CN116338162B - Sample application liquid, protein chip prepared by using sample application liquid and preparation method of protein chip - Google Patents
Sample application liquid, protein chip prepared by using sample application liquid and preparation method of protein chip Download PDFInfo
- Publication number
- CN116338162B CN116338162B CN202310277631.7A CN202310277631A CN116338162B CN 116338162 B CN116338162 B CN 116338162B CN 202310277631 A CN202310277631 A CN 202310277631A CN 116338162 B CN116338162 B CN 116338162B
- Authority
- CN
- China
- Prior art keywords
- sample application
- application liquid
- protein
- sample
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000007788 liquid Substances 0.000 title claims abstract description 79
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 70
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims description 10
- 239000000243 solution Substances 0.000 claims abstract description 67
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000003906 humectant Substances 0.000 claims abstract description 22
- 239000003223 protective agent Substances 0.000 claims abstract description 21
- 239000004094 surface-active agent Substances 0.000 claims abstract description 20
- 239000006177 biological buffer Substances 0.000 claims abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 20
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 20
- 229960003237 betaine Drugs 0.000 claims description 20
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 19
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 18
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 17
- 239000007995 HEPES buffer Substances 0.000 claims description 17
- 239000013504 Triton X-100 Substances 0.000 claims description 14
- 229920004890 Triton X-100 Polymers 0.000 claims description 14
- 239000000758 substrate Substances 0.000 claims description 14
- 238000007789 sealing Methods 0.000 claims description 12
- 241000283707 Capra Species 0.000 claims description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 7
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 229920002674 hyaluronan Polymers 0.000 claims description 6
- 229960003160 hyaluronic acid Drugs 0.000 claims description 6
- 239000007993 MOPS buffer Substances 0.000 claims description 5
- 229920002307 Dextran Polymers 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 239000005862 Whey Substances 0.000 claims description 3
- 102000007544 Whey Proteins Human genes 0.000 claims description 3
- 108010046377 Whey Proteins Proteins 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 238000005842 biochemical reaction Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 230000014509 gene expression Effects 0.000 claims description 2
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 125000000647 trehalose group Chemical group 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 98
- 230000000052 comparative effect Effects 0.000 description 52
- 238000006243 chemical reaction Methods 0.000 description 22
- 238000001514 detection method Methods 0.000 description 21
- 102000011683 Centromere Protein B Human genes 0.000 description 15
- 108010076305 Centromere Protein B Proteins 0.000 description 15
- 238000000018 DNA microarray Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 239000012530 fluid Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 239000008118 PEG 6000 Substances 0.000 description 3
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 3
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000000891 luminescent agent Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000661 sodium alginate Substances 0.000 description 3
- 235000010413 sodium alginate Nutrition 0.000 description 3
- 229940005550 sodium alginate Drugs 0.000 description 3
- ZPFAVCIQZKRBGF-UHFFFAOYSA-N 1,3,2-dioxathiolane 2,2-dioxide Chemical compound O=S1(=O)OCCO1 ZPFAVCIQZKRBGF-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000005281 excited state Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003234 fluorescent labeling method Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229960005150 glycerol Drugs 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000004377 microelectronic Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- -1 protectant Substances 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005303 weighing Methods 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
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a sample application liquid, and relates to the technical field of immunoassay. The sample application liquid comprises a surfactant, a protective agent, a humectant and a biological buffer solution. The invention also provides a method for preparing the protein chip by using the sample application liquid. According to the invention, the sample application liquid is prepared by mixing the surfactant, the protective agent, the humectant and the biological buffer solution, the protein chip is prepared by adopting the prepared sample application liquid, when the prepared protein chip is used for detecting the substance to be detected, the sample information displayed in the picture collected by the CCD is very clear, the hollow problem is avoided, and the accuracy of the immunodetection reagent is improved.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to sample application liquid, a protein chip prepared by using the sample application liquid and a preparation method thereof.
Background
The biochip technology is a high-new technology formed by the comprehensive intersection of physics, microelectronics and molecular biology, and integrates a discontinuous analysis process in the life science field on a micro biochemical analysis system on the surface of a silicon chip or a glass chip according to the principle of specific interaction between molecules by a micro technology, and thousands of information molecules related to life can be integrated on a centimeter square chip by a micro processing technology, so that the high-efficiency and rapid test and analysis of bioactive substances such as genes, ligands, antigens and the like are realized. By using the biochip technology, a plurality of indexes of the object to be detected can be checked at a time. Depending on the biological material solidified on the chip, the biochip can be classified into a gene chip, a protein chip, a polysaccharide chip, and a neuron chip.
Protein chip is to fix trace protein onto specific solid carrier in certain array, to react with biological sample to be detected, such as tissue, cell, body fluid, etc. and to combine some specific protein with the protein on the surface of the chip, and to combine the combined protein with some signal amplifying matter and to detect signal via instrument to obtain the result. Compared with the traditional detection method, the method has the advantages of high throughput and parallel analysis, can provide a large amount of information in one experiment, can comprehensively and accurately analyze a group of related proteins, and meanwhile, has a very small required sample size, which is incomparable with the traditional single protein detection method.
The spotting method is the most commonly used method for preparing protein chips, i.e. the immobilized probes are synthesized before spotting, and the synthesized samples are transferred to a substrate by a spotting instrument. The sample application method is divided into contact sample application and non-contact sample application, wherein the non-contact sample application method is to directly spray the sample to the surface of the substrate through a capillary tube by using a piezoelectric principle, and has the advantages of high sample application precision and high speed; the contact sample application method is to dip the sample needle directly into the sample pool, and when the needle contacts with the surface substrate, the sample will adhere to the surface of the substrate due to the change of the surface energy among the needle, the liquid and the substrate, the contact sample application has wide applicability, many samples can be applied, and the sample application process is safe.
The conventional method for detecting a sample by using a protein chip mainly comprises a chemiluminescent method, a fluorescent labeling method and a chromogenic method, wherein chemiluminescent refers to the phenomenon that a luminescent agent absorbs chemical energy generated in the reaction process during a chemical reaction, electrons in a product molecule or an intermediate molecule of the reaction are transited to an excited state, and when the electrons return to a ground state from the excited state, energy is released in the form of emitted photons to generate luminescence. The chemiluminescence method is to mark a luminescent agent on an antigen or an antibody, enable the luminescent agent to emit photons through direct reaction or enzyme catalysis, detect a reaction system through collection of luminescent signals, collect detection results by using CCD, and judge the concentration of a specific marker antibody in a detected sample through the intensity of the optical signals. The chemiluminescence method has the advantages of high flux, high sensitivity, microminiaturization, small sample consumption and the like, is clinically accepted gradually in screening, auxiliary diagnosis and treatment monitoring of tumors, infectious diseases, autoimmune diseases and allergic diseases, and has wide application prospect.
When the chemiluminescence method is used for detecting the sample, the protein can be diluted by the sample application liquid, the sample application liquid containing the protein is subjected to sample application on the chip matrix to manufacture a protein chip, the manufactured protein chip is used for detecting the detected sample, and finally the CCD is used for collecting the displayed picture. However, when the sample is detected by using the method in practice, due to factors such as the fact that the existing spotting liquid is volatile, the protein property or the conformation changes, the display picture collected by the CCD often has a hollow problem, namely the recognition degree of the edge area of the displayed picture is higher, and the recognition degree of the middle area is lower, so that the result is abnormal. Therefore, the problem of hollowing out the display image in the detection result of the protein chip becomes a problem to be solved.
Disclosure of Invention
The invention aims to provide a sample application liquid and a protein chip manufactured by using the sample application liquid. Because the sample application liquid has stable properties, the protein chip prepared by using the sample application liquid has better stability, so that the sample application liquid is not easy to volatilize when the protein chip is used for detecting a sample, and the picture displayed by the detection result is regular and clear, thereby solving the problem of hollowness in the display result.
One of the purposes of the present invention is to provide a spotting fluid comprising a surfactant, a protectant, a humectant and a biological buffer. The addition of the surfactant is helpful for forming better shapes of the points on the chip substrate and enhancing the morphology of the points; the presence of the protective agent is helpful for protecting the protein during the preparation of the protein chip, the addition of the humectant can prolong the shelf life of the protein chip, and the buffer solution is mainly used for stabilizing the pH value, and can also play a role in protecting and stabilizing the protein during the preparation of the protein chip.
In one embodiment of the present invention, in the spotting liquid, the volume fraction of the surfactant is 0.05 to 0.5%, the volume fraction of the protective agent is 0.1 to 1%, the volume fraction of the humectant is 0.1 to 10%, and the volume fraction of the biological buffer is 0.05 to 0.5%.
In one embodiment of the invention, the surfactant is selected from one or more of triton X-100, tween-20, NP-40, surfactant S9, CA-630.
In one embodiment of the invention, the surfactant is selected from the group consisting of triton X-100.
In one embodiment of the invention, the protecting agent is selected from one or more of PEG-2000, PEG-4000, PEG-6000, sucrose, trehalose, dextran and dimethyl sulfoxide.
In one embodiment of the invention, the protective agent comprises at least one of PEG-2000, PEG-4000 and PEG-6000, and the volume fraction of the PEG-2000, the PEG-4000 or the PEG-6000 in the sample application liquid is 0.1-1%.
In one embodiment of the invention, the protective agent comprises sucrose and/or trehalose, wherein the volume fraction of the sucrose and/or the trehalose in the sample application liquid is 1-10%, and the concentration of the sucrose and/or the trehalose is 1.5-5 mol/L.
In one embodiment of the present invention, the humectant is selected from at least one of glycerin, betaine, hyaluronic acid, preferably betaine.
In one embodiment of the invention, in the sample application liquid, the volume fraction of glycerol is 1-10%, the volume fraction of betaine is 0.1-1%, and the concentration of betaine is 5-10 mol/L.
In one embodiment of the invention, the biological buffer is selected from one or more of Tris buffer, carbonate buffer, phosphate buffer and HEPES buffer, preferably HEPES buffer.
In one embodiment of the present invention, the biological buffer is present in the spotting fluid in a volume fraction of 0.05% to 0.5% and in a concentration of 0.01 to 0.1mol/L.
In one embodiment of the present invention, the pH of the spotting liquid is 7.0 to 9.5.
The invention also discloses application of the sample application liquid in the aspect of preparing a gene chip.
The third object of the present invention is to provide a method for preparing a protein chip using the spotting liquid, comprising the steps of:
(1) Preparing sample application liquid: mixing surfactant, protectant, humectant and biological buffer solution uniformly to obtain sample application liquid;
(2) Spotting: diluting the protein with the sample application liquid prepared in the step (1), and then applying sample on a chip substrate;
(3) And (3) sealing: and (3) sealing the chip after the sample application in the step (2) by using a sealing liquid.
In one embodiment of the present invention, in step (2), the spotting is performed using a non-contact spotting method.
In one embodiment of the invention, the sealing liquid comprises 1-10% of whey, 4-20% of goat serum, 9%o of physiological saline, 300%o of Proclin and the balance of pure water according to volume fraction.
The fourth object of the present invention is to provide the use of the protein chip prepared according to the above method in screening gene expression, detecting specific antigen and antibody, screening protein, detecting biochemical reaction, screening medicine and diagnosing diseases.
The invention has the beneficial effects that:
according to the invention, the sample application liquid is prepared by mixing the surfactant, the protective agent, the humectant and the biological buffer solution, the protein chip is prepared by adopting the prepared sample application liquid, when the prepared protein chip is used for detecting the substance to be detected, the sample information displayed in the picture collected by the CCD is very clear, the hollow problem is avoided, and the accuracy of the immunodetection reagent is improved.
Drawings
FIG. 1 shows the detection results of CENP B antibody after reaction of sample 1 with the protein chips prepared by the spotting liquids in examples 1 to 5 and comparative examples 1 to 8;
FIG. 2 shows the detection results of CENP B antibody after reaction of sample 2 with the protein chips prepared by the spotting liquids in examples 1 to 5 and comparative examples 1 to 8;
FIG. 3 shows the detection results of CENP B antibody after the reaction of sample 3 with the protein chips prepared by the spotting liquids in examples 1 to 5 and comparative examples 1 to 8;
FIG. 4 shows the detection results of CENP B antibody after the reaction of sample 4 with the protein chips prepared by the spotting liquids in examples 1 to 5 and comparative examples 1 to 8;
FIG. 5 shows the detection results of CENP B antibody after the reaction of sample 5 with the protein chips prepared by the spotting liquids in examples 1 to 5 and comparative examples 1 to 8;
FIG. 6 shows the detection results of CENP B antibody after reaction of sample 1 with the protein chips prepared by the spotting liquids in comparative examples 9 to 10 and examples 6 to 7;
FIG. 7 shows the detection results of CENP B antibody after reaction of sample 2 with the protein chips prepared by the spotting liquids in comparative examples 9 to 10 and examples 6 to 7;
FIG. 8 shows the detection results of CENP B antibody after reaction of sample 3 with the protein chips prepared by the spotting liquids in comparative examples 9 to 10 and examples 6 to 7.
FIG. 9 shows the detection results of SSB antibodies after reaction of sample 6 with the protein chips prepared in examples 1 to 5 and comparative examples 1 to 8;
FIG. 10 shows the detection results of SSB antibodies after reaction of sample 7 with the protein chips prepared in examples 1 to 5 and comparative examples 1 to 8;
FIG. 11 shows the detection results of SSB antibodies after sample 8 has reacted with the protein chips prepared in examples 1 to 5 and comparative examples 1 to 8.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for better illustration of the invention, and should not be construed as limiting the invention.
The treantom X-100 used in the embodiment of the invention is a 30188928 model product produced by Shanghai Dongshi chemical reagent Co., ltd., trehalose is a trehalose solution with a concentration of 1.5mol/L, betaine is a betaine solution with a concentration of 5mol/L, HEPES buffer is a solution with a concentration of 0.01mol/L prepared by a laboratory, polyethylene glycol sulfate is purchased from Anhuizhen Chemie Co., ltd., tween is a model T104863 product produced by Shanghai Dongshi chemical Co., ltd., CA-630 and dimethyl sulfoxide are purchased from Shanghai Donghai Dongshi Chemie Co., ltd., ethylenediamine tetraacetic acid solution with a concentration of 0.5mol/L, sodium alginate solution is 2g/L, sorbitol solution is 0.4g/L, hyaluronic acid solution is 3.6g/L, MOPS buffer is 0.01mol/L, dextran solution is 2g/L, and sucrose buffer is 0.01 g/L.
Example 1
The sample application liquid comprises, by volume fraction, 0.05% of triton X-100, 1% of trehalose solution, 0.1% of betaine solution, 0.05% of HEPES buffer solution, and the balance of pure water.
Example 2
The sample application liquid comprises, by volume fraction, 0.5% of triton X-100, 1% of trehalose solution, 0.1% of betaine solution, 0.05% of HEPES buffer solution, and the balance of pure water.
Example 3
The sample application liquid comprises, by volume fraction, 0.05% of triton X-100, 10% of trehalose solution, 0.1% of betaine solution, 0.05% of HEPES buffer solution, and the balance of pure water.
Example 4
The sample application liquid comprises, by volume fraction, 0.05% of triton X-100, 5% of trehalose solution, 0.5% of betaine solution, 0.05% of HEPES buffer solution, and the balance of pure water.
Example 5
The sample application liquid comprises, by volume fraction, 0.05% of triton X-100, 1% of trehalose solution, 1% of betaine solution, 0.5% of HEPES buffer solution, and the balance of pure water.
Example 6
The sample application liquid comprises, by volume fraction, 0.05% Tween, 5% dextran solution, 1% hyaluronic acid solution, 0.5% MOPS buffer solution, and the balance pure water.
Example 7
The sample application liquid comprises, by volume, 0.05% of CA-630, 1% of dimethyl sulfoxide, 1% of hyaluronic acid solution, 1% of Tris buffer solution, and the balance of pure water.
Example 8
A preparation method of a protein chip comprises the following steps:
(1) Preparing sample application liquid: weighing triton X-100, trehalose solution, betaine solution and HEPES buffer solution according to the component proportion of the embodiment 1, adding the components into purified water, and uniformly mixing to obtain sample application liquid;
(2) Spotting: diluting the antigen with the sample application liquid prepared in the step (1), and then spraying the antigen on the corresponding position of the glass substrate by using a piezoelectric sample application instrument, wherein the sample application time is 30min;
(3) Preparing a sealing liquid: uniformly mixing 1% of whey, 4% of goat serum, 9%o of physiological saline and 1%o of Proclin300 to prepare a sealing liquid;
(4) And (3) sealing: and (3) sealing the chip after the sample application in the step (2) by using the sealing liquid prepared in the step (3).
Comparative example 1
A spotting solution comprises 0.05% HEPES buffer solution and 99.5% pure water by volume percent.
Comparative example 2
A sample application liquid comprises 1% betaine solution, 0.05% HEPES buffer solution, and pure water by volume.
Comparative example 3
The sample application liquid comprises 1% trehalose solution, 0.1% betaine solution, 0.05% HEPES buffer solution, and pure water.
Comparative example 4
The sample application liquid comprises, by volume fraction, 0.05% of triton X-100, 0.1% of betaine solution, 0.05% of HEPES buffer solution, and the balance of pure water.
Comparative example 5
The sample application liquid comprises, by volume fraction, 0.05% of triton X-100, 1% of trehalose solution, 0.05% of HEPES buffer solution, and the balance of pure water.
Comparative example 6
The sample application liquid comprises, by volume fraction, 0.05% of triton X-100, 1% of trehalose solution, 0.1% of betaine solution, and the balance of pure water.
Comparative example 7
A spotting solution comprises, by volume fraction, 0.05% of triton X-100, 0.5% of trehalose solution, 0.1% of betaine solution and 0.05% of HEPES buffer.
Comparative example 8
A spotting solution comprises, by volume fraction, 0.05% of triton X-100, 1% of trehalose solution, 0.05% of betaine solution and 0.05% of HEPES buffer.
Comparative example 9
The sample application liquid comprises, by volume, 0.05% polyethylene glycol sulfate, 1% sodium alginate solution, 1% sorbitol, 0.5% MOPS buffer solution, and the balance being pure water.
Comparative example 10
The sample application liquid comprises, by volume, 0.5% of ethylenediamine tetraacetic acid, 1% of sodium alginate solution, 0.5% of hyaluronic acid, 0.5% of MOPS buffer solution, and the balance of pure water.
Signal value test:
centrifuging the collected antibody positive blood sample, taking supernatant, adding the supernatant into a protein chip to react with the protein chip, adding goat anti-human IgG antibody marked with HRP to react, finally adding chemiluminescent substrate luminol, and testing the signal intensity of the sample by using a biochip analyzer.
The detection method of the antibody comprises the following steps:
centrifuging the collected antibody positive blood sample, taking supernatant, adding the supernatant into a protein chip to react with the protein chip, adding goat anti-human IgG antibody marked with HRP to react, finally adding chemiluminescent substrate luminol, and observing pictures after photographing by a biochip analyzer; the biochip analyzer used was a triple biological full-automatic biochip analyzer SLXP-001B. The picture taken is shown in figure 1.
5 different CENP B index positive samples (samples 1 to 5) are selected, protein chips are prepared by using sample application liquids of examples 1 to 5 and comparative examples 1 to 8 and reacted with the sample application liquids, after the reaction, goat anti-human IgG antibodies marked with HRP are added to the sample application liquids for reaction, finally chemiluminescent substrates luminol are added, the preparation method of the protein chips is described in example 8, signal values are tested, pictures are taken, the signal values are shown in table 1, and the pictures after the reaction of the samples 1 to 5 are shown in figures 1 to 5.
TABLE 1 detection of Signal values of CENP B samples Using protein chips prepared by the spotting fluids of examples 1 to 5 and comparative examples 1 to 8
| / | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 |
| Example 1 | 7581.1 | 6706.7 | 7525.6 | 7384.4 | 7203.3 |
| Example 2 | 5844.4 | 6230.0 | 6161.1 | 5800.0 | 5994.4 |
| Example 3 | 5588.9 | 6441.1 | 6368.9 | 5830.0 | 5896.7 |
| Example 4 | 6282.2 | 5866.7 | 6547.8 | 6031.1 | 6382.2 |
| Example 5 | 6192.2 | 6213.3 | 5891.1 | 5613.3 | 6000.0 |
| Comparative example 1 | 3107.8 | 2804.4 | 2273.3 | 3137.8 | 3016.7 |
| Comparative example 2 | 2620.0 | 3322.2 | 2670.0 | 2352.2 | 2591.1 |
| Comparative example 3 | 4384.4 | 4416.7 | 3873.3 | 4370.0 | 3976.7 |
| Comparative example 4 | 3631.1 | 4222.2 | 3765.6 | 4434.4 | 4035.6 |
| Comparative example 5 | 3615.6 | 4178.9 | 4396.7 | 3743.3 | 3816.7 |
| Comparative example 6 | 3556.7 | 4088.9 | 4357.8 | 3700.0 | 4217.8 |
| Comparative example 7 | 3436.7 | 3978.9 | 4166.7 | 3881.1 | 3901.1 |
| Comparative example 8 | 3740.0 | 3652.2 | 3902.2 | 4177.8 | 3800.0 |
The results show that: the comparative example 1 only contains biological buffer, the comparative example 2 only contains humectant and biological buffer, the comparative examples 3 to 6 lack one of surfactant, protective agent, humectant and biological buffer respectively, the chip results of the above comparative examples for testing CENP B antibody all show hollow phenomenon and have lower signal, the comparative examples 7 to 8 respectively change the amounts of protective agent and humectant, and the amounts of protective agent and humectant are lower than the amounts of protective agent and humectant corresponding to the example 1, and as a result, the chip also shows hollow problem. The spotting solution in example 1 comprises a surfactant, a protective agent, a humectant and a biological buffer, the minimum addition amount of each component is determined, and in examples 2 to 5, the preferred higher addition amount is obtained by adjusting the content of one or more components of the surfactant, the protective agent, the humectant and the biological buffer, and in examples 1 to 5, the problem of hollow chips can be effectively solved, and the detection sensitivity is improved.
3 different CENP B index positive samples (samples 1 to 3) are selected, protein chips are prepared by using sample application liquids of examples 6 to 7 and comparative examples 9 to 10 to react with the sample application liquids, after the reaction, goat anti-human IgG antibodies marked with HRP are added to the sample application liquids to react, finally chemiluminescent substrates luminol are added, the preparation method of the protein chips is described in example 8, signal values are tested, pictures are taken, the signal values are shown in table 2, and the pictures after the reaction of the samples 1 to 3 are shown in fig. 6 to 8.
TABLE 2 detection of Signal values of CENP B samples Using the protein chips prepared by the spotting fluids of examples 6 to 7 and comparative examples 9 to 10
The results show that: comparative example 9 and comparative example 10 test CENP B antibodies, the signal value was high and a hollow phenomenon occurred, and example 6 and example 7 test CENP B antibodies, as a result, the signal value was low but a hollow phenomenon did not occur, thereby indicating that the signal value did not directly relate to whether or not the picture was hollow.
3 different SSB index positive samples (samples 6-8) are selected, protein chips are prepared by using sample application liquids of examples 1-3, 6-7 and comparative examples 1-3 and 9-10 to react with the sample application liquids, after the reaction, goat anti-human IgG antibodies marked with HRP are added to react, finally chemiluminescent substrates luminol are added, the protein chip preparation method is referred to in example 8, signal values are tested, pictures are taken, the signal values are shown in table 3, and the pictures after the 6-8 sample reaction are shown in figures 9-11.
TABLE 3 detection of signal values of SSB samples using protein chips prepared from spotting liquids of examples 1 to 3, 6 to 7 and comparative examples 1 to 3, 9 to 10
| / | Sample 6 | Sample 7 | Sample 8 |
| Example 1 | 4871.1 | 5223.3 | 6405.6 |
| Example 2 | 5862.2 | 6494.4 | 6026.7 |
| Example 3 | 6490.0 | 6202.2 | 6321.1 |
| Example 6 | 4058.9 | 5240.0 | 4230.0 |
| Example 7 | 4150.0 | 4140.0 | 3373.3 |
| Comparative example 1 | 4167.8 | 4412.2 | 3997.8 |
| Comparative example 2 | 3896.7 | 4428.9 | 4410.0 |
| Comparative example 3 | 4781.1 | 5412.2 | 5632.2 |
| Comparative example 9 | 6490.0 | 6415.6 | 6335.6 |
| Comparative example 10 | 5985.6 | 6310.0 | 5816.7 |
The results show that: the comparative example 1 only contains biological buffer, the comparative example 2 only contains humectant and biological buffer, the comparative example 3 lacks surfactant, the chip results of the test of SSB antibody in the above comparative examples all show hollow phenomena, and the comparative examples 9-10 change the components and contents of surfactant, protective agent, humectant and buffer, have higher signals, but the results still show hollow phenomena. The sample application liquid in the embodiment 1 of the application comprises a surfactant, a protective agent, a humectant and a biological buffer solution, wherein in the embodiments 2 to 3, the preferable higher addition amount is obtained by adjusting the content of one or more components of the surfactant and the protective agent, and the sample application liquid used in the embodiments 6 to 7 has lower result signals, but the problem of hollow pictures is solved, and the problem of hollow chips can be effectively solved in the above embodiments, so that the detection sensitivity is improved. And from the result, there is no direct relation between the signal value and whether the picture is empty.
Claims (7)
1. A method for preparing protein chip is characterized in that protein is diluted by sample application liquid and then sample application is carried out on a chip substrate, and sealing liquid is used for sealing the sample application chip; wherein, according to the volume fraction, the sample application liquid comprises 0.05-0.5% of surfactant, 1-10% of protective agent, 0.1-1% of humectant, 0.05-0.5% of buffer solution and the balance of water, the surfactant is at least one of triton X-100, tween-20 and CA-630, the protective agent is at least one of trehalose, dextran and dimethyl sulfoxide, the humectant is at least one of betaine solution and hyaluronic acid, and the buffer solution is at least one of Tris buffer solution, MOPS buffer solution and HEPES buffer solution; the preparation process of the sample application liquid comprises the step of uniformly mixing a surfactant, a protective agent, a humectant and a biological buffer solution, wherein the pH value of the sample application liquid is 7.0-9.5.
2. The method according to claim 1, wherein the protecting agent is a trehalose solution having a concentration of 1.5 to 5mol/L.
3. The method according to claim 1, wherein the humectant is a betaine solution having a concentration of 5 to 10mol/L.
4. The method according to claim 1, wherein the concentration of the buffer is 0.01 to 0.1mol/L.
5. The method according to claim 1, wherein the sealing liquid comprises, by volume fraction, 1-10% whey, 4-20% goat serum, 9%o physiological saline, 300%o Proclin, and the balance pure water.
6. A protein chip prepared by the method according to any one of claims 1 to 5.
7. The use of the protein chip prepared by the method of claim 6 in the aspects of screening gene expression, detecting specific antigen and antibody, screening protein, detecting biochemical reaction, screening medicine and preparing products for diagnosing diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310277631.7A CN116338162B (en) | 2023-03-21 | 2023-03-21 | Sample application liquid, protein chip prepared by using sample application liquid and preparation method of protein chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310277631.7A CN116338162B (en) | 2023-03-21 | 2023-03-21 | Sample application liquid, protein chip prepared by using sample application liquid and preparation method of protein chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116338162A CN116338162A (en) | 2023-06-27 |
| CN116338162B true CN116338162B (en) | 2024-01-12 |
Family
ID=86876917
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310277631.7A Active CN116338162B (en) | 2023-03-21 | 2023-03-21 | Sample application liquid, protein chip prepared by using sample application liquid and preparation method of protein chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116338162B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0205856D0 (en) * | 2002-03-13 | 2002-04-24 | Amersham Biosciences Uk Ltd | The use of organic solvents in the preparation of arrays |
| CN203798811U (en) * | 2014-04-04 | 2014-08-27 | 首都医科大学附属北京天坛医院 | Hypophysoma multi-hormone combined detection chip |
| CN108484756A (en) * | 2018-04-28 | 2018-09-04 | 河北鑫辉生物科技有限公司 | A kind of antibody preserves liquid and preparation method thereof |
| CN109613248A (en) * | 2018-12-26 | 2019-04-12 | 郑州安图生物工程股份有限公司 | Detect the kit of lipoprotein phospholipase A2 protein concentration |
| CN113030479A (en) * | 2019-12-25 | 2021-06-25 | 洛阳中科生物芯片技术有限公司 | Spotting solution of protein chip |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8877514B2 (en) * | 2004-02-26 | 2014-11-04 | Candor Bioscience Gmbh | Aqueous solution for use as medium for the specific binding reaction of a binding pair |
| EP1794581A2 (en) * | 2004-09-15 | 2007-06-13 | Microchip Biotechnologies, Inc. | Microfluidic devices |
-
2023
- 2023-03-21 CN CN202310277631.7A patent/CN116338162B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0205856D0 (en) * | 2002-03-13 | 2002-04-24 | Amersham Biosciences Uk Ltd | The use of organic solvents in the preparation of arrays |
| CN203798811U (en) * | 2014-04-04 | 2014-08-27 | 首都医科大学附属北京天坛医院 | Hypophysoma multi-hormone combined detection chip |
| CN108484756A (en) * | 2018-04-28 | 2018-09-04 | 河北鑫辉生物科技有限公司 | A kind of antibody preserves liquid and preparation method thereof |
| CN109613248A (en) * | 2018-12-26 | 2019-04-12 | 郑州安图生物工程股份有限公司 | Detect the kit of lipoprotein phospholipase A2 protein concentration |
| CN113030479A (en) * | 2019-12-25 | 2021-06-25 | 洛阳中科生物芯片技术有限公司 | Spotting solution of protein chip |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116338162A (en) | 2023-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7855068B2 (en) | Methods and kits for detecting a target cell | |
| JP3471753B2 (en) | Biosensor array containing cell populations confined in micropores | |
| WO2017016281A1 (en) | Method of detecting target molecule concentration | |
| CN1380976A (en) | Biosensor | |
| JP6437009B2 (en) | Method for detecting circulating tumor cells (CTC) | |
| CN111551712A (en) | Novel test strip and kit for quickly detecting coronavirus IgM/IgG two-in-one and preparation method of test strip and kit | |
| CN107328931A (en) | A kind of quick continuous detection technique based on nano-probe and magnetic micro-nano granules | |
| CN106370848A (en) | Immune lateral chromatography detection system and preparation method thereof | |
| CN103033619A (en) | Protein chip reagent kit and method for comprehensively detecting lung cancer marker | |
| CN101833009A (en) | Double antibody complex retinol-binding protein assay kit | |
| CN1866012A (en) | Quantitative and quick immune detection method and special apparatus therefor | |
| CN111505285A (en) | SARS-CoV-2 detecting chip and its application | |
| CN113933502B (en) | Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography | |
| CN107656079A (en) | A kind of method using nanoparticle time-resolved fluorescence probe in detecting sulfa antibiotics | |
| CN116338162B (en) | Sample application liquid, protein chip prepared by using sample application liquid and preparation method of protein chip | |
| EP1336107B1 (en) | Preparation of spheres for diagnostic tests | |
| RU2298796C2 (en) | Diagnostic system containing protein chips having features for concurrently determining a plurality of indices | |
| CN107966563A (en) | A kind of antimyeloperoxidase antibody IgG chemiluminescence immunoassay kits and preparation method thereof | |
| CN111735962B (en) | Novel coronavirus IgM/IgG antibody joint detection immunochromatography test strip | |
| CN115494240A (en) | Method and reagent combination capable of simultaneously detecting different subtype immune globulin | |
| CN101639444B (en) | Method for nano particle reinforced fluorescence polarization analysis | |
| KR20200144459A (en) | Apparatus for edtecting analyte and detection method using the same | |
| CN110672834A (en) | Kit for rapidly detecting lead content in sample | |
| CN110887822A (en) | Method for detecting embryo secretory protein | |
| CN110221083A (en) | A kind of excretion body identification apparatus and excretion body identification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |