CN107167613B - Albumen spotting buffer for plasma gold chip - Google Patents
Albumen spotting buffer for plasma gold chip Download PDFInfo
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- CN107167613B CN107167613B CN201710482189.6A CN201710482189A CN107167613B CN 107167613 B CN107167613 B CN 107167613B CN 201710482189 A CN201710482189 A CN 201710482189A CN 107167613 B CN107167613 B CN 107167613B
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 239000010931 gold Substances 0.000 title claims abstract description 34
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 34
- 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
- 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
- 239000001509 sodium citrate Substances 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 14
- 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
- 239000000758 substrate Substances 0.000 claims description 4
- 238000011534 incubation Methods 0.000 abstract description 11
- 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 11
- 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
- 238000005516 engineering process Methods 0.000 description 7
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 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
- 239000000463 material Substances 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
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 3
- 238000000151 deposition Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000000034 method 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
- 230000003139 buffering effect Effects 0.000 description 2
- 238000004140 cleaning Methods 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
- 230000007774 longterm Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002090 nanochannel Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical group [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 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
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006378 damage 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
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- 230000004048 modification Effects 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 230000009871 nonspecific binding 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
- 238000004904 shortening Methods 0.000 description 1
- 238000003860 storage Methods 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
- 238000005406 washing Methods 0.000 description 1
- 210000004885 white matter Anatomy 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
- G01N33/68—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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- 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)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the albumen spotting buffer for plasma gold chip, which includes trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.The present invention can keep being fixed the bioactivity of albumen in a long time for the albumen spotting buffer of plasma gold chip, shorten point sample after incubation time, improve crystallized ability of the albumen on chip.
Description
Technical field
The invention belongs to biotechnologies, in particular to the albumen point sample buffering for plasma gold chip
Liquid.
Background technology
Plasma material is commonly referred to as noble metal (such as gold) and its compound, the light in particular range of wavelengths
There is unique surface plasmon resonance effect according to lower.Because having specific structure and surface chemical property, it is this it is equal 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, it can be achieved that biomarker detection.
Currently, 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.Since micro biology is living
Property substance (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 keeps the stability of protein chip.Therefore, how research keeps the stability of protein chip to have highly important meaning
Justice.
Invention content
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, the present invention
One purpose is to propose that the albumen spotting buffer for plasma gold chip, the buffer solution can be protected in a long time
It holds the incubation time after the bioactivity for being fixed albumen, shortening point sample, improve crystallized ability of the albumen on chip.
The present invention is based on the finding that proposing：Currently used plasma gold chip albumen spotting buffer is sweet
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 can not keep its bioactivity long-term effectively.Keep protein on chip active
Method is unable to freeze thawing typically by chip long-term preservation in -20 DEG C of environment below, and in 2-8 DEG C of refrigerator storage one
As soon as week or 37 DEG C of destructions day, bioactivity has lost 50% or more.In addition, when with conventional method point sample, protein needs
Incubation 12~could be fixed on chip for 24 hours.Inventors be surprised to learn that by using trehalose, glycine, isoleucine, sulphur
The albumen spotting buffer that sour ammonium and sodium citrate configure effectively can solve to have protein in the related technology in chip
On crystallized ability it is poor, and fixed protein be easy loss of activity the problem of.
As a result, according to an aspect of the present invention, the present invention proposes a kind of protein site for plasma gold chip
Sample buffer solution, the albumen spotting buffer include trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.
The present invention includes trehalose, glycine, isoleucine, sulphur for the albumen spotting buffer of plasma gold chip
Sour ammonium and sodium citrate.The buffer solution can not only make albumen and chip base compared with current common glycerine or phosphate buffer
The combination at bottom is more secured, moreover it is possible to reduce the incubation time after point sample, and keep the biology for being fixed albumen living in a long time
Property.
In some embodiments of the invention, a concentration of 10-20g/ of trehalose described in the albumen spotting buffer
L, a concentration of 5-10g/L of the glycine, a concentration of 5-10g/L of the isoleucine, the ammonium sulfate it is a concentration of
0.2-1mol/L, a concentration of 0.05-0.2mol/L of the sodium citrate.Thus, it is possible to further increase albumen on chip
Crystallized ability, reduce the incubation time after point sample and holding in a long time is fixed the bioactivity of albumen.
In some embodiments of the invention, a concentration of 10g/L of trehalose described in the albumen spotting buffer, institute
A concentration of 5g/L of glycine, a concentration of 5g/L of the isoleucine, a concentration of 0.2mol/L of the ammonium sulfate are stated, it is described
A concentration of 0.05mol/L of sodium citrate.Thus, it is possible to further increase crystallized ability, reduction point sample of the albumen on chip
Incubation time and holding in a long time afterwards is fixed the bioactivity of albumen.
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 the way that sodium hydroxide solution is added
And/or hydrochloric acid solution adjusts acquisition.Thus, it is possible to the effectively pH of regulatory protein spotting buffer.
In some embodiments of the invention, the plasma gold chip by substrate of glass and is formed in the glass base
The nano gold layer of bottom surface forms.Thus, it is possible to by deposition techniques by specific albumen by scheduled sequence be fixed to it is equal from
On daughter gold chip, facilitate the detection for realizing biomarker.
Description of the drawings
Fig. 1 is after CEA antibody preserves 1 day, 3 days, 5 days, 7 days at 37 DEG C after point sample according to an embodiment of the invention
Fluorescent scanning comparison diagram.
Fig. 2 is after CEA antibody preserves 1 day, 3 days, 5 days, 7 days at 37 DEG C after point sample according to an embodiment of the invention
Fluorescence intensity block diagram.
Fig. 3 is after CEA antibody is incubated 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 after CEA antibody is incubated 0.5h, 6h and 12h at 30 DEG C after point sample according to an embodiment of the invention
Fluorescence intensity block diagram.
Specific implementation mode
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and is 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 plasma gold chip
Liquid, the albumen spotting buffer include trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.
The present invention includes trehalose, glycine, isoleucine, sulphur for the albumen spotting buffer of plasma gold chip
Sour ammonium and sodium citrate.The buffer solution can make albumen and chip base compared with current common glycerine or phosphate buffer
Combination it is more secured, moreover it is possible to reduce the incubation time after point sample, and keep the bioactivity for being fixed albumen in a long time.
According to an embodiment of the invention, albumen spotting buffer can be by containing trehalose, glycine, isoleucine, sulphur
The aqueous solution of sour ammonium and sodium citrate forms.It is proposed by the present invention only by trehalose, glycine, isoleucine, ammonium sulfate as a result,
With the albumen spotting buffer of sodium citrate several aqueous solutions composition for plasma gold chip with commonly use at present glycerine 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 inactivates, and can also make the incubation time of the combination of albumen and chip base more securely, after reduction point sample, and in the long period
The interior bioactivity for keeping being fixed albumen.
According to a particular embodiment of the invention, the concentration of trehalose described in the albumen spotting buffer can be 10-
The concentration of 20g/L, 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.As a result, in the present invention
The albumen spotting buffer configured by using said ratio can further increase albumen on plasma gold chip
Crystallized ability, shorten the incubation time after point sample, and keep being fixed the bioactivity of albumen in a long time.
According to a particular embodiment of the invention, the concentration of trehalose described in the albumen spotting buffer can be 10g/
The concentration of L, 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.As a result, by using said ratio in the present invention
Obtained albumen spotting buffer is configured, crystallized ability of the albumen on plasma gold chip can be further increased, reduced
Incubation time and holding in a long time after point sample are fixed the bioactivity of albumen.
According to a particular embodiment of the invention, the pH value of the albumen spotting buffer is 6.0-7.2.It is of the invention as a result,
In by using above-mentioned pH value albumen spotting buffer, can further keep the biology for being fixed albumen in a long time
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 the way that sodium hydroxide is added
Liquid and/or hydrochloric acid solution adjust acquisition.Sodium hydroxide solution can be added by control in the present invention as a result, and/or hydrochloric acid is molten
The concentration and addition of liquid carry out the pH of effective regulatory protein spotting buffer, and the present invention is made to be used for the albumen of plasma gold chip
Spotting buffer has suitable pH value.
According to a particular embodiment of the invention, the plasma gold chip can be by substrate of glass and being formed in the glass
The nano gold layer of glass substrate surface forms.Thus, it is possible to which specific albumen is fixed to by scheduled sequence by deposition techniques
On plasma gold chip, facilitate the detection for realizing biomarker.
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 can specifically foreshorten to incubation time 0.5 hour in the crystallized ability of plasma gold chip.
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 80% or more of initial value.
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, below in conjunction with the accompanying drawings and specific real
Example is applied to be described in detail the specific implementation mode 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 without violating the connotation 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, with 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 to CEA antibody on chip of embodiment 1, comparative example 1 and comparative example 2 is verified
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 diluted CEA captures antibody of the spotting buffer of ratio 2 print to first row on plasma gold chip per hole, second row and
Third is arranged.Capture antibody is repeated 5 times printing according to each points of 3nL, each point, the final round spot for obtaining about 400 microns of diameter
Point.
2) detection method
Chip after point sample is incubated at room temperature 0.5h, then 37 DEG C of vacuum preserve, respectively preserve 1 day, 3 days, 5
It, respectively take a piece of be detected after 7 days.
It is as follows：Chip after point sample is shaken 1 hour in the PBS containing 1%BSA and is closed, to reduce non-spy
The opposite sex combines.After being cleaned with the PBST (0.05% polysorbas20) in 150 holes μ L/, 100 microlitres of CEA antigens (10ng/ is added per hole
ML), shake 2 hours.Chip is washed three times with PBST, and the detection antibody of fluorescent dye IRDye800 labels is then added
(4nmol/L) shakes dyeing half an hour in the dark, after successively with PBST wash three times, pure water clean primary, centrifuge dries.
Fluorescence measurement and analysis
With the MidaScan scanner scannings chip, 800 nanochannels, laser intensity is selected to be set as 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.Selection grid array analysis pattern measures the intensity each put, and 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 are positive correlations between fluorescence intensity in CEA capture antibody activities and acquired image.
Testing result is as shown in table 1, Fig. 1 and Fig. 2.Wherein, table 1 is that the antibody of different spotting buffer printings is protected at 37 DEG C
The fluorescence intensity table measured after depositing 1 day, 3 days, 5 days, 7 days, 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 fluorescence intensity that the antibody of 1 spotting buffer of table printing measures after being preserved 1 day, 3 days, 5 days, 7 days at 37 DEG C
(2) crystallized ability of the spotting buffer to CEA antibody on chip of embodiment 1, comparative example 1 and comparative example 2 is verified：
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 diluted CEA captures antibody of the spotting buffer of ratio 2 print to first row on plasma gold chip per hole, second row and
Third is arranged.Capture antibody is repeated 5 times printing according to each points of 3nL, each point, the final round 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, respectively takes a piece of be detected.
It is as follows：Chip after point sample is incubated 0.5h, 4h or 12h respectively at 30 DEG C, immediately after containing
It shakes 1 hour and closes in the PBS of 1%BSA, to reduce non-specific binding.With the PBST (0.05% polysorbas20) in 150 holes μ L/
After cleaning, 100 microlitres of CEA antigens (10ng/mL) are added per hole, shake 2 hours.Chip is washed three times with PBST, is then added
The detection antibody (4nmol/L) for entering fluorescent dye IRDye800 label shakes dye half an hour in the dark, after use PBST successively
Washing three times, pure water cleaning it is primary, centrifuge drying.
Fluorescence measurement and analysis
With MidaScan scanner scanning chips, 800 nanochannels, laser intensity is selected to be set as the setting of 7.0, resolution ratio
It is 20 microns.16 gray level images are obtained after scanning.It is analyzed with MidaScan Software V1.0.0 or more highest version software
The image.Selection grid array analysis pattern measures the intensity each put, and 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 are positive correlations between fluorescence intensity in CEA capture antibody activities and acquired image.
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
It is incubated the fluorescence intensity table measured after 0.5h, 6h and 12h, Fig. 3 and Fig. 4 are that the CEA antibody after point sample is incubated at 30 DEG C respectively
Fluorescent scanning comparison diagram after 0.5h, 6h and 12h and fluorescence intensity block diagram.
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 be obtained from table 1, Fig. 1 and Fig. 2 to draw a conclusion：The spotting buffer of embodiment 1 can be protected effectively
The activity for holding antibody on chip preserves 7 days at 37 DEG C, and activity can still be maintained at 80% of initial value or more, and at this time
Comparative example 1 and 2 antibody of comparative example almost inactivate.(2) it can be obtained from table 2, Fig. 3 and Fig. 4 to draw a conclusion：Embodiment 1
Spotting buffer can effectively enhance crystallized ability of the antibody on plasma gold chip, 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 specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
It can be combined in any suitable manner in a or multiple embodiments or example.In addition, without conflicting with each other, the technology of this field
The feature of different embodiments or examples described in this specification and different embodiments or examples can be combined by personnel
And combination.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (6)
1. a kind of albumen spotting buffer for plasma gold chip, which is characterized in that the albumen spotting buffer packet
Containing trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate,
Wherein, a concentration of 10-20g/L of trehalose described in the albumen spotting buffer, a concentration of 5- of the glycine
10g/L, a concentration of 5-10g/L of the isoleucine, a concentration of 0.2-1mol/L of the ammonium sulfate, the sodium citrate
A concentration of 0.05-0.2mol/L.
2. being used for the albumen spotting buffer of plasma gold chip according to claim 1, which is characterized in that the albumen
A concentration of 10g/L of trehalose described in spotting buffer, a concentration of 5g/L of the glycine, the concentration of the isoleucine
For 5g/L, a concentration of 0.2mol/L of the ammonium sulfate, a concentration of 0.05mol/L of the sodium citrate.
3. being used for the albumen spotting buffer of plasma gold chip according to claim 2, which is characterized in that the albumen
The pH value of spotting buffer is 6.0-7.2.
4. being used for the albumen spotting buffer of plasma gold chip according to claim 3, which is characterized in that the albumen
The pH value of spotting buffer is 6.0.
5. being used for the albumen spotting buffer of plasma gold chip according to claim 4, which is characterized in that the albumen
The pH value of spotting buffer is adjusted and is obtained by the way that sodium hydroxide solution and/or hydrochloric acid solution is added.
6. being used for the albumen spotting buffer of plasma gold chip according to claim 1, which is characterized in that it is described it is equal from
Daughter gold chip is made of substrate of glass with the nano gold layer for being formed in the glass basic surface.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 DISSOCIATION BUFFER AND USE THEREOF IN PREPARING A PROTEIN CHIP |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710482189.6A CN107167613B (en) | 2017-06-22 | 2017-06-22 | Albumen spotting buffer for plasma gold chip |
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| Publication Number | Publication Date |
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| CN107167613A CN107167613A (en) | 2017-09-15 |
| CN107167613B true CN107167613B (en) | 2018-10-02 |
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| CN201710482189.6A Active CN107167613B (en) | 2017-06-22 | 2017-06-22 | Albumen spotting buffer for plasma gold chip |
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| CN (1) | CN107167613B (en) |
| WO (1) | WO2018233328A1 (en) |
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| CN107167613B (en) * | 2017-06-22 | 2018-10-02 | 深圳清华大学研究院 | Albumen spotting buffer for plasma gold chip |
| 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 |
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| US6165981A (en) * | 1995-03-07 | 2000-12-26 | Dade Behring Inc. | Stabilizing solutions for proteins and peptides |
| CN1544943A (en) * | 2003-11-21 | 2004-11-10 | 中国计量学院 | A method for preparing an antibody chip |
| CN1699585A (en) * | 2004-05-20 | 2005-11-23 | 中国科学院成都生物研究所 | A kind of preparation method of biological antibacterial agent |
| US20080090278A1 (en) * | 2006-03-31 | 2008-04-17 | Toyo Boseki Kabushiki Kaisha | Method for enhancing stability of a composition comprising soluble glucose dehydrogenase (gdh) |
| GB0700523D0 (en) * | 2007-01-11 | 2007-02-21 | Insense Ltd | The Stabilisation Of Proteins |
| US20110223208A1 (en) * | 2010-03-09 | 2011-09-15 | Beth Hill | Non-Aqueous High Concentration Reduced Viscosity Suspension Formulations |
| EP2576589A4 (en) * | 2010-05-27 | 2014-10-15 | Ge Healthcare Bio Sciences Ab | PROCESS AND NECESSARY FOR MARKING PROTEINS |
| JP5758174B2 (en) * | 2011-03-31 | 2015-08-05 | 株式会社ナリス化粧品 | Antioxidants and antioxidant cosmetics |
| AR093297A1 (en) * | 2012-10-31 | 2015-05-27 | Amgen Res Munich Gmbh | LIQUID FORMULATION THAT INCLUDES A GM-CSF NEUTRALIZING COMPOUND |
| CN104459147A (en) * | 2013-09-18 | 2015-03-25 | 深圳迈瑞生物医疗电子股份有限公司 | Biomarker preserving liquid, biomarker reagent and method |
| CN103869059A (en) * | 2014-03-27 | 2014-06-18 | 上海奥普生物医药有限公司 | Sample applying method of diagnostic kit |
| BR112016025126B1 (en) * | 2014-05-07 | 2024-02-15 | Takeda Pharmaceutical Company Limited | AQUEOUS COMPOSITION COMPRISING GMCSF NEUTRALIZING ANTIBODY, AND USE THEREOF |
| JP2018500380A (en) * | 2014-12-31 | 2018-01-11 | ノベルメド セラピューティクス，インコーポレーテッド | Aglycosylated therapeutic antibody formulations |
| CN105524894B (en) * | 2016-02-26 | 2019-03-26 | 福建华灿制药有限公司 | The preparation method of factor XIII |
| CN107167613B (en) * | 2017-06-22 | 2018-10-02 | 深圳清华大学研究院 | Albumen spotting buffer for plasma gold chip |
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- 2017-06-22 CN CN201710482189.6A patent/CN107167613B/en active Active
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| CN107167613A (en) | 2017-09-15 |
| WO2018233328A1 (en) | 2018-12-27 |
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