CN110361370A - A kind of single embryo's secretory protein quantitative detecting method based on Microfluidic droplet - Google Patents
A kind of single embryo's secretory protein quantitative detecting method based on Microfluidic droplet Download PDFInfo
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- CN110361370A CN110361370A CN201910702915.XA CN201910702915A CN110361370A CN 110361370 A CN110361370 A CN 110361370A CN 201910702915 A CN201910702915 A CN 201910702915A CN 110361370 A CN110361370 A CN 110361370A
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- secretory protein
- single embryo
- microfluidic droplet
- immunomagnetic beads
- detecting method
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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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
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
Abstract
The invention belongs to detection method fields, are specifically related to a kind of single embryo's secretory protein quantitative detecting method based on Microfluidic droplet.Detection method of the invention includes the following steps: the capture antibody and labelled antibody that take single embryo's secretory protein to be measured, capture antibody is connected on immunomagnetic beads, labelled antibody biotin labeling；Then immunomagnetic beads are mixed and is incubated for single embryo's secretory protein to be measured, then it mixes and is incubated for the beta galactosidase of labelled antibody and marked by streptavidin after cleaning, immunomagnetic beads are resuspended with PBS buffer solution, finally by laser induced fluorescence detector fluorescence intensity, the content of single embryo's secretory protein to be measured is obtained.The present invention generates fluorescent material, it can be achieved that the signal of testing protein amplifies using the continuous catalysis substrate reaction of enzymatic reaction；The introducing of Microfluidic droplet technology simultaneously, can fast enriching fluorescent material reach and can survey concentration, there is higher detection sensitivity, the sample volume required for detecting is smaller.
Description
Technical field
The invention belongs to detection method fields, and it is fixed to be specifically related to a kind of single embryo's secretory protein based on Microfluidic droplet
Quantity measuring method.
Background technique
Multiple protein can be secreted during embryo cutting, these albumen with it is a variety of in embryonic development and implantation process
Function is closely related.Single embryo refers to an embryo, detects the content pair of the secretory protein of single embryo in single embryo medium
In understanding embryonic development situation, explores embryonic development potential etc. and be of great significance.Single embryo's secretory protein refers to individually
The protein content of secretory protein in embryo medium.However, the protein concentration of single embryo's secretion is extremely low, nutrient solution volume
Only 20~30 μ L, while protein can carry out signal amplification by amplification without nucleic acid substances such as image of Buddha DNA, therefore to detection
The sensitivity of method has high requirement.Immunomagnetic beads (ImpetiCbead, IMB abbreviation magnetic bead), are developed in recent years
One new immunological technique, the high degree of specificity of the peculiar advantage of solidified reagents and immunological response is incorporated into one by it,
Based on immunology, the every field such as pathology, physiology, pharmacology, microorganism, biochemistry and molecular genetics are penetrated into,
Immune detection, cell separation, biological macromolecule purifying and molecular biology etc. are more and more widely used.With
It is good and can be into that the synthetic method of core-shell structure copolymer synthesizes the composite material of the macromolecule surface covering containing superparamagnetism substance, stability
The substance of row later period label carries out the covalent of antibody using the functional group such as amino, carboxyl and sulfydryl etc. of these material surfaces
Or non-covalent associations, it can be used for combining corresponding antigen or antibody, displacement can be done under the attraction of externally-applied magnetic field in this way,
To reach separation, detect, the purpose of purified genes, protein, cell and microorganism etc..Determine that different embryos has difference
Antibody pair, i.e. capture antibody and labelled antibody, these antibody to that can buy in the market, for detecting secretory protein
Content.
In the prior art, the detection method for embryo's secretory protein includes that mass spectrography is related to enzyme linked immunological (ELESA)
Detection method.Mass spectrography is widely used in the research of embryo's secretion group, however due to the content of added proteins in culture solution
High and type is abundant, affects the detection of embryo's secretory protein, therefore each research institute survey result difference is huge.ELISA is related
Detection method can be applied, but presently relevant to avoid the interference of added proteins in the quantitative detection of embryo's secretory protein
Method is difficult to meet single embryo detection of most embryo's secretory proteins, even for human soluble leukocyte antigen G (sHLA-G)
The equal higher albumen of embryos' secretory volume still has research to think effectively detect its content in culture solution.
The above analytic explanation, conventional protein detection method is difficult to realize the quantitative inspection of single embryo's secretory protein at present
It surveys, multiple documents explicitly point out, and lacking effective detection method is to restrict the major reason of embryo's secretory protein correlative study development.
Summary of the invention
The present invention in order to solve in above-mentioned background technique the technical issues of can not accurately detecting single embryo's secretory protein,
A kind of single embryo's secretory protein quantitative detection based on Microfluidic droplet that can accurately detect single embryo's secretory protein is provided
Method.
The technical scheme to solve the above technical problems is that a kind of single embryo based on Microfluidic droplet secretes egg
White quantitative detecting method, comprising the following steps:
A, capture antibody is connected to immunomagnetic beads by the capture antibody and labelled antibody for taking single embryo's secretory protein to be measured
On, by labelled antibody biotin labeling；
B, the immunomagnetic beads in step a are mixed and is incubated for single embryo's secretory protein to be measured, then take immunomagnetic beads
It cleans 3~5 times out, the labelled antibody and Streptavidin with biotin labeling that immunomagnetic beads, the step a after cleaning are obtained
It after the beta galactosidase mixing of label, is incubated for, immunomagnetic beads is then taken out into cleaning 3~5 times, then be resuspended with PBS buffer solution
Immunomagnetic beads；
C, the magnetic bead after respin that step b is obtained is placed in the first sample well of Microfluidic droplet, fluorogenic substrate is set
In the second sample well, drop formation oil is placed in oily phase sample holes, it is by syringe that fluorogenic substrate and drop formation is oily
It is added to formation Water-In-Oil drop in the first sample well, the Water-In-Oil drop of generation is collected with capillary, collects and completes
It is afterwards that capillary is closed at both ends, it is incubated for；
D, the Water-In-Oil drop being incubated in the capillary of completion obtained in step c is imported into detection chip, in sheath stream
Under effect, when Water-In-Oil drop window after testing, laser induced fluorescence detector detects to obtain the fluorescence of Water-In-Oil drop
Intensity is to get the content for arriving single embryo's secretory protein to be measured.
The beneficial effects of the present invention are: the secretory protein due to determining single embryo has determining antibody pair, that is, capture
Antibody and labelled antibody as label labelled antibody and utilize its fluorescein 2- β-D- pyrans using beta galactosidase (β GAL)
The signal amplification of testing protein is realized in enzymatic reaction between galactoside (FDG).In step a, it will be covered on immunomagnetic beads
The capture antibody of list embryo's secretory protein to be measured.In step b, immunomagnetic beads mix acquisition with single embryo's secretory protein to be measured, should
Invention marks single embryo's secretory protein to be measured on immunomagnetic beads to be measured using beta galactosidase, the immunomagnetic beads after cleaning with
The beta galactosidase of labelled antibody and marked by streptavidin is mixed and is incubated for be reacted using the continuous catalysis substrate of enzymatic reaction
Fluorescent material is generated, can effectively realize the signal amplification of single embryo's secretory protein to be measured.The introducing of Microfluidic droplet technology simultaneously,
Can fast enriching fluorescent material reach and can survey concentration.More particle fluorescence can be achieved in the use of highly sensitive laser induced fluorescence detector
The detection of substance can reduce the reaction time to a certain extent, and artificial subjective factor can be reduced compared with fluorescence imaging
Interference keeps the signal read more objective.More importantly being detected compared to currently used for embryo's secretory protein in culture solution
Technology, the invention have higher detection sensitivity, detect required for sample volume it is smaller.This method has one simultaneously
Fixed versatility, different single embryo's secretory proteins have different antibody to i.e. capture albumen and labelled protein, can be to not
Congener embryo's secretory protein carries out quantitative detection.
Based on the above technical solution, the present invention can also be improved as follows.
Further, in step a, the diameter of the immunomagnetic beads is 5~10um, the function on the surface of the immunomagnetic beads
Group is one of carboxyl or n-hydroxysuccinimide.
Beneficial effect using above-mentioned further scheme is, immunomagnetic beads determine diameter and functional group can more preferably and its
He combines reactant, plays better suction-operated.
Further, in stepb, the temperature of the incubation is 37 DEG C, and the time of the incubation is 25~35min；
The cleaning is all made of pure water；The volume of the PBS buffer solution is 5~20uL.
Beneficial effect using above-mentioned further scheme is, the operating condition of above-mentioned determination can allow last testing result
It is more acurrate.
Further, the time of the incubation is 30min；The volume of the PBS buffer solution is 10uL.
Beneficial effect using above-mentioned further scheme is more accurate reaction condition, can allow detection of the invention
Method is more accurate.
Further, in step c, the fluorogenic substrate is fluorescein 2- β-D- galactopyranoside or resorufin -2- β -
One kind of D- galactopyranoside.
Further, when fluorogenic substrate is fluorescein 2- β-D- galactopyranoside, in step d, the induced with laser
The excitation wavelength of fluorescence detector is one of 473nm or 488nm, and phosphor collection wave band is 510~530nm.
Further, when fluorogenic substrate is resorufin -2- β-D- galactopyranoside, in step d, the induced with laser
The excitation wavelength of fluorescence detector is 558nm, and phosphor collection wave band is 560~580nm.
Beneficial effect using above-mentioned further scheme is to determine the inspection of fluorogenic substrate and laser induced fluorescence detector
It surveys, the content of more accurate single embryo's secretory protein to be measured can be obtained.
Further, in step c, the drop formation oil is any one in fluorocarbon oil, mineral oil and paraffin oil, described
The temperature of incubation is 37 DEG C, and the time of the incubation is 1-3h.
Beneficial effect using above-mentioned further scheme is determining drop together with fluorogenic substrate and magnetic bead, preferably real
The formation of existing Water-In-Oil drop.
Detailed description of the invention
Fig. 1 is the quantitation curves figure of the embodiment of the present invention；
Fig. 2 is the detection fluorescence signal figure of single embryo's secretory protein to be detected of the embodiment of the present invention；
Fig. 3 is the detection fluorescence signal figure of the blank culture solution of the embodiment of the present invention.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
Embodiment 1,
As shown in Figure 1, a kind of single embryo's secretory protein quantitative detecting method based on Microfluidic droplet, which is characterized in that
The following steps are included:
A, the capture antibody and labelled antibody of single blastula embryo secretion human chorionic gonadotrophin (β HCG) are taken, it will
Capture antibody is connected on immunomagnetic beads, labelled antibody biotin labeling；
B, the immunomagnetic beads in step a are mixed with single blastula embryo secretion human chorionic gonadotrophin (β HCG)
It is incubated for 30min at 37 DEG C, immunomagnetic beads are then taken out into cleaning 3~5 times, immunomagnetic beads and labelled antibody and chain after cleaning
The beta galactosidase of mould Avidin label mixes and is incubated for 30min at 37 DEG C, and immunomagnetic beads are then taken out cleaning 3~5
Time, immunomagnetic beads are resuspended with the PBS buffer solution of 10uL after cleaning；
C, the magnetic bead after respin in step b is placed in the first sample well, fluorescein 2- β-D- galactopyranoside is placed in
Two sample wells, are placed in oily phase sample holes for fluorocarbon oil, and fluorogenic substrate and drop are added to shape in the first sample well by syringe
At Water-In-Oil drop, then the Water-In-Oil drop of generation is collected with capillary, by two end seal of capillary after the completion of collection
It closes in 37 DEG C of incubation 2h；
D, the Water-In-Oil drop being incubated in the capillary of completion in step c is imported into detection chip, under the action of sheath stream,
When Water-In-Oil drop individually window after testing, laser induced fluorescence detector detects the fluorescence intensity of Water-In-Oil drop, swashs
Hair wavelength is 488nm, and phosphor collection wave band obtains the content of single embryo's secretory protein to be measured in 520~530nm.
Then it takes blank culture solution to substitute single blastula embryo secretion human chorionic gonadotrophin, repeats above-mentioned step
Suddenly, the fluorescence detection of single blastula embryo secretion human chorionic gonadotrophin and blank culture solution as shown in Figure 2 is obtained
Signal.
Fig. 1 is the embodiment quantitation curves.Embodiment detection is limited to 0.03mIU/mL, and quantitation curves are linear
Range is 0.10~5.00mIU/mL.Relatively clinically used chemiluminescence particulate immunodetection is high by 10 at present for detection sensitivity
Times, and required sample volume is small (20 μ L) in detection process, for embryo medium sample without dilution.
Fig. 2 and Fig. 3 is respectively the fluorescent assay signal of single blastocyst culture liquid and blank culture solution, blank culture in Fig. 3
No positive signal in liquid, and have apparent Positive fluorescence in single blastula embryo secretion human chorionic gonadotrophin in Fig. 2
Signal.It is 0.86mIU/mL according to the protein content of β HCG in Fig. 1 standard curve calculated result blastocyst culture liquid.
In the description of this specification, the description of term " one embodiment ", " some embodiments ", " specific embodiment " etc.
Mean that particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are contained at least one reality of the invention
It applies in example or example.In the present specification, schematic expression of the above terms are not necessarily referring to identical embodiment or reality
Example.Moreover, description particular features, structures, materials, or characteristics can in any one or more of the embodiments or examples with
Suitable mode combines.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of single embryo's secretory protein quantitative detecting method based on Microfluidic droplet, which comprises the following steps:
A, the capture antibody and labelled antibody for taking single embryo's secretory protein to be measured, capture antibody is connected on immunomagnetic beads, will
Labelled antibody biotin labeling；
B, the immunomagnetic beads in step a are mixed and is incubated for single embryo's secretory protein to be measured, then taken out immunomagnetic beads clear
It washes 3~5 times, the labelled antibody and marked by streptavidin with biotin labeling that immunomagnetic beads, the step a after cleaning are obtained
Beta galactosidase mixing after, be incubated for, immunomagnetic beads then taken out into cleaning 3~5 times, then be resuspended with PBS buffer solution immune
Magnetic bead；
C, the magnetic bead after respin that step b is obtained is placed in the first sample well of Microfluidic droplet, fluorogenic substrate is placed in
In two sample wells, drop formation oil is placed in oily phase sample holes, fluorogenic substrate and drop formation oil are added by syringe
Water-In-Oil drop is formed into the first sample well, and the Water-In-Oil drop of generation is collected with capillary, it will after the completion of collecting
Capillary is closed at both ends, is incubated for；
D, the Water-In-Oil drop being incubated in the capillary of completion obtained in step c is imported into detection chip, in the effect of sheath stream
Under, when Water-In-Oil drop window after testing, laser induced fluorescence detector detects to obtain the fluorescence intensity of Water-In-Oil drop,
Obtain the content of single embryo's secretory protein to be measured.
2. single embryo's secretory protein quantitative detecting method according to claim 1 based on Microfluidic droplet, feature exist
In, in step a, the diameters of the immunomagnetic beads is 5~10um, the functional group on the surface of the immunomagnetic beads be carboxyl or
One of n-hydroxysuccinimide.
3. single embryo's secretory protein quantitative detecting method according to claim 1 based on Microfluidic droplet, feature exist
In in stepb, the temperature of the incubation is 37 DEG C, and the time of the incubation is 25~35min；The cleaning is adopted
Use pure water；The volume of the PBS buffer solution is 5~20uL.
4. single embryo's secretory protein quantitative detecting method according to claim 3 based on Microfluidic droplet, feature exist
In the time of the incubation is 30min；The volume of the PBS buffer solution is 10uL.
5. single embryo's secretory protein quantitative detecting method according to claim 1 based on Microfluidic droplet, feature exist
In in step c, the fluorogenic substrate is fluorescein 2- β-D- galactopyranoside or resorufin -2- β-D- galactopyranose
One kind of glycosides.
6. single embryo's secretory protein quantitative detecting method according to claim 5 based on Microfluidic droplet, feature exist
In, when fluorogenic substrate is fluorescein 2- β-D- galactopyranoside, in step d, the laser induced fluorescence detector
Excitation wavelength is one of 473nm or 488nm, and phosphor collection wave band is 510~530nm.
7. single embryo's secretory protein quantitative detecting method according to claim 5 based on Microfluidic droplet, feature exist
In, when fluorogenic substrate is resorufin -2- β-D- galactopyranoside, in step d, the laser induced fluorescence detector
Excitation wavelength is 558nm, and phosphor collection wave band is 560~580nm.
8. single embryo's secretory protein quantitative detecting method according to claim 1 based on Microfluidic droplet, feature exist
In in step c, the drop formation oil is any one in fluorocarbon oil, mineral oil and paraffin oil, and the temperature of the incubation is
37 DEG C, the time of the incubation is 1-3h.
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Cited By (6)
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| CN110887822A (en) * | 2019-11-13 | 2020-03-17 | 深圳中山泌尿外科医院 | Method for detecting embryo secretory protein |
| CN111381029A (en) * | 2020-03-20 | 2020-07-07 | 江苏师范大学 | Single-molecule multi-component digital immunoassay method |
| WO2021135991A1 (en) * | 2019-12-31 | 2021-07-08 | 深圳市帝迈生物技术有限公司 | Detection system for implementing classification and quantitative analysis, and detection method for immunological multi-joint inspection |
| CN113791207A (en) * | 2021-08-06 | 2021-12-14 | 南方科技大学 | High-sensitivity immunoassay method and application thereof |
| CN114713299A (en) * | 2022-01-05 | 2022-07-08 | 宁波大学 | Microfluidic chip and exosome detection method |
| CN117388227A (en) * | 2023-10-18 | 2024-01-12 | 博瑞生物医药（苏州）股份有限公司 | Method, computer readable medium and analysis device for determining concentration of target molecules in sample using detection microspheres |
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| CN110887822A (en) * | 2019-11-13 | 2020-03-17 | 深圳中山泌尿外科医院 | Method for detecting embryo secretory protein |
| WO2021135991A1 (en) * | 2019-12-31 | 2021-07-08 | 深圳市帝迈生物技术有限公司 | Detection system for implementing classification and quantitative analysis, and detection method for immunological multi-joint inspection |
| CN111381029A (en) * | 2020-03-20 | 2020-07-07 | 江苏师范大学 | Single-molecule multi-component digital immunoassay method |
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| CN114713299A (en) * | 2022-01-05 | 2022-07-08 | 宁波大学 | Microfluidic chip and exosome detection method |
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| CN117388227A (en) * | 2023-10-18 | 2024-01-12 | 博瑞生物医药（苏州）股份有限公司 | Method, computer readable medium and analysis device for determining concentration of target molecules in sample using detection microspheres |
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