CN1645147A - Protein chip for immune analysis - Google Patents
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- CN1645147A CN1645147A CN 200510013108 CN200510013108A CN1645147A CN 1645147 A CN1645147 A CN 1645147A CN 200510013108 CN200510013108 CN 200510013108 CN 200510013108 A CN200510013108 A CN 200510013108A CN 1645147 A CN1645147 A CN 1645147A
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Abstract
A protein chip is prepared as plating a metal film on glass substrate and coating a layer of photosensitive glue on it, obtaining micropattern and decorating glass pattern to let it have active functional group-amino, covering antigen egg white on substrate surface, acting protein with amino and then fixing them on glass substrate through chemical bond, labeling protein sample molecule with fluorescent label matter and detecting protein sample with fluorescent microscopic analyzing system of chip.
Description
Technical field
The invention belongs to life science, the protein-chip of particularly a kind of immunology detection, analysis and diagnosis.
Background technology
Protein-chip is a kind of at solid substrate surface fixing protein or polypeptide microarray, by immunology principle protein example is detected, discerns, analyzes and diagnoses, can be applicable to life science, medicine and pharmacology, and the association areas such as development of biology sensor.
Traditional immune analysis method mainly contains immunofluorescence method (Fluorescent Immunoassay at present, FIA), enzyme-linked immunosorbent assay (Enzyme-linked Immunoassay, EIA) and radiating immuning analysis technology (RadioactiveImmunoassay, RIA).These technology have higher sensitivity and specificity, have brought into play vital role in clinical medicine and biological research.Yet traditional technique in measuring speed is slow, and efficient is low, can not satisfy the especially requirement of life science develop rapidly of medical science.For example, FIA and the sensitivity of EIA method are lower, though and RIA sensitivity is higher, its radioactivity can damage operating personnel's health again.
The appearance of biochip has brought new development opportunity to immuno analytical method.Modern biochip is meant the array that is that is included in the highdensity DNA of surface of solid phase carriers stationary arrangement, antigen, antibody or cell, utilize the ability of the specific recognition that has between the biomacromolecule, react with test sample or biomacromolecule or hybridize, can obtain a large amount of useful biological informations by automatic checkout equipment.
Protein-chip is on a kind of solid phase carrier substrate, with protein or the direct point sample of polypeptide thereon, makes it and the substrate surface strong bonded, forms protein microarray.Antigen-antibody mostly is a protein, and the combination between them is again specific.Therefore, the protein that is fixed on substrate surface can carry out specific immune response with the protein molecule that has special marking (antibody or antigen), both in conjunction with after, realize the mutual inspection of antigen-antibody being used to detect corresponding antibody or antigen by detection to the antigen-antibody complex of mark.The characteristics of this technology are high flux, microminiaturization and robotization.Can be widely used in the research in fields such as clinical diagnosis, drug screening and proteomics.
Protein-chip can be divided into two types of chemical type and biochemicals.The chemical type protein-chip is based on classical chromatographic media, and its ultimate principle is: spread specific medium on the protein-chip surface, hydrophobic force, electrostatic force and covalent bond etc. by medium are in conjunction with the protein in the sample.Use specific eluent flush away impurity albumen then, analyze and keep particular proteins.Bio-chemical protein chip is that bioactive molecule (as antigen, antibody or acceptor, part etc.) is fixed to chip surface, is used for the complementary target albumen in conjunction with sample.Because bio-chemical protein chip has the higher specificity and the diversity of bioactive molecule, so its range of application and prospect are all considerably beyond the chemical type protein-chip.The preparation of protein-chip must guarantee correct location and its bioactive maintenance of protein.But traditional protein-chip requires high to experimental implementation, the reaction system strictness, because the error that human factor produces is bigger, unstable result, and its detecting instrument equipment price costliness detect the cost height, therefore can't produce in enormous quantities and use.
Summary of the invention
The purpose of this invention is to provide a kind of protein-chip that is used for immune analysis, the present invention is sensitivity and the higher bio-chemical protein chip of specificity, method for making is easy, be suitable for producing in batches, and can carry out check and analysis to the multiple protein quality sample.
The concrete technical scheme that the present invention is used for the protein-chip of immune analysis is:
1) on clean glass substrate, plates the back photoresist spinner of layer of metal film (as crome metal film or aluminium film) and be coated with one deck light-sensitive emulsion in the above with high speed rotating;
2) mask that will have covers on substrate, and photoetching under the ultraviolet photolithographic machine obtains the metallic substrates pattern behind the developing fixing;
3) metal film that will expose again erodes, and exposes the substrate of glass pattern, after removing photoresist, modifies the substrate of glass pattern that exposes, and makes it have activity functional groups---amino;
4) remove remaining metal film and clean and dry after, antigen protein solution is covered in substrate surface, protein is connected on the microslide by chemical bond-linking with amino effect back and is fixed; Described protein example becomes microarray;
5) utilize fluorescent marker that the protein example molecule is carried out mark, utilize the chip fluorescent microscope that protein example is carried out check and analysis at last.
Described protein example microarray is square shape dot matrix or strip array.
The number of each square shape microarray mid point of described square shape microarray is 40 * 40-400 * 400.The diameter of protein example point is 50 μ m in the described square shape microarray, and the clearance distance between the point is 50 μ m.
Be 10-100 group, every group of 4 bars in the described strip microarray.The width of described strip microarray discal patch is followed successively by 30 μ m, 50 μ m, 80 μ m and 120 μ m, and the clearance distance between the bar is 50 μ m.The length of bar is 0.2-2.0cm.
The manufacture craft that is used for the protein-chip of immune analysis is taked following steps:
1) plates crome metal film or the aluminium film of the about 150-250nm of a bed thickness in the slide surface of cleaning, be coated with the thick ultraviolet eurymeric photoresist of one deck 500-700nm with the photoresist spinner of high speed rotating in the above then;
2) dry post bake 30-60min down at 60-80 ℃, optical cement and metal film are combined closely;
3) by making the mask with microarray pattern of the fabrication techniques of circuit version in the microelectronics; Perhaps design and print the microarray pattern in advance, make photographic negative by the reproduction technology, with it as mask;
4) mask is covered on the slide of gluing photoetching under the ultraviolet photolithographic machine;
5) the NaOH solution with 0.5-1% develops, and removes the photoresist of exposing patterns part, exposes the metallic substrates pattern;
6) erode the chromium layer that exposes or, obtain glass pattern with ceric ammonium nitrate solution of the phosphoric acid solution corrosion of aluminium film with 1.6mol/L; NaOH solution with 1-10% removes remaining photoresist.
Take following method to modify glass surface:
1) with glass sheet 1: 1: 5 25%NH of volume ratio
3, 30%H
2O
2And H
2O solution cleans; Use volume ratio to be 37%HCl, 30%H at 1: 1: 5 then
2O
2And H
2O solution-treated 5-10 minute; The glass sheet of aluminizing is then used instead 30% H
2O
2Handle; With its water respectively, alcohol and acetone clean again, make it places 100-120 ℃ again after drying under air baking oven inner drying 0.5-1 hour;
2) glass sheet was modified 3-10 minute with the toluene solution of 3-amido-propyl group-triethoxysilane (APTES) of 1-5%, is cleaned it with toluene and acetone again, then 100-120 ℃ dry 0.5-1 hour down, make on the glass sheet surface band amino;
3) erode remaining chromium layer with the ammonium ceric nitrate corrosive liquid; Or with phosphoric acid solution corrosion aluminium film; And dry after cleaning fully with distilled water.
Described ammonium ceric nitrate corrosion liquid formula: ammonium ceric nitrate 100 grams, acetic acid 17.5mL adds distilled water and is diluted to 500mL; Described phosphoric acid solution is the phosphoric acid solution of 1.6mol/L.
The using method that is used for the protein-chip of immune analysis is through following step:
1) antigen (chicken IgG) for preparing is added to glass substrate surface, makes the about 10-30min of amino effect on protein and surface, be connected on the microslide by chemical bond-linking and be fixed through modifying; Then with pH value phosphate buffer between 7.2-7.4: the Na of 0.2mol/L
2HPO
481.0mL, the NaH of 0.2mol/L
2PO
419.0mL mix and distilled water is distinguished the rinse substrate surface, dry after washing unnecessary antigen off;
2) will be added on the glass sheet surface that is fixed with antigen (chicken IgG) with fluorescently-labeled testing sample (as the chicken Anti-IgG of FITC mark) solution, keep 5-10min at normal temperatures, antigen and antibody are fully reacted;
3) wash glass sheet successively with the phosphate buffer and the distilled water of pH value between 7.2-7.4, the sample of flush away remnants, and dry at normal temperatures;
4) under fluorescent microscope, chip is carried out check and analysis, or after utilizing the CCD system to take pictures its fluorescence intensity is analyzed.
The present invention is used for the protein-chip of immune analysis, and with the research of semiconductor fabrication process and microelectric technique introducing immunoassay, it is integrated that the protein-chip microarray is realized first.The present invention is sensitivity and the higher bio-chemical protein chip of specificity, and method for making is easy, and detection method is quick more sensitive, and detection efficiency is higher.Be suitable for producing in batches, and can carry out check and analysis the multiple protein quality sample.
Description of drawings
Fig. 1 is the mask photo that makes by the reproduction technology.
Fig. 2 is the little pattern fluorescence micrograph of being clapped after the immune response of protein.
Fig. 3 is the light micrograph of the square shape microarray figure of protein-chip.
Fig. 4 is the stereoscan photograph of the square shape microarray figure of protein-chip.
Fig. 5 is that protein-chip carries out the square shape protein microarray fluorescence micrograph clapped after the immune response.
Fig. 6 is the light micrograph of the strip microarray figure of protein-chip.
Fig. 7 is the stereoscan photograph of the strip microarray figure of protein-chip.
Fig. 8 is that protein-chip carries out the strip protein microarray fluorescence micrograph clapped after the immune response.
Embodiment
Example 1: the method for making of protein microarray chip (chromium plating film):
1) uses coating machine (coating machine, the DM-450C type, Beijing instrument plant), heating chromium silk is at the crome metal film of the about 154nm of slide surface evaporation one bed thickness of cleaning under the vacuum, (glue is spared the glue clean work station to be coated with the thick eurymeric photoetching of one deck 600nm with the photoresist spinner of high speed rotating in the above then, model: SW-CJ-9B, Suzhou Decontamination Equipment Plant; Ultraviolet eurymeric photoresist, model: BP212, Beijing Inst. of Chemical Reagent);
2) dry post bake 30min down at 80 ℃, optical cement and metal film are combined closely;
3) design and print the microarray pattern, as shown in Figure 1; Fig. 1 is the mask photo that makes by the reproduction technology, and the diameter of circle and pentagram is respectively 150-250 and does not wait among Fig. 1.Spacing is 150 μ m, and the number of microarray mid point is 48 * 60, and makes photographic negative by the reproduction technology, with it as mask.
4) mask is covered on the slide of gluing photoetching under the ultraviolet photolithographic machine (semi-automatic litho machine, JKG-1 type, Shanghai optical-mechanical factory);
5) develop with 0.5% NaOH solution, remove the photoresist of exposing patterns part, expose the pattern of chromium;
6) erode the chromium layer that exposes with ceric ammonium nitrate solution, obtain the substrate of glass pattern, remove remaining photoresist with 5% NaOH solution then;
7) with glass sheet 25%NH
3, 30%H
2O
2, and H
2O solution (volume ratio 1: 1: 5) cleans a moment; Use 37%HCl then, 30%H
2O
2And H
2O solution (volume ratio 1: 1: 5) was handled 5-10 minute; With its water respectively, alcohol and acetone clean again, make it places 100 ℃ again after drying under air baking oven inner drying 0.5 hour;
8) glass sheet was modified 3-10 minute with APTES (3-amido-propyl group-triethoxysilane) toluene solution (3-aminopropyltriethoxysilane) of 1-5%, clean it with toluene and acetone again, descended dry 1 hour at 100 ℃ then, make on the glass sheet surface band amino;
9) with ceric ammonium nitrate solution (prescription: ammonium ceric nitrate 100 gram, acetic acid 17.5mL. add distilled water and be diluted to 500mL) erode remaining chromium layer, and dry after cleaning fully with distilled water.
10) antigen (chicken IgG) for preparing is added to glass substrate surface, makes the about 10-30min of amino effect on protein and surface, be connected on the microslide by chemical bond-linking and be fixed through modifying; Then with pH value phosphate buffer (PBS, Na of 0.2mol/L between 7.2-7.4
2HPO
481.0mL, the NaH of 0.2mol/L
2PO
419.0mL mix) and distilled water distinguish the rinse substrate surface, dry after washing unnecessary antigen off;
11) will be added on the glass sheet surface that is fixed with chicken IgG with the chicken Anti-IgG solution of fluorescently-labeled testing sample FITC mark, keep 5-10min at normal temperatures, antigen and antibody are fully reacted;
12) wash glass sheet with the pH value successively at 7.4 phosphate buffer (PBS) and distilled water, the sample of flush away remnants, and dry at normal temperatures;
13) under fluorescent microscope chip is carried out check and analysis, or after utilizing the CCD system to take pictures its fluorescence intensity is analyzed, as shown in Figure 2, Fig. 2 is the little pattern fluorescence micrograph of being clapped after the immune response of protein.By the immune response between antigen and the fluorescently-labeled antibody, can obtain green fluorescence pattern clearly, the diameter of circle and pentagram is respectively 150-250 and does not wait among the figure.Spacing is 150 μ m, and the number of microarray mid point is 48 * 60.This explanation have only have functional group (amino) through chemical modification but zone conjugated antigen, and not modified zone is basically in conjunction with albumen, and then by immune response combined with fluorescent albumen specifically, forms green little pattern.The gained pattern is very clear as can be seen, and this illustrates that this bio-chemical protein chip has higher sensitivity and specificity.And method for making is easy, and detection method is quick more sensitive, and detection efficiency is higher.
Example 2: the method for making of square shape protein microarray chip (aluminizer):
1) uses coating machine, the heating aluminium wire is at the aluminium film of the about 200nm of slide surface evaporation one bed thickness of cleaning under the vacuum, (glue is spared glue clean work station, model: SW-CJ-9B, Suzhou Decontamination Equipment Plant to be coated with the thick eurymeric photoresist of one deck 600nm with the photoresist spinner of high speed rotating in the above then; Ultraviolet eurymeric photoresist, model: BP212, Beijing Inst. of Chemical Reagent);
2) dry post bake 30min down at 80 ℃, photoresist and metal film are combined closely;
3) make square microarray mask by the plate-making technology of making circuit in the microelectronics;
4) mask is covered on the slide of gluing photoetching under the ultraviolet photolithographic machine (semi-automatic litho machine, JKG-1 type, Shanghai optical-mechanical factory);
5) develop with 0.5% NaOH solution, remove the photoresist of exposing patterns part, expose the pattern of chromium;
6) erode the aluminium lamination that exposes with phosphoric acid solution (1.6mol/L), obtain the substrate of glass pattern, as shown in Figure 3, Fig. 3 is the light micrograph of the square shape microarray figure of protein-chip.Remove remaining photoresist with 5% NaOH solution then, and under scanning electron microscope observation pattern pattern, as shown in Figure 4, Fig. 4 is the stereoscan photograph of the square shape microarray figure of protein-chip.
7) with glass sheet 25%NH
3, 30%H
2O
2, and H
2O solution (volume ratio 1: 1: 5) cleans a moment; Use 30%H then
2O
2Handled 10 minutes; With its water respectively, alcohol and acetone clean again, make it places 100 ℃ again after drying under air baking oven inner drying 0.5 hour;
8) glass sheet was modified 5 minutes with 5% APTES (3-amido-propyl group-triethoxysilane) toluene solution (3-aminopropyltriethoxysilane), clean it with toluene and acetone again, descended dry 1 hour at 100 ℃ then, make on the glass sheet surface band amino;
9) erode remaining aluminium lamination with phosphoric acid solution (1.6mol/L), and dry after cleaning fully with distilled water.
10) antigen (chicken IgG) for preparing is added to glass substrate surface, makes the about 10min of amino effect on protein and surface, be connected on the microslide by chemical bond-linking and be fixed through modifying; Then with pH value phosphate buffer (PBS, Na of 0.2mol/L between 7.2-7.4
2HPO
481.0mL, the NaH of 0.2mol/L
2PO
419.0mL mix) and distilled water distinguish the rinse substrate surface, dry after washing unnecessary antigen off;
11) will be added on the glass sheet surface that is fixed with chicken IgG with the chicken Anti-IgG solution of fluorescently-labeled testing sample FITC mark, keep 5min at normal temperatures, antigen and antibody are fully reacted;
12) wash glass sheet with the pH value successively at 7.4 phosphate buffer (PBS) and distilled water, the sample of flush away remnants, and dry at normal temperatures;
13) under fluorescent microscope chip is carried out check and analysis, or utilize the CCD system back of taking pictures that its fluorescence intensity is analyzed, as shown in Figure 5, Fig. 5 is that protein-chip carries out the square shape protein microarray fluorescence micrograph clapped after the immune response.By the immune response between antigen and the fluorescently-labeled antibody, can obtain green fluorescence pattern clearly, the number of each square shape microarray mid point of square shape microarray is 200 * 200, and the diameter of protein example point is 50 μ m, and the clearance distance between the point is 50 μ m.This explanation have only have functional group (amino) through chemical modification but zone conjugated antigen, and not modified zone is basically in conjunction with albumen, and then by immune response combined with fluorescent albumen specifically, forms the little pattern of square shape green.The gained pattern is very clear as can be seen, and this illustrates that this bio-chemical protein chip has higher sensitivity and specificity.And method for making is easy, and detection method is quick more sensitive, and detection efficiency is higher.
Example 3: the method for making of strip protein microarray chip (aluminizer):
1) uses coating machine (coating machine, the DM-450C type, Beijing instrument plant), the heating aluminium wire is at the aluminium film of the about 200nm of slide surface evaporation one bed thickness of cleaning under the vacuum, (glue is spared the glue clean work station to be coated with the thick eurymeric photoresist of one deck 600nm with the photoresist spinner of high speed rotating in the above then, model: SW-CJ-9B, Suzhou Decontamination Equipment Plant; Ultraviolet eurymeric photoresist, model: BP212, Beijing Inst. of Chemical Reagent);
2) dry post bake 30min down at 80 ℃, photoresist and metal film are combined closely;
3) make square microarray mask by the plate-making technology of making circuit in the microelectronics;
4) mask is covered on the slide of gluing photoetching under the ultraviolet photolithographic machine (semi-automatic litho machine, JKG-1 type, Shanghai optical-mechanical factory);
5) develop with 0.5% NaOH solution, remove the photoresist of exposing patterns part, expose the pattern of chromium;
6) erode the aluminium lamination that exposes with phosphoric acid solution (1.6mol/L), obtain the substrate of glass pattern, as shown in Figure 6, Fig. 6 is the light micrograph of the strip microarray figure of protein-chip.Remove remaining photoresist with 5% NaOH solution then, and under scanning electron microscope observation pattern pattern, as shown in Figure 7, Fig. 7 is the stereoscan photograph of the strip microarray figure of protein-chip.。
7) with glass sheet 25%NH
3, 30%H
2O
2, and H
2O solution (volume ratio 1: 1: 5) cleans a moment; Use 30%H then
2O
2Handled 10 minutes; With its water respectively, alcohol and acetone clean again, make it places 100 ℃ again after drying under air baking oven inner drying 0.5 hour;
8) glass sheet was modified 5 minutes with 5% APTES (3-amido-propyl group-triethoxysilane) toluene solution (3-aminopropyltriethoxysilane), clean it with toluene and acetone again, descended dry 1 hour at 100 ℃ then, make on the glass sheet surface band amino;
9) erode remaining aluminium lamination with phosphoric acid solution (1.6mol/L), and dry after cleaning fully with distilled water.
10) the chicken IgG solution for preparing is added to glass substrate surface, makes the about 10min of amino effect on protein and surface, be connected on the microslide by chemical bond-linking and be fixed through modifying; Then with pH value phosphate buffer (PBS, Na of 0.2mol/L between 7.2-7.4
2HPO
481.0mL, the NaH of 0.2mol/L
2PO
419.0mL mix) and distilled water distinguish the rinse substrate surface, dry after washing unnecessary antigen off;
11) will be added on the glass sheet surface that is fixed with antigen chicken IgG with the chicken Anti-IgG solution of fluorescently-labeled testing sample FITC mark, keep 5min at normal temperatures, antigen and antibody are fully reacted;
12) phosphate buffer (PBS) and the distilled water with pH value 7.4 washes glass sheet successively, the sample of flush away remnants, and dry at normal temperatures;
13) under fluorescent microscope chip is carried out check and analysis, or utilize the CCD system back of taking pictures that its fluorescence intensity is analyzed, as shown in Figure 8, Fig. 8 is that protein-chip carries out the strip protein microarray fluorescence micrograph clapped after the immune response.By the immune response between antigen and the fluorescently-labeled antibody, can obtain green fluorescence pattern clearly, have 60 groups in the strip microarray, every group of 4 bars, the width of strip microarray discal patch is followed successively by 30 μ m, 50 μ m, 80 μ m and 120 μ m, the length of bar is 2cm, and the clearance distance between the bar is 50 μ m.This explanation have only have functional group (amino) through chemical modification but zone conjugated antigen, and not modified zone is basically in conjunction with albumen, and then by immune response combined with fluorescent albumen specifically, forms the little pattern of strip green.The gained pattern is very clear as can be seen, and this illustrates that this bio-chemical protein chip has higher sensitivity and specificity.And method for making is easy, and detection method is quick more sensitive, and detection efficiency is higher.
Claims (10)
1, a kind of protein-chip that is used for immune analysis is characterized in that:
1) photoresist spinner with high speed rotating is coated with one deck light-sensitive emulsion in the above after plating layer of metal film on the clean glass substrate;
2) mask that will have covers on substrate, and photoetching under the ultraviolet photolithographic machine obtains the metallic substrates pattern behind the developing fixing;
3) metal film that will expose again erodes, and exposes the substrate of glass pattern, after removing photoresist, modifies the substrate of glass pattern that exposes, and makes it have activity functional groups---amino;
4) remove remaining metal film and clean and dry after, antigen protein solution is covered in substrate surface, protein is connected on the microslide by chemical bond-linking with amino effect back and is fixed; Described protein example becomes microarray;
5) utilize fluorescent marker that the protein example molecule is carried out mark, utilize the chip fluorescent microscope that protein example is carried out check and analysis at last.
2, the protein-chip that is used for immune analysis according to claim 1 is characterized in that described protein example microarray is square shape dot matrix or strip array.
3, the protein-chip that is used for immune analysis according to claim 2, the number that it is characterized in that each square shape microarray mid point of described square shape microarray are 40 * 40-400 * 400.
4, the protein-chip that is used for immune analysis according to claim 3, the diameter that it is characterized in that protein example point in the described square shape microarray is 50 μ m, the clearance distance between the point is 50 μ m.
5, the protein-chip that is used for immune analysis according to claim 2 is characterized in that being in the described strip microarray 10-100 group, every group of 4 bars.
6, the protein-chip that is used for immune analysis according to claim 5 is characterized in that the width of described strip microarray discal patch is followed successively by 30 μ m, 50 μ m, 80 μ m and 120 μ m, and the clearance distance between the bar is 50 μ m.
7, the manufacture craft that is used for the protein-chip of immune analysis according to claim 1 is characterized in that taking following steps:
1) plates crome metal film or the aluminium film of the about 150-250nm of a bed thickness in the slide surface of cleaning, be coated with the thick ultraviolet eurymeric photoresist of one deck 500-700nm with the photoresist spinner of high speed rotating in the above then;
2) dry post bake 30-60min down at 60-80 ℃, optical cement and metal film are combined closely;
3) by making the mask with microarray pattern of the fabrication techniques of circuit version in the microelectronics; Perhaps design and print the microarray pattern in advance, make photographic negative by the reproduction technology, with it as mask;
4) mask is covered on the slide of gluing photoetching under the ultraviolet photolithographic machine;
5) the NaOH solution with 0.5-1% develops, and removes the photoresist of exposing patterns part, exposes the metallic substrates pattern;
6) erode the chromium layer that exposes or, obtain glass pattern with ceric ammonium nitrate solution of the phosphoric acid solution corrosion of aluminium film with 1.6mol/L; NaOH solution with 1-10% removes remaining photoresist.
8, the manufacture craft that is used for the protein-chip of immune analysis according to claim 1 is characterized in that taking following method to modify glass surface:
1) with glass sheet 1: 1: 5 25%NH of volume ratio
3, 30%H
2O
2And H
2O solution cleans; Use volume ratio to be 37%HCl, 30%H at 1: 1: 5 then
2O
2And H
2O solution-treated 5-10 minute; The glass sheet of aluminizing is then used instead 30% H
2O
2Handle; With its water respectively, alcohol and acetone clean again, make it places 100-120 ℃ again after drying under air baking oven inner drying 0.5-1 hour;
2) glass sheet was modified 3-10 minute with the toluene solution of 3-amido-propyl group-triethoxysilane (APTES) of 1-5%, is cleaned it with toluene and acetone again, then 100-120 ℃ dry 0.5-1 hour down, make on the glass sheet surface band amino;
3) erode remaining chromium layer with the ammonium ceric nitrate corrosive liquid; Or with phosphoric acid solution corrosion aluminium film; And dry after cleaning fully with distilled water.
9, the manufacture craft that is used for the protein-chip of immune analysis according to claim 8 is characterized in that described ammonium ceric nitrate corrosion liquid formula: ammonium ceric nitrate 100 grams, and acetic acid 17.5mL adds distilled water and is diluted to 500mL; Described phosphoric acid solution is the phosphoric acid solution of 1.6mol/L.
10, the described using method that is used for the protein-chip of immune analysis of claim 1 is characterized in that it is through following step:
1) antigen for preparing is added to glass substrate surface, makes the about 10-30min of amino effect on protein and surface, be connected on the microslide by chemical bond-linking and be fixed through modifying; Then with pH value phosphate buffer between 7.2-7.4: the Na of 0.2mol/L
2HPO
481.0mL, the NaH of 0.2mol/L
2PO
419.0mL mix and distilled water is distinguished the rinse substrate surface, dry after washing unnecessary antigen off;
2) will be added on the glass sheet surface that is fixed with antigen with fluorescently-labeled testing sample solution, keep 5-10min at normal temperatures, antigen and antibody are fully reacted;
3) wash glass sheet successively with the phosphate buffer and the distilled water of pH value between 7.2-7.4, the sample of flush away remnants, and dry at normal temperatures;
4) under fluorescent microscope, chip is carried out check and analysis, or after utilizing the CCD system to take pictures its fluorescence intensity is analyzed.
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|---|---|---|---|
| CNB2005100131085A CN1327225C (en) | 2005-01-18 | 2005-01-18 | Protein chip for immune analysis |
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|---|---|---|---|
| CNB2005100131085A CN1327225C (en) | 2005-01-18 | 2005-01-18 | Protein chip for immune analysis |
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| CN1109109C (en) * | 1999-09-20 | 2003-05-21 | 中国科学院力学研究所 | Multielement protein chip and its preparation and application |
| CN1159581C (en) * | 2001-03-28 | 2004-07-28 | 上海晶泰生物技术有限公司 | Protein chip for immunological analysis |
| CN1131428C (en) * | 2001-04-23 | 2003-12-17 | 清华大学 | Process for preparing capillary electrophoresis chip used in chemical analysis |
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| CN102246042A (en) * | 2008-12-11 | 2011-11-16 | 皇家飞利浦电子股份有限公司 | Sensing device for detecting target elements in a fluid |
| CN102246042B (en) * | 2008-12-11 | 2015-04-29 | 皇家飞利浦电子股份有限公司 | Sensing device for detecting target elements in a fluid |
| CN103868862A (en) * | 2012-06-06 | 2014-06-18 | 林世明 | Sensor for detection of target of interest |
| CN110318099A (en) * | 2019-07-23 | 2019-10-11 | 武汉新芯集成电路制造有限公司 | CIS genetic chip and preparation method thereof |
| CN110318099B (en) * | 2019-07-23 | 2022-08-09 | 武汉新芯集成电路制造有限公司 | CIS gene chip and manufacturing method thereof |
| CN112611861A (en) * | 2020-11-23 | 2021-04-06 | 武汉世纪康敏生物科技有限公司 | Fluorescence immunoassay chip and preparation method thereof |
| CN112611861B (en) * | 2020-11-23 | 2024-03-29 | 武汉世纪康敏生物科技有限公司 | Fluorescent immunodetection chip and preparation method thereof |
| CN113311160A (en) * | 2021-06-17 | 2021-08-27 | 山东科讯生物芯片技术有限公司 | Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody |
| CN113976058A (en) * | 2021-11-17 | 2022-01-28 | 深圳市华云智能健康有限公司 | Photoetching synthesis method of high-density polypeptide array chip |
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