CN107167613A - Albumen spotting buffer for the golden chip of plasma - Google Patents
Albumen spotting buffer for the golden chip of plasma Download PDFInfo
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- CN107167613A CN107167613A CN201710482189.6A CN201710482189A CN107167613A CN 107167613 A CN107167613 A CN 107167613A CN 201710482189 A CN201710482189 A CN 201710482189A CN 107167613 A CN107167613 A CN 107167613A
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- 239000000872 buffer Substances 0.000 title claims abstract description 60
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000004471 Glycine Substances 0.000 claims abstract description 16
- 239000001509 sodium citrate Substances 0.000 claims abstract description 16
- 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 abstract description 15
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims abstract description 15
- 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 abstract description 15
- 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 abstract description 15
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229960000310 isoleucine Drugs 0.000 claims abstract description 15
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 15
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 239000011521 glass Substances 0.000 claims description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 6
- 239000010931 gold Substances 0.000 claims description 6
- 229910052737 gold Inorganic materials 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 4
- 102000008102 Ankyrins Human genes 0.000 abstract description 10
- 108010049777 Ankyrins Proteins 0.000 abstract description 10
- 238000011534 incubation Methods 0.000 abstract description 10
- 239000000523 sample Substances 0.000 description 21
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 235000011083 sodium citrates Nutrition 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- 238000007639 printing Methods 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 4
- 239000005864 Sulphur Substances 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 102000034356 gene-regulatory proteins Human genes 0.000 description 2
- 108091006104 gene-regulatory proteins Proteins 0.000 description 2
- 235000014705 isoleucine Nutrition 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002090 nanochannel Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 150000003625 trehaloses Chemical class 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
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- 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
-
- 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
- G01N33/531—Production of immunochemical test materials
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
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- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
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- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention discloses the albumen spotting buffer for the golden chip of plasma, the albumen spotting buffer includes trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.The present invention can in a long time keep by the bioactivity of ankyrin for the albumen spotting buffer of the golden chip of plasma, shorten the crystallized ability of the incubation time after point sample, raising albumen on chip.
Description
Technical field
The invention belongs to biological technical field, buffered in particular to the albumen point sample for the golden chip of plasma
Liquid.
Background technology
Plasma material is commonly referred to as noble metal (such as golden) and its compound, its light in particular range of wavelengths
There is unique surface plasmon resonance effect according to lower.Because with specific structure and surface chemical property, this grade from
Daughter material has enhancement effect of fluorescence near infrared region (NIR-FE, 650-1700nm), by micro-sampling technology this
Specific albumen (antibody or antigen) is fixed on material, the detection of biomarker can be achieved.
At present, clinical field application immune detection side mainly have immunoturbidimetry, immunochromatography, enzyme linked immunological (Elisa),
Chemiluminescence immune assay (CLIA), and development and application in recent years protein chip-immunofluorescence technique.Due to micro biological living
Property material (such as antigen or antibody) be fixed on chip after be easy to loss of activity, protein chip be directed to one it is crucial
Technology, that is, keep the stability of protein chip.Therefore, how research keeps the stability of protein chip to have highly important meaning
Justice.
The content of the invention
It is contemplated that at least solving one of technical problem in correlation technique to a certain extent.Therefore, the present invention
One purpose is that proposition is used for the albumen spotting buffer of the golden chip of plasma, and the buffer solution can be protected in a long time
Hold by the bioactivity of ankyrin, shorten the crystallized ability of the incubation time after point sample, raising albumen on chip.
The present invention is based on the finding that proposing：The golden chip albumen spotting buffer of conventional plasma is sweet at present
Oil or phosphate buffer, this kind of buffer solution can only play moisture-keeping function to the protein on chip, be prevented in the short time because excessively
Protein inactivation caused by drying, its bioactivity can not be kept long-term effectively.Keep protein active on chip
Method is typically to be stored in chip in less than -20 DEG C of environment for a long time, and is unable to freeze thawing, and in 2-8 DEG C of refrigerator storage one
Week or 37 DEG C destroy one day, its bioactivity just have lost more than 50%.In addition, during with conventional method point sample, protein needs
Being incubated 12~24h could be fixed on chip.Inventors be surprised to learn that, by using trehalose, glycine, isoleucine, sulphur
The albumen spotting buffer that sour ammonium and sodium citrate configuration is obtained, effectively can solve to have protein in correlation technique in chip
On crystallized ability it is poor, and the problem of loss of activity easy by fixed protein.
Thus, according to an aspect of the present invention, the present invention proposes a kind of protein site for the golden chip of plasma
Sample buffer solution, the albumen spotting buffer includes trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.
The present invention includes trehalose, glycine, isoleucine, sulphur for the albumen spotting buffer of the golden chip of plasma
Sour ammonium and sodium citrate.The buffer solution can not only make albumen and chip base compared with current conventional glycerine or phosphate buffer
The combination at bottom is more firm, moreover it is possible to reduce the incubation time after point sample, and keeps living by the biology of ankyrin in a long time
Property.
In some embodiments of the invention, the concentration of trehalose described in the albumen spotting buffer is 10-20g/
L, the concentration of the glycine is 5-10g/L, and the concentration of the isoleucine is 5-10g/L, and the concentration of the ammonium sulfate is
0.2-1mol/L, the concentration of the sodium citrate is 0.05-0.2mol/L.Thus, it is possible to further improve albumen on chip
Crystallized ability, reduce point sample after incubation time and keep in a long time by the bioactivity of ankyrin.
In some embodiments of the invention, the concentration of trehalose described in the albumen spotting buffer is 10g/L, institute
The concentration for stating glycine is 5g/L, and the concentration of the isoleucine is 5g/L, and the concentration of the ammonium sulfate is 0.2mol/L, described
The concentration of sodium citrate is 0.05mol/L.Thus, it is possible to further improve crystallized ability, reduction point sample of the albumen on chip
Rear incubation time is simultaneously kept by the bioactivity of ankyrin in a long time.
In some embodiments of the invention, the pH value of the albumen spotting buffer is 6.0-7.2.
In some embodiments of the invention, the pH value of the albumen spotting buffer is 6.0.
In some embodiments of the invention, the pH value of the albumen spotting buffer is by adding sodium hydroxide solution
And/or hydrochloric acid solution regulation acquisition.Thus, it is possible to the effectively pH of regulatory protein spotting buffer.
In some embodiments of the invention, the golden chip of the plasma is by substrate of glass and is formed in the glass base
The nano gold layer composition of basal surface.Thus, it is possible to by deposition techniques by specific albumen by predetermined sequence be fixed to wait from
On daughter gold chip, the detection of biomarker is conveniently realized.
Brief description of the drawings
Fig. 1 be after point sample according to an embodiment of the invention CEA antibody after 37 DEG C preserve 1 day, 3 days, 5 days, 7 days
Fluorescent scanning comparison diagram.
Fig. 2 be after point sample according to an embodiment of the invention CEA antibody after 37 DEG C preserve 1 day, 3 days, 5 days, 7 days
Fluorescence intensity block diagram.
Fig. 3 is that CEA antibody is incubated after 0.5h, 6h and 12h at 30 DEG C after point sample according to an embodiment of the invention
Fluorescent scanning comparison diagram.
Fig. 4 is that CEA antibody is incubated after 0.5h, 6h and 12h at 30 DEG C after point sample according to an embodiment of the invention
Fluorescence intensity block diagram.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and be not considered as limiting the invention.
According to an aspect of the present invention, the present invention proposes a kind of albumen point sample buffering for the golden chip of plasma
Liquid, the albumen spotting buffer includes trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.
The present invention includes trehalose, glycine, isoleucine, sulphur for the albumen spotting buffer of the golden chip of plasma
Sour ammonium and sodium citrate.The buffer solution can make albumen and chip base compared with current conventional glycerine or phosphate buffer
Combination it is more firm, moreover it is possible to reduce the incubation time after point sample, and keep in a long time by the bioactivity of ankyrin.
Embodiments in accordance with the present invention, albumen spotting buffer can be by containing trehalose, glycine, isoleucine, sulphur
The aqueous solution composition of sour ammonium and sodium citrate.Thus, it is proposed by the present invention only by trehalose, glycine, isoleucine, ammonium sulfate
The albumen spotting buffer for being used for the golden chip of plasma constituted with several aqueous solution of sodium citrate and conventional glycerine at present or
Phosphate buffer is compared, and not only can be played moisture-keeping function to the protein on chip, be prevented the egg because caused by over-drying
White matter is inactivated, can also make the combination of albumen and chip base more firmly, the incubation time after point sample is reduced, and in the long period
Interior holding is by the bioactivity of ankyrin.
According to a particular embodiment of the invention, the concentration of trehalose described in the albumen spotting buffer can be 10-
20g/L, the concentration of the glycine can be 5-10g/L, and the concentration of the isoleucine can be 5-10g/L, the sulfuric acid
The concentration of ammonium can be 0.2-1mol/L, and the concentration of the sodium citrate can be 0.05-0.2mol/L.Thus, in the present invention
Obtained albumen spotting buffer is configured by using said ratio, albumen can be further improved on the golden chip of plasma
Crystallized ability, shorten the incubation time after point sample, and keep in a long time by the bioactivity of ankyrin.
According to a particular embodiment of the invention, the concentration of trehalose described in the albumen spotting buffer can be 10g/
L, the concentration of the glycine can be 5g/L, and the concentration of the isoleucine can be 5g/L, and the concentration of the ammonium sulfate can
Think 0.2mol/L, the concentration of the sodium citrate can be 0.05mol/L.Thus, by using said ratio in the present invention
Obtained albumen spotting buffer is configured, crystallized ability of the albumen on the golden chip of plasma can be further improved, reduce
Incubation time after point sample is simultaneously kept by the bioactivity of ankyrin in a long time.
According to a particular embodiment of the invention, the pH value of the albumen spotting buffer is 6.0-7.2.Thus, it is of the invention
In by using above-mentioned pH value albumen spotting buffer, further can keep in a long time by the biology of ankyrin
Activity.
According to a particular embodiment of the invention, the pH value of the albumen spotting buffer can be 6.0.
According to a particular embodiment of the invention, the pH value of the albumen spotting buffer can be molten by adding sodium hydroxide
What liquid and/or hydrochloric acid solution regulation were obtained.Thus, the present invention can be by controlling addition sodium hydroxide solution and/or hydrochloric acid molten
The concentration and addition of liquid carry out the pH of effective regulatory protein spotting buffer, make albumen of the present invention for the golden chip of plasma
Spotting buffer has suitable pH value.
According to a particular embodiment of the invention, the golden chip of the plasma by substrate of glass and can be formed in the glass
The nano gold layer composition of glass substrate surface.Thus, it is possible to which specific albumen is fixed to by predetermined sequence by deposition techniques
On plasma gold chip, the detection of biomarker is conveniently realized.
According to a particular embodiment of the invention, can have by using the albumen spotting buffer of the above embodiment of the present invention
Effect enhancing specific protein specifically, can foreshorten to incubation time 0.5 hour in the crystallized ability of the golden chip of plasma.
Meanwhile, the albumen spotting buffer can also effectively keep the activity of specific protein on chip, specifically, be preserved 7 days at 37 DEG C,
The activity of its specific protein is still maintained at more than the 80% of initial value.
In order to facilitate the understanding of the purposes, features and advantages of the present invention, it is below in conjunction with the accompanying drawings and specific real
Example is applied to be described in detail the embodiment of the present invention.Elaborate in the following description many details in order to
Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology
Personnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is not by following public specific implementation
Limitation.
Embodiment 1
Albumen spotting buffer is configured according to the final concentration of following components：10g/L trehaloses, 5g/L glycine, 5g/L are different
Leucine, 0.2mol/L ammonium sulfate, 0.05mol/L sodium citrates.With 1mol/L sodium hydroxide solutions or 1mol/L hydrochloric acid solutions
PH value is adjusted to 6.0,2-8 DEG C of preservation.
Comparative example 1
Albumen spotting buffer is configured according to the final concentration of following components：5% (v/v) glycerine, 10mM PBS, use 1mol/L
Sodium hydroxide solution or 1mol/L hydrochloric acid solutions adjust pH value to 6.0,2-8 DEG C of preservation.
Comparative example 2
Albumen spotting buffer is configured according to the final concentration of following components：10mM PBS, use 1mol/L sodium hydroxide solutions
Or 1mol/L hydrochloric acid solutions adjust pH value to 7.2,2-8 DEG C of preservation.
(1) active protection of the spotting buffer of checking embodiment 1, comparative example 1 and comparative example 2 to CEA antibody on chip
Effect：
1) spotting methods
CEA capture antibody is diluted to 0.2mg/ with the spotting buffer of embodiment 1, comparative example 1 and comparative example 2 respectively
ML, and embodiment 1, comparative example 1 and right will be utilized by trace of albumin printing instrument GeSim Nano-Plotter TM 2.1 respectively
The CEA capture antibody of the spotting buffer dilution of ratio 2 print to first row on the golden chip of plasma per hole, second row and
3rd row.Capture antibody is repeated 5 times printing according to each points of 3nL, each point, the final circular spot for obtaining about 400 microns of diameter
Point.
2) detection method
Chip after point sample is incubated 0.5h at room temperature, then 37 DEG C of vacuum are preserved, respectively preserve 1 day, 3 days, 5
My god, after 7 days respectively take a piece of detected.
Comprise the following steps that：Chip after point sample is shaken to closing in 1 hour in the PBS containing 1%BSA, to reduce non-spy
The opposite sex is combined.After being cleaned with the PBST (0.05% polysorbas20) in 150 μ L/ holes, 100 microlitres of CEA antigens (10ng/ is added per hole
ML), shake 2 hours.Chip is washed three times with PBST, is subsequently added the detection antibody of fluorescent dye IRDye800 marks
(4nmol/L) in the dark shake dyeing half an hour, after wash successively with PBST three times, pure water cleaning once, centrifuge drying.
Fluorescence measurement and analysis
With the MidaScan scanner scannings chip, 800 nanochannels of selection, laser intensity are set to 7.0, resolution ratio and set
It is set to 20 microns.16 gray level images are obtained after scanning.With MidaScan Software V1.0.0 or more highest version software point
Analyse the image.The intensity that the measurement of selection grid array analysis pattern is each put, the pattern of dot matrix is by automatic program identification.Each put
Intensity is obtained by selected areas total signal strength divided by area.The average fluorescent strength of 5 parallel points is defined on image
For the intensity of test.There is positive correlation between capturing the fluorescence intensity in antibody activity and acquired image in CEA.
Testing result is as shown in table 1, Fig. 1 and Fig. 2.Wherein, table 1 is the antibody of different spotting buffer printings in 37 DEG C of guarantors
The fluorescence intensity table measured after 1 day, 3 days, 5 days, 7 days is deposited, Fig. 1 and Fig. 2 are preserved 1 day, 3 days, 5 days and 7 at 37 DEG C respectively
The fluorescent scanning comparison diagram and fluorescence intensity block diagram of CEA antibody after it point sample.
The antibody of the spotting buffer of table 1 printing is in 37 DEG C of fluorescence intensities measured after preserving 1 day, 3 days, 5 days, 7 days
(2) crystallized ability of the spotting buffer of checking embodiment 1, comparative example 1 and comparative example 2 to CEA antibody on chip：
1) spotting methods
CEA capture antibody is diluted to 0.2mg/ with the spotting buffer of embodiment 1, comparative example 1 and comparative example 2 respectively
ML, and embodiment 1, comparative example 1 and right will be utilized by trace of albumin printing instrument GeSim Nano-Plotter TM 2.1 respectively
The CEA capture antibody of the spotting buffer dilution of ratio 2 print to first row on the golden chip of plasma per hole, second row and
3rd row.Capture antibody is repeated 5 times printing according to each points of 3nL, each point, the final circular spot for obtaining about 400 microns of diameter
Point.
2) detection method
Chip after point sample is incubated 0.5h, 6h and 12h respectively at 30 DEG C, a piece of detected respectively is taken.
Comprise the following steps that：Chip after point sample is incubated 0.5h, 4h or 12h respectively at 30 DEG C, immediately after containing
Shake 1 hour and close in 1%BSA PBS, to reduce non-specific binding.With the PBST (0.05% polysorbas20) in 150 μ L/ holes
After cleaning, 100 microlitres of CEA antigens (10ng/mL) are added per hole, are shaken 2 hours.Chip is washed three times with PBST, is then added
Enter fluorescent dye IRDye800 mark detection antibody (4nmol/L) shakes in the dark dye half an hour, after use PBST successively
Once, centrifuge is dried for washing three times, pure water cleaning.
Fluorescence measurement and analysis
With MidaScan scanner scanning chips, 800 nanochannels of selection, laser intensity are set to the setting of 7.0, resolution ratio
For 20 microns.16 gray level images are obtained after scanning.Analyzed with MidaScan Software V1.0.0 or more highest version software
The image.The intensity that the measurement of selection grid array analysis pattern is each put, the pattern of dot matrix is by automatic program identification.That each puts is strong
Degree is obtained by selected areas total signal strength divided by area.The average fluorescent strength of 5 parallel points is defined as on image
The intensity of test.There is positive correlation between capturing the fluorescence intensity in antibody activity and acquired image in CEA.
Testing result is as shown in table 2, Fig. 3 and Fig. 4.Wherein, table 2 is the antibody of different spotting buffer printings at 30 DEG C
The fluorescence intensity table measured after 0.5h, 6h and 12h is incubated, Fig. 3 and Fig. 4 are that the CEA antibody after point sample is incubated at 30 DEG C respectively
Fluorescent scanning comparison diagram and fluorescence intensity block diagram after 0.5h, 6h and 12h.
The antibody of the different spotting buffer printings of table 2 is incubated the fluorescence intensity table measured after 0.5h, 6h and 12h at 30 DEG C
Conclusion：(1) it can draw to draw a conclusion from table 1, Fig. 1 and Fig. 2：The spotting buffer of embodiment 1 can be protected effectively
The activity of antibody on chip is held, is preserved 7 days at 37 DEG C, its activity can still be maintained at more than the 80% of initial value, and now
Comparative example 1 and the antibody of comparative example 2 almost complete deactivation.(2) it can draw to draw a conclusion from table 2, Fig. 3 and Fig. 4：Embodiment 1
Spotting buffer can effectively strengthen crystallized ability of the antibody on the golden chip of plasma, shorten the set time.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be any
Combined in an appropriate manner in individual or multiple embodiments or example.In addition, in the case of not conflicting, the technology of this area
Not be the same as Example or the feature of example and non-be the same as Example or example described in this specification can be combined by personnel
And combination.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (7)
1. a kind of albumen spotting buffer for the golden chip of plasma, it is characterised in that the albumen spotting buffer bag
Containing trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.
2. it is used for the albumen spotting buffer of the golden chip of plasma according to claim 1, it is characterised in that the albumen
The concentration of trehalose described in spotting buffer is 10-20g/L, and the concentration of the glycine is 5-10g/L, the isoleucine
Concentration be 5-10g/L, the concentration of the ammonium sulfate is 0.2-1mol/L, and the concentration of the sodium citrate is 0.05-0.2mol/
L。
3. it is used for the albumen spotting buffer of the golden chip of plasma according to claim 2, it is characterised in that the albumen
The concentration of trehalose described in spotting buffer is 10g/L, and the concentration of the glycine is 5g/L, the concentration of the isoleucine
For 5g/L, the concentration of the ammonium sulfate is 0.2mol/L, and the concentration of the sodium citrate is 0.05mol/L.
4. it is used for the albumen spotting buffer of the golden chip of plasma according to claim 3, it is characterised in that the albumen
The pH value of spotting buffer is 6.0-7.2.
5. it is used for the albumen spotting buffer of the golden chip of plasma according to claim 4, it is characterised in that the albumen
The pH value of spotting buffer is 6.0.
6. it is used for the albumen spotting buffer of the golden chip of plasma according to claim 5, it is characterised in that the albumen
The pH value of spotting buffer is obtained by adding sodium hydroxide solution and/or hydrochloric acid solution regulation.
7. be used for the albumen spotting buffer of the golden chip of plasma according to claim 1, it is characterised in that the grade from
Daughter gold chip is made up of substrate of glass with the nano gold layer formed in the glass basic surface.
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| CN201710482189.6A CN107167613B (en) | 2017-06-22 | 2017-06-22 | Albumen spotting buffer for plasma gold chip |
| PCT/CN2018/079494 WO2018233328A1 (en) | 2017-06-22 | 2018-03-19 | Protein loading buffer and use thereof in preparation of protein chip |
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| CN201710482189.6A CN107167613B (en) | 2017-06-22 | 2017-06-22 | Albumen spotting buffer for plasma gold chip |
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| CN107167613B CN107167613B (en) | 2018-10-02 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108896752A (en) * | 2018-06-08 | 2018-11-27 | 深圳清华大学研究院 | A kind of Block buffer for plasma gold chip |
| WO2018233328A1 (en) * | 2017-06-22 | 2018-12-27 | Wwhs Biotech, Inc | Protein loading buffer and use thereof in preparation of protein chip |
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| CN107167613B (en) | 2018-10-02 |
| WO2018233328A1 (en) | 2018-12-27 |
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