
- 235000021240 caseins Nutrition 0.000 claims description 7
- 235000013922 glutamic acid Nutrition 0.000 claims description 7
- 239000004220 glutamic acid Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000000565 sealant Substances 0.000 claims description 7
- 239000004475 Arginine Substances 0.000 claims description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 6
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 claims description 5
- 241000251468 Actinopterygii Species 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 5
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 239000003761 preservation solution Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical group [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 3
- 238000004166 bioassay Methods 0.000 claims description 2
- 230000012447 hatching Effects 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 18
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 108010017384 Blood Proteins Proteins 0.000 abstract description 2
- 102000004506 Blood Proteins Human genes 0.000 abstract description 2
- 239000004005 microsphere Substances 0.000 description 31
- 239000006148 magnetic separator Substances 0.000 description 21
- 238000005516 engineering process Methods 0.000 description 19
- 239000003153 chemical reaction reagent Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 238000001514 detection method Methods 0.000 description 10
- 102000004987 Troponin T Human genes 0.000 description 7
- 108090001108 Troponin T Proteins 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000007836 KH2PO4 Substances 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000019800 disodium phosphate Nutrition 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a sealing liquid for a liquid chip, which comprises: phosphate buffer, protein, amino acid, surfactant, and preservative. According to the invention, a sealing link of coated antibody magnetic beads is added in the traditional liquid chip magnetic bead coating process, and amino acid and protein in sealing liquid can be well combined with gaps on the magnetic beads, so that nonspecific combination of serum protein is reduced, the reaction background is reduced, and the sensitivity and precision of the reaction are improved.
Description
Technical Field
The invention relates to the technical field of biological detection reagents, in particular to sealing liquid, a sealing method and application for a liquid chip technology.
Background
The liquid chip technology is also called liquid phase suspension chip technology, multifunctional flow type lattice technology, liquid phase chip analysis technology, flow type fluorescence detection technology, multifunctional multi-index parallel analysis technology, suspension array technology, etc. The method organically integrates a plurality of scientific and technological achievements such as a fluorescent coding microsphere technology, a laser analysis technology, a flow cytometry technology, a high-speed digital signal processing technology, a computer algorithm and the like, has the advantages of free combination, high flux, high speed, low cost, high accuracy, good repeatability, high sensitivity, wide linear range, no need of washing, simple and convenient operation, capability of completing the detection of protein and nucleic acid on the same platform and the like, represents the development direction of basic research of life science and medical diagnosis technology, and is evaluated as one of trend technologies of clinical diagnosis by international industry experts.
The analysis system of liquid chip technology uses carboxylated microballoons as the reaction substrate, and the system can utilize a small amount of sample in a test tube or a micro-porous plate to simultaneously carry out a plurality of different immunoassays. The microspheres used in the system are 5.6 mu m plastic microspheres or 6.5 mu m magnetic microspheres produced by Luminex company, a plurality of microspheres with different codes are obtained by mixing two or three different red fluorescein molecules in different proportions, the microspheres are combined with a reporter molecule with another fluorescein in the reaction process, and the two fluoresceins on the microspheres can be detected by a multifunctional flow type dot-matrix instrument of the Luminex company, so that the aim of simultaneously detecting a plurality of target molecules is fulfilled. There are currently 500 different microspheres that can detect up to 500 different analytes simultaneously.
At present, in the conventional magnetic bead coating method, after the microspheres are activated by NHS and EDC, the microspheres are combined and coupled with protein through self carboxyl to obtain magnetic beads coated with antibodies, the coated microspheres are directly stored in phosphate buffer solution containing 1% BSA to play a role in sealing and storing, and the sealing of the coated magnetic beads is not carried out independently, so that in the reaction process, gaps on the microspheres cannot be completely and fully sealed, the background of the reaction is higher, the sensitivity of the reaction is influenced, and meanwhile, the precision, the accuracy and the accuracy of the reaction are influenced to a certain extent.
Disclosure of Invention
Therefore, the present invention is directed to overcome the drawbacks of the prior art, and to provide a sealing liquid for liquid chip, a sealing method and applications thereof.
In order to achieve the above object, a first aspect of the present invention provides a sealing liquid for a liquid chip, wherein the sealing liquid comprises: phosphate buffer solution, protein, amino acid, surfactant and preservative. The blocking solution according to the first aspect of the present invention, wherein the phosphate buffer solution has a pH of 7.0 to 7.4;
preferably, the concentration of the phosphate buffer is 0.005M to 0.05M, preferably 0.01 to 0.05M, most preferably 0.01M.
A sealant fluid according to the first aspect of the present invention, wherein the protein is selected from one or more of: bovine serum albumin, casein, fish gelatin; preferably bovine serum albumin;
more preferably, the concentration of the protein is 1-10% by mass, preferably 3% by mass.
A sealant fluid according to the first aspect of the present invention, wherein the amino acid is selected from one or more of: glycine, glutamic acid, arginine, lysine; preferably glycine;
more preferably, the mass percentage concentration of the amino acid is 1-10%, preferably 2%.
The sealant fluid according to the first aspect of the present invention, wherein the surfactant is selected from one or more of: tween-20, Triton x-100, BRIJ 35; preferably Tween-20;
more preferably, the mass percentage concentration of the surfactant is 0.05-0.1%, preferably 0.05%.
The sealing liquid according to the first aspect of the present invention, wherein the preservative is a liquid preservative and/or a solid preservative;
preferably, the liquid preservative is proclin 300, and the solid preservative is merthiolate and/or sodium azide;
more preferably, the volume percentage concentration of the liquid preservative is 0.05-0.2%, preferably 0.1%, and the mass percentage concentration of the solid preservative is 0.05-0.1%.
The second aspect of the present invention provides a magnetic bead coating method for a liquid chip, the method comprising the steps of:
(1) washing the magnetic beads with water;
(2) activating the magnetic beads;
(3) coating antibody and hatching;
(4) sealing the magnetic beads obtained in the step (3) with the sealing liquid described in the first aspect;
(5) and (4) preserving the magnetic beads obtained in the step (4) by using a preservation solution.
The method according to the second aspect of the present invention, wherein the magnetic bead preservation solution comprises: 10mM phosphate buffer pH7.4, 1% BSA by mass and 0.05% Tween 20 by mass.
In a third aspect of the invention, a kit is provided, which comprises a blocking solution as described in the first aspect and fluorescent magnetic beads.
The fourth aspect of the present invention provides the use of the blocking solution of the first aspect and/or the kit of the third aspect for the preparation of a liquid chip product for bioassay
Aiming at the defects in the prior art, the invention aims to provide a preparation method of a sealing liquid for improving the magnetic bead performance of an antibody coated by a liquid chip technology, which reduces the reaction background of a detection method by increasing the sealing after the antibody is coated by magnetic beads, thereby improving the sensitivity of the method and simultaneously improving the precision and the accuracy of the reaction.
The invention also aims to provide a preparation method of the confining liquid for improving the performance of the magnetic beads of the antibody coated by the liquid chip technology, which is simple and convenient to operate and suitable for large-scale popularization and application.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a confining liquid for improving the performance of antibody-coated magnetic beads in a liquid chip technology comprises the following components: phosphate buffer, protein, amino acid, surfactant, liquid preservative or solid preservative.
Preferably, the pH of the phosphate buffer is 7.0-7.4.
More preferably, the concentration of the phosphate buffer is 0.01-0.05M.
Preferably, the protein is at least one of bovine serum albumin, casein, fish gelatin and the like.
More preferably, the protein concentration is 1-10%.
Preferably, the amino acid is one of glycine, glutamic acid, arginine and lysine.
More preferably, the concentration of the amino acid is 1-10%.
Preferably, the surfactant is one of Tween-20, Triton x-100 and BRIJ 35.
More specifically, the concentration of the surfactant is 0.05-0.1%.
Preferably, the liquid preservative is proclin 300. The solid antiseptic is selected from sodium thiomersalate, sodium azide, etc.
More preferably, the concentration of the liquid preservative is 0.05-0.2%, and the concentration of the solid preservative is 0.05-0.1%.
In order to improve the performance of magnetic beads coated with antibodies, the invention provides a preparation method of a confining liquid, which comprises the components of 0.01-0.05M phosphate buffer, 1-5% of protein, 1-10% of amino acid, 0.05-0.1% of surfactant, 0.05-0.2% of liquid preservative or 0.05-0.1% of solid preservative.
As a further scheme, the confining liquid for improving the performance of the coated antibody magnetic beads in the liquid chip technology comprises 0.01M phosphate buffer, 3.0% of protein, 2% of amino acid, 0.05% of surfactant and 0.1% of proclin 300.
As a further development, the phosphate buffer according to the invention preferably has a pH of 7.0 to 7.4.
As a further research scheme, the protein provided by the invention is at least one of bovine serum albumin, casein, fish gelatin and analogues thereof. Bovine serum albumin is preferred.
As a further research scheme, the amino acid is one of glycine, glutamic acid, arginine and lysine, and glycine is preferred.
As a further research scheme, the surfactant is one of Tween-20, Triton x-100 and BRIJ 35. Tween 20 is preferred.
As a further embodiment, the phosphate buffer of the present invention is 0.01M phosphate buffer pH7.4 comprising 0.358g Na2HPO4 & 12H2O, 0.024g KH2PO4, 0.8g NaCl, 0.02g KCl, 100ml purified water.
The sealant fluid of the present invention may have, but is not limited to, the following beneficial effects:
according to the invention, a sealing link of coated antibody magnetic beads is added in the traditional liquid chip magnetic bead coating process, and amino acid and protein in sealing liquid can be well combined with gaps on the magnetic beads, so that nonspecific combination of serum protein is reduced, the reaction background is reduced, and the sensitivity and precision of the reaction are improved.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
The reagents and instrumentation used in the following examples are as follows:
reagent:
Na2HPO4·12H2O，KH2PO4NaCl, KCl were purchased from chemical reagents of national drug group, Inc.;
bovine serum albumin was purchased from Proliant;
glycine was purchased from Amresco;
tween 20 was purchased from national drug group chemical reagents, Inc.;
proclin 300 was purchased from sigma.
The instrument comprises the following steps:
a multi-functional flow dot matrix Luminex200 is available from Luminex.
Example 1
This example illustrates the preparation of phosphate buffer.
Preparing a phosphate buffer solution, accurately weighing 0.358g of Na2HPO4 & 12H2O, 0.024g of KH2PO4, 0.8g of NaCl and 0.02g of KCl reagent by using an electronic balance, putting the mixture into a 100ml beaker, adding 80% of purified water, uniformly stirring, adjusting the pH value to 7.4, and then fixing the volume for later use.
Example 2
This example illustrates the formulation of a sealant according to the present invention.
Preparing confining liquid for improving the performance of coated antibody magnetic beads, weighing 3g of bovine serum albumin and 2g of glycine, weighing 0.05ml of Tween 20 and 0.1ml of proclin 300 by using a pipette, adding a reagent into 80ml of phosphate buffer, and after complete dissolution, using a volumetric flask to fix the volume to 100ml by using the prepared phosphate buffer.
Example 3
This example is intended to illustrate the blocking method of the present invention, and the blocking solution in example 2 is taken as an example.
Antibody coating process of magnetic beads:
3.1 materials for experiments:
magnetic beads: purchased from Luminex corporation 1.25X107/ml,1mL。
Activating reagent: 100mM sodium dihydrogen phosphate pH 6.3.
Reaction reagents: 50mM MES, pH 5.0.
Magnetic bead preservation solution: 10mM phosphate buffer, pH7.4, 1% BSA, 0.05% Tween 20.
3.2 coating process:
3.2.1 washing of microspheres
(1) Resuspending the magnetic microspheres, shaking at 2000rpm for 15min, transferring 5.0X 106Magnetic microspheres were put into centrifuge tubes.
(2) And (3) placing the centrifuge tube containing the magnetic beads into a magnetic separator, standing for 30-60 seconds, removing the supernatant, and paying attention not to touch the magnetic beads.
(3) The tube was removed from the magnetic separator, 200. mu.L of purified water was added, vortexed for 20 seconds, and sonicated for approximately 10 seconds
(4) The centrifuge tube containing the magnetic beads was placed in a magnetic separator and allowed to stand for 60 seconds, the tube was placed in a magnetic separator, and the supernatant was removed. The microspheres were not sucked, and if they were sucked, they were immediately pumped back into the centrifuge tube and allowed to stand for 60 seconds.
(5) Repeating the steps 5, 6 and 7 for 2 times.
3.2.2 activated microspheres
(1) The tube was removed from the magnetic separator, 80 μ L of activating reagent was added, vortexed for approximately 20 seconds, and sonicated for 10 seconds.
(2) Add 10. mu.L of Sulfo-NHS to the microsphere solution and vortex for 20 seconds.
(3) Add 10. mu.L EDC to the microsphere solution and vortex mix for 20 seconds.
(4) The tube was mixed well on a shaker at 1500rpm for 20 minutes, and the reaction was carefully protected from light.
(5) The centrifuge tube containing the magnetic beads was placed in a magnetic separator and allowed to stand for 60 seconds, the tube was placed in a magnetic separator, and the supernatant was removed. Care was taken not to suck the microspheres.
3.2.3 coating antibodies
(1) The tube was removed from the magnetic separator, the microspheres were resuspended using 250. mu.L of reaction reagent, vortexed for approximately 20 seconds, and sonicated for 10 seconds.
(2) Placing the centrifuge tube containing the magnetic beads into a magnetic separator and standing for 60 seconds
(3) The tube was placed in a magnetic separator and the supernatant removed. Care was taken not to suck the microspheres.
(4) Repeating the steps 1, 2 and 3 for 2 times.
(5) The tube was removed from the magnetic separator, and the microspheres were resuspended using 100. mu.L of reaction reagent, vortexed for approximately 20 seconds, and sonicated for 10 seconds.
(6) Add the corresponding concentration of antibody to the microsphere solution, then add the reagent solution to a final volume of 500. mu.L, vortex mix.
(7) Incubate with mixing (shaking) at room temperature, 1500rpm, shake for 2h in the dark.
(8) The centrifuge tube containing the antibody magnetic beads was placed in a magnetic separator and allowed to stand for 60 seconds. The tube was still placed in the magnetic separator and the supernatant was removed. Care was taken not to suck the microspheres.
3.2.4 sealing
(1) The tube was removed from the magnetic separator, the microspheres were resuspended using 500. mu.L of magnetic bead stock solution, and vortexed for approximately 20 seconds.
(2) The centrifuge tube containing the antibody magnetic beads was placed in a magnetic separator and allowed to stand for 60 seconds. The tube was still placed in the magnetic separator and the supernatant was removed. Care was taken not to suck the microspheres.
(3) And (5) repeating the steps 1, 2 and 3.
(4) The tube was removed from the magnetic separator and the microspheres were resuspended using 1mL of blocking solution prepared in example 2 and shaken for 1 h.
3.2.5 storing
(1) The tube was removed from the magnetic separator, the microspheres were resuspended using 500. mu.L of magnetic bead stock solution, and vortexed for approximately 20 seconds.
(2) The centrifuge tube containing the antibody magnetic beads was placed in a magnetic separator and allowed to stand for 60 seconds. The tube was still placed in the magnetic separator and the supernatant was removed. Care was taken not to suck the microspheres.
(3) And (5) repeating the steps 1, 2 and 3.
(4) The tube was removed from the magnetic separator, and the microspheres were resuspended in 1mL of magnetic bead storage solution and stored at 2-8 ℃.
Test example 1
In the multifunctional flow type dot matrix instrument detection system, troponin T is taken as a detection target.
Troponin T antibodies were coated on magnetic beads using the method of example 3. The blocking solution used was that obtained in example 2.
Comparative example 1
Comparative example 1 is different from example 3 in that there is no blocking step in the comparative example and the beads are directly stored after coating.
Comparative example 2
Comparative example 2 the coating process was identical to that of example 3, the blocking liquids used were as follows: after 500uL of blocking solution reagent A (100 mM lysine (Amresco) dissolved in PBS at pH 7.4) and 500uL of blocking solution reagent B (100 mM 3-Thiyl propionic acid (Thermo) dissolved in PBS at pH 7.4) were mixed well, incubation and shaking were performed for 1 hour using a 50mM Tris buffer in a magnetic bead stock solution containing: casein (sigma): 1 wt%, BSA (prolitant): 0.5 wt%, Tween 20 (national medicine): 0.5 wt%, EDTA-K2 (Chinese medicine, 100 g): 10mM, PEG (Shanghai future reagent, 250 g): 1 wt%, proclin 300(sigma, 400 ml): 0.1% (v/v).
A10 ng/ml calibrator was prepared, diluted to various concentrations, and the fluorescence values were measured, and the results are shown in Table 1.
TABLE 1 fluorescence values of calibrators of different concentrations
| Concentration of calibrator (ng/ml) | Application example 1 | Comparative example 1 | Comparative example 2 |
| 0 | 19.5 | 32 | 28 |
| 0.1 | 131 | 93.5 | 87 |
| 0.5 | 597 | 428 | 511.5 |
| 1 | 1123.5 | 876 | 1021 |
| 2 | 2014 | 1574.5 | 1897 |
| 5 | 3876.5 | 2838 | 3246 |
| 10 | 5634 | 4251 | 3957 |
TABLE 2 comparison of fluorescence values of blank spots measured in test example 1 and comparative examples 1 and 2
Table 3 comparison of precision and accuracy of high and low quality control materials measured in test example 1 and comparative examples 1 and 2
From tables 1 and 2, it can be seen that the background of the curve in test example 1 after the addition of the blocking step is the lowest, and when the blank limit is measured, the blank limit in test example 1 is significantly lower than that in the two comparative examples, and the sensitivity of troponin T to the used marker has a large influence on the detection result, so that it can be seen that the sensitivity of the antibody coated by the magnetic beads is greatly improved by using the method of the present invention, while the sensitivity of the product can be improved by using the blocking solution of comparative example 2, but the influence on the curve is large, and when the concentration reaches 5ng/ml, the curve tends to a plateau stage and is not suitable for the blocking of the magnetic beads in the technical field.
As can be seen from the data in table 3, the repeatability and relative deviation of the low-value quality control and the high-value quality control in test example 1 are significantly lower than those in the two comparative examples, which indicates that after the addition of the blocking step, the nonspecific reaction in the serum is reduced, the reaction is more stable, and the precision and accuracy of the reaction are improved. Therefore, the confining liquid can effectively improve the sensitivity and precision of the product in a liquid chip system.
Test example 2
In the multifunctional flow type dot matrix instrument detection system, troponin T is taken as a detection target.
Troponin T antibodies were coated on magnetic beads using the method of example 3.
In this test example, the preparation procedure of the blocking solution was substantially the same as that in example 2, except that 3g of bovine serum albumin, 2g of glutamic acid, 0.05ml of Tween 20 and 0.1ml of proclin 300 were added to the blocking solution, and the volume was finally adjusted to 100 ml. The results are shown in tables 4 and 5:
TABLE 4 blank limit determination
TABLE 5 repeatability determination
As can be seen from tables 4 and 5, glutamic acid and lysine were found to be almost effective, and both the sensitivity and accuracy of the product were improved.
Test example 3
In the multifunctional flow type dot matrix instrument detection system, troponin T is taken as a detection target.
Troponin T antibodies were coated on magnetic beads using the method of example 3.
In this test example, a blocking solution was prepared in substantially the same manner as in example 2, except that 1g of casein, 2g of arginine, 0.05ml of Triton x-100, and 0.1ml of proclin 300 were added to the blocking solution to a volume of 100 ml. The results are shown in tables 6 and 7:
TABLE 6 blank limit determination
TABLE 7 repeatability determination
As can be seen from tables 6 and 7, the blank limit of the coated magnetic beads is significantly lower than that of comparative example 1 by using the blocking solution of the formulation, which shows that the blocking solution can increase the sensitivity and accuracy of the magnetic beads.
In the above examples and alternatives of the blocking solution, the protein may be bovine serum albumin, casein or fish gelatin.
The amino acids in the above examples and alternatives of the blocking solution may be glycine, glutamic acid, arginine, lysine.
The surfactant in the above embodiments and alternatives of the blocking solution may be Tween-20, Triton x-100, BRIJ 35.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Claims (10)
1. A confining liquid for a liquid chip, the confining liquid comprising: phosphate buffer solution, protein, amino acid, surfactant and preservative.
2. The sealing solution according to claim 1, wherein the phosphate buffer has a pH of 7.0 to 7.4;
preferably, the concentration of the phosphate buffer is 0.005M to 0.05M, preferably 0.01 to 0.05M, most preferably 0.01M.
3. The sealant fluid according to claim 1 or 2, wherein the protein is selected from one or more of the following: bovine serum albumin, casein, fish gelatin; preferably bovine serum albumin;
more preferably, the concentration of the protein is 1-5% by mass, preferably 3% by mass.
4. The confining liquid according to any one of claims 1 to 3, wherein the amino acid is selected from one or more of the following: glycine, glutamic acid, arginine, lysine; preferably glycine;
more preferably, the mass percentage concentration of the amino acid is 1-10%, preferably 2%.
5. The sealant fluid according to any one of claims 1 to 4, wherein the surfactant is selected from one or more of: tween-20, Triton x-100, BRIJ 35; preferably Tween-20;
more preferably, the mass percentage concentration of the surfactant is 0.05-0.1%, preferably 0.05%.
6. The sealing liquid according to any one of claims 1 to 5, wherein the preservative is a liquid preservative and/or a solid preservative;
preferably, the liquid preservative is proclin 300, and the solid preservative is merthiolate and/or sodium azide;
more preferably, the volume percentage concentration of the liquid preservative is 0.05-0.2%, preferably 0.1%, and the mass percentage concentration of the solid preservative is 0.05-0.1%.
7. A magnetic bead coating method for a liquid chip is characterized by comprising the following steps:
(1) washing the magnetic beads with water;
(2) activating the magnetic beads;
(3) coating antibody and hatching;
(4) blocking the magnetic beads obtained in the step (3) by using the blocking solution of any one of claims 1 to 6;
(5) and (4) preserving the magnetic beads obtained in the step (4) by using a preservation solution.
8. The method of claim 7, wherein the magnetic bead preservation solution comprises: 10mM phosphate buffer pH7.4, 1% BSA by mass and 0.05% Tween 20 by mass.
9. A kit comprising the blocking solution of any one of claims 1 to 6 and fluorescent magnetic beads.
10. Use of the blocking solution of any one of claims 1 to 6 and/or the kit of claim 9 for the preparation of a liquid chip product for bioassay.
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| CN113311154A (en) * | 2021-05-28 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Coupling method, diluent and application of immunomagnetic particles |
| CN117949272A (en) * | 2024-01-31 | 2024-04-30 | 宁波瑞源生物科技有限公司 | Pretreatment method of streptavidin magnetic beads for electrochemiluminescence detection |
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