CN107478837A - Micro-fluidic chemiluminescence detection system and its application based on magnetic particle - Google Patents
Micro-fluidic chemiluminescence detection system and its application based on magnetic particle Download PDFInfo
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- CN107478837A CN107478837A CN201710779749.4A CN201710779749A CN107478837A CN 107478837 A CN107478837 A CN 107478837A CN 201710779749 A CN201710779749 A CN 201710779749A CN 107478837 A CN107478837 A CN 107478837A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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Abstract
The invention discloses a kind of micro-fluidic chemiluminescence detection system based on magnetic particle, including chassis and upper strata chip；Upper strata chip is included positioned at the sample application zone of upper strata chip center and at least one micro-fluidic reaction detection passage connected with sample application zone；Micro-fluidic reaction detection passage includes：Sample Disengagement zone, it is connected with sample application zone；Antibody is coated with area, and it is connected by capillary microchannels with sample Disengagement zone；Magnetic particle is coated with area, and it is coated with area with antibody by capillary microchannels and connected；Waste collection area, it is coated with area with magnetic particle by capillary microchannels and connected；Wherein, antibody coating area is coated with another strain antibody of the determinand of a strain antibody of the determinand of luminescent substance mark and a mark in a pair of materials with pathoklisis；Magnetic particle coating area is coated with the magnetic particle of another mark in a pair of materials with pathoklisis；Chassis is arranged at the lower section of upper strata chip, and the position that chassis corresponds to magnetic particle coating area is provided with magnet.
Description
Technical field
The invention belongs to field of medical examination, more particularly to a kind of micro-fluidic chemiluminescence detection system based on magnetic particle
System.
Background technology
In recent years, bioassay technique field has obtained quick development, many important research directions occurs.Miniflow
It is wherein most active one to control chip analysis technology, and extensive attention is all obtained in scientific research and application field.Micro-fluidic core
Piece has the advantages that high flux, integrated, portable, easy to operate, inexpensive, as a kind of new analysis test platform
Through being widely used in various fields.
The sample that current chemiluminescence detection adapts to is serum, needs to carry out sample centrifugation point during detection
From serum is obtained, process is complicated cumbersome and take.Lack fully elution, interference caused by eliminate non-specific adsorption.
Magnetic particle currently used for biochemical analysis has the characteristics that：1) superpower paramagnetism, depositing in magnetic field is just referred to
It can assemble rapidly under, leaving magnetic field can be dispersed, occurs without aggregation and shows phenomenon；2) suitable particle diameter and particle diameter point
Cloth narrow range, microballoon is had sufficiently strong magnetic responsiveness, will not be settled again because particle diameter is too big；3) there is abundant table
Face active group, so that microballoon can be coupled with biochemical substances, and realized and dividing by testing sample in the presence of external magnetic field
From.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of micro-fluidic chemiluminescence detection system based on magnetic particle
System, it includes chassis and upper strata chip；
The upper strata chip is included positioned at the sample application zone of upper strata chip center and at least one miniflow connected with the sample application zone
Control reaction detection passage；
The micro-fluidic reaction detection passage includes：
Sample Disengagement zone, it is connected with sample application zone；Antibody is coated with area, and it is connected by capillary microchannels and the sample Disengagement zone
It is logical；Magnetic particle is coated with area, and it is coated with area with the antibody by capillary microchannels and connected；Waste collection area, it passes through capillary
Pipe microchannel connects with magnetic particle coating area；Wherein, the antibody coating area is coated with the determinand of luminescent substance mark
A strain antibody and with pathoklisis a pair of materials in one mark determinand another strain antibody；The magnetic is micro-
Grain coating area is coated with the magnetic particle of another mark in a pair of materials with pathoklisis；
Using when, the chassis is arranged at the lower section of upper strata chip, and the position that the chassis corresponds to the magnetic particle coating area is set
There is magnet, the magnet is permanent magnet or electromagnet.
As optimal technical scheme, the sample application zone is connected by Loading channel with the sample Disengagement zone；The sample-adding
Passage is the circular passage formed around sample application zone, and one end of Loading channel connects with the sample application zone, the side wall of Loading channel
Connect the sample Disengagement zone.
As optimal technical scheme, the luminescent substance is horseradish peroxidase, alkaline phosphatase, glucose oxidase
Or acridinium ester；A pair of materials with pathoklisis are biotin and Streptavidin, biotin and Avidin, or
For fluorescein and anti-fluorescein.
As optimal technical scheme, covered with anti-erythrocyte hemofiltration film, anti-erythrocyte filter above the aperture of the sample application zone
Covered with blood lid in blood film.
As optimal technical scheme, the average grain diameter of magnetic particle is at 0.5 ~ 2 μm.
As optimal technical scheme, a strain antibody of the determinand of luminescent substance mark and with pathoklisis
The mol ratio of another strain antibody of the determinand of a mark in a pair of materials is 4 ~ 1:1.
As optimal technical scheme, the antibody is coated with entering for the capillary microchannels between area and the sample Disengagement zone
Mouth diameter is less than outlet diameter；The entrance of capillary microchannels between the magnetic particle coating area and antibody coating area is straight
Footpath is less than outlet diameter；The inlet diameter of capillary microchannels between the waste collection area and magnetic particle coating area is small
In outlet diameter；Inlet diameter, the magnetic of capillary microchannels between the antibody coating area and the sample Disengagement zone
Particulate be coated with area and the antibody coating area between capillary microchannels inlet diameter and the waste collection area with it is described
The inlet diameter of capillary microchannels between magnetic particle coating area reduces successively.
Or the inlet diameter of the capillary microchannels between the antibody coating area and the sample Disengagement zone is more than
Mouth diameter；It is straight that the inlet diameter of capillary microchannels between the magnetic particle coating area and antibody coating area is more than outlet
Footpath；The inlet diameter of capillary microchannels between the waste collection area and magnetic particle coating area is more than outlet diameter；
The outlet diameters of capillary microchannels between antibody coating area and the sample Disengagement zone, magnetic particle coating area with
The outlet diameter of capillary microchannels between the antibody coating area and the waste collection area and magnetic particle coating area
Between the outlet diameters of capillary microchannels reduce successively.
As optimal technical scheme, the upper strata chip is that shape identical is circular with the chassis, the sample-adding position
In the center of circle of upper strata chip, radial direction of the micro-fluidic reaction detection passage along the upper strata chip is formed；In chassis
The heart is provided with through hole.
As optimal technical scheme, the micro-fluidic reaction detection passage is multiple；Permanent magnet on the chassis is circle
Annular, it is corresponding positioned at the lower section in magnetic particle coating area.
The present invention also provides the preparation method of the above-mentioned micro-fluidic chemiluminescence detection system based on magnetic particle, including such as
Lower step：
1）The sample application zone and the micro-fluidic reaction detection passage are opened up on chip substrate；
2）By a strain antibody of the determinand of luminescent substance mark and there is a mark in a pair of materials of pathoklisis
The solution of another strain antibody of determinand be overlying on antibody coating area, dry；
3）The solution that surface markers are had to another the magnetic particle in a pair of materials with pathoklisis is overlying on magnetic particle
Area is coated with, is dried；
4）The position that chassis corresponds to the magnetic particle coating area sets permanent magnet or electromagnet.
The present invention also provides the application process of the above-mentioned micro-fluidic chemiluminescence detection system based on magnetic particle, including such as
Lower step：
1）Upper strata chip is fixed on the axis of rotation top of detecting instrument；When the magnet on chassis is permanent magnet, chassis is arranged
In the bottom of the axis of rotation away from upper strata die bottom surface；When the magnet on chassis is electromagnet, chassis is attached to upper strata chip bottom
Face, electromagnet is correspondingly positioned at the magnetic particle coating area lower section of upper strata chip, electromagnet no power；Whole blood is added from sample application zone
This, startup instrument, axis of rotation rotation, under the influence of centrifugal force, the configured anti-erythrocyte hemofiltration film mistake in sample application zone of whole blood
Filter, into sample Disengagement zone；
2）After whole blood sample flowing is stable, make sample Disengagement zone by increasing axis of rotation rotation rotating speed to increase centrifugal action
Blood sample sample breaks through the capillary micro-valve effect of capillary microchannels and flows to antibody coating area, the determinand in blood sample sample with
The determinand of a mark in one strain antibody of the determinand that luminescent substance marks, a pair of materials with pathoklisis
Another strain antibody combines to form immune complex；
3）By increase again centrifugal force make immune complex break through capillary microchannels capillary micro-valve effect enter magnetic
Particulate be coated with area, mark in a pair of materials with pathoklisis on magnetic particle surface another with immune complex
A pair of materials with pathoklisis in a quick immune complex to be formed with reference to magnetic particle that reacts, so
Afterwards, the chassis with permanent magnet is moved upward to the bottom surface of fitting upper strata chip, magnetic of the permanent magnet correspondingly positioned at upper strata chip is micro-
Below grain coating area；Or the electromagnet energization to the chassis with electromagnet makes it rich in magnetic；Due to the magnetic attraction of magnet, knot
Close magnetic particle immune complex be enriched under the influence of a magnetic field magnetic particle coating area bottom, then by increase again from
Mental and physical efforts make the sample for not participating in reaction flow to waste collection area through capillary microchannels；
4）Immune complex of the cleaning fluid washing with reference to magnetic particle is added from sample application zone, cleaning fluid is moved to magnetic particle coating area
When, the chassis with permanent magnet is moved down away from into upper strata chip, or disconnecting power device makes electromagnet lose magnetism, ultrasound
Vibration, is fully washed with reference to the immune complex of magnetic particle, then moves on the chassis with permanent magnet at the chip of upper strata,
Or the electromagnet energization to the chassis with electromagnet makes it that the immune complex for combining magnetic particle are enriched into magnetic rich in magnetic
Particulate is coated with the bottom in area, cleaning fluid is flowed into waste collection area by increasing centrifugal force；
5）Luminous substrate liquid is added from sample application zone, luminous substrate liquid is transferred into magnetic particle by centrifugation is coated with area, by carry magnet
Chassis move down away from upper strata chip, or disconnecting power device makes electromagnet lose magnetism, after sonic oscillation, instrument inspection
Examining system detects the intensity of luminous signal, so as to realize the quantitative detection of determinand.
The present invention can reach following effect：
1st, the present invention dexterously combines microflow control technique and magnetic microparticle chemiluminescence technology, realizes the quick, high of target substance
Spend sensitive, accurate quantitative analysis detection.
2nd, separated and reacted using magnetic particle immunity enrichment, simplified separation process, improve the sensitivity of sample detection.Magnetic is micro-
The separation effect of grain, low concentration testing sample in sample to be tested is effectively caught, with reference to chemiluminescence detection mode, makes sensitivity big
Amplitude improves.
3rd, the micro-fluidic chemiluminescence detection system based on magnetic particle of the invention can carry out multinomial detection simultaneously, during saving
Between, improve efficiency.
4th, the micro-fluidic chemiluminescence detection system based on magnetic particle of the invention can be used for whole blood test, overcome biography
System chemiluminescence can only carry out Virus monitory, and the defects of whole blood test can not be carried out, simplify operating process.
5th, during reaction and washing by the way of sonic oscillation, reaction speed and elimination are effectively improved
Interference caused by non-specific adsorption.
Brief description of the drawings
Figure 1A is the upper strata chip structure schematic diagram one of the micro-fluidic chemiluminescence detection system based on magnetic particle.
Figure 1B is the upper strata chip structure schematic diagram two of the micro-fluidic chemiluminescence detection system based on magnetic particle.
Fig. 2 is the chassis portion structural representation of the micro-fluidic chemiluminescence detection system based on magnetic particle.
Fig. 3 is that the micro-fluidic reaction detection channel cross-section structure of the micro-fluidic chemiluminescence detection system based on magnetic particle is shown
It is intended to.
Fig. 4 is the process signal of the step 1 of the application process of the micro-fluidic chemiluminescence detection system based on magnetic particle
Figure.
Fig. 5 is the process schematic of the step 2 of the application process of the micro-fluidic chemiluminescence detection system based on magnetic particle.
Fig. 6 is the process schematic of the step 3 of the application process of the micro-fluidic chemiluminescence detection system based on magnetic particle.
Fig. 7 is step 4 process schematic of the application process of the micro-fluidic chemiluminescence detection system based on magnetic particle.
Fig. 8 is step 5 process schematic of the application process of the micro-fluidic chemiluminescence detection system based on magnetic particle.
Embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings, so that those skilled in the art can be with
It is better understood from the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
With reference to shown in Figure 1A ~ 3, a kind of micro-fluidic chemiluminescence detection system based on magnetic particle provided by the invention, bag
Include chassis 2 and upper strata chip 1；
Upper strata chip 1 include positioned at the center of upper strata chip 1 sample application zone 10 and it is at least one connected with sample application zone it is micro-fluidic instead
Answer sense channel 11；In a preferred embodiment, can be covered with anti-erythrocyte hemofiltration film on the aperture of sample application zone 10（In figure
It is not shown）, can be covered with blood lid on anti-erythrocyte hemofiltration film（Not shown in figure）；
Micro-fluidic reaction detection passage 11 includes：
Sample Disengagement zone 110, it is connected with sample application zone 10；Antibody is coated with area 111, and it passes through capillary microchannels 114 and sample
Disengagement zone 110 connects；Magnetic particle is coated with area 112, and it is coated with area 111 with antibody by capillary microchannels 115 and connected；Waste liquid is received
Collect area 113, it is coated with area 112 with magnetic particle by capillary microchannels 116 and connected；Wherein, antibody coating area 111 is coated with hair
The determinand of one strain antibody of the determinand of stimulative substance mark and a mark in a pair of materials with pathoklisis
Another strain antibody；The magnetic that magnetic particle coating area 112 is coated with another mark in a pair of materials with pathoklisis is micro-
Grain；Wherein, the average grain diameter of magnetic particle is at 0.5 ~ 2 μm；
Chassis 2 is arranged at the lower section of upper strata chip, chassis（Vinyl disc）The position in corresponding magnetic particle coating area is provided with magnet 20, magnetic
Iron 20 can be permanent magnet or electromagnet.
In a preferred embodiment of the invention, upper strata chip 1 is that shape identical is circular with chassis 2, sample application zone 10
In the center of circle of upper strata chip, radial direction of the micro-fluidic reaction detection passage 11 along upper strata chip 1 is formed；The center on chassis 2 is set
There is through hole 21.
With reference to shown in Fig. 3, the capillary micro-valve effect that is formed in centrifugation, antibody coating area 111 and sample Disengagement zone 110 it
Between the inlet diameter A1 of capillary microchannels 114 be less than outlet diameter B1；Magnetic particle is coated with area 112 and antibody coating area 111
Between the inlet diameter A2 of capillary microchannels 115 be less than outlet diameter B2；Waste collection area 113 and magnetic particle coating area
The inlet diameter A3 of capillary microchannels 116 between 112 is less than outlet diameter B3；Antibody is coated with area 111 and sample Disengagement zone
Inlet diameter A1, magnetic particle coating area 112 and the antibody of capillary microchannels 114 between 110 are coated with the capillary between area 111
Capillary microchannels 116 between the inlet diameter A2 of pipe microchannel 115 and waste collection area 113 and magnetic particle coating area 112
Inlet diameter A3 reduce successively.
Or in another embodiment（Not shown in figure）, between antibody coating area 111 and sample Disengagement zone 110
The inlet diameter A1 of capillary microchannels 114 is more than outlet diameter B1；Magnetic particle is coated between area 112 and antibody coating area 111
The inlet diameter A2 of capillary microchannels 115 be more than outlet diameter B2；Waste collection area 113 and magnetic particle coating area 112 it
Between the inlet diameter A3 of capillary microchannels 116 be more than outlet diameter B3；Antibody be coated with area 111 and sample Disengagement zone 110 it
Between capillary microchannels 114 outlet diameter B1, magnetic particle coating area 112 and antibody coating area 111 between capillary it is micro-
The outlet diameter B2 of passage 115 and waste collection area 113 and magnetic particle are coated with going out for the capillary microchannels 116 between area 112
Mouth diameter B3 reduces successively.
With reference to Figure 1B, sample application zone 10 is connected by Loading channel 12 with sample Disengagement zone 110；Loading channel 12 is around sample-adding
The circular passage that area 10 is formed, one end of Loading channel 12 connect with sample application zone 10, the side wall connection sample Disengagement zone of Loading channel.
Micro-fluidic reaction detection passage 11 can be multiple；Permanent magnet on chassis 2 is annular, corresponding to be located at magnetic particle bag
By the lower section in area 112.
In a preferred embodiment of the invention, enzyme is horseradish peroxidase, alkaline phosphatase, glucose oxidase；
A pair of materials with pathoklisis are biotin and Streptavidin, biotin and Avidin, or are fluorescein and anti-
Fluorescein.
In a preferred embodiment of the invention, the antibody of one plant of determined antigen of luminescent substance mark and with special parent
Mol ratio with the antibody of another plant of determined antigen of a mark in a pair of materials of property is 4 ~ 1:1.
The preparation method of the micro-fluidic chemiluminescence detection system based on magnetic particle of the present invention, comprises the following steps：
1）Sample application zone 10 and micro-fluidic reaction detection passage 11 are opened up on chip substrate；
2）By the antibody of one plant of determined antigen of luminescent substance mark and there is a mark in a pair of materials of pathoklisis
The solution of the antibody of another plant of determined antigen of note is overlying on antibody coating area 111, dries；
3）The solution that surface markers are had to another the magnetic particle in a pair of materials with pathoklisis is overlying on magnetic particle
Area 112 is coated with, is dried；
4）The position in the corresponding magnetic particle coating area 112 in chassis 2 sets magnet 20.
With reference to Fig. 4 ~ 8, the application process of the micro-fluidic chemiluminescence detection system of the invention based on magnetic particle, including such as
Lower step：
1）The axis of rotation 3 of detecting instrument passes through the through hole 21 at the center of chassis 2 with reference to shown in Fig. 2, and chassis is set under the axis of rotation 3
Side, upper strata chip 1 are fixed on the top of the axis of rotation 3 of detecting instrument；When the magnet on chassis is permanent magnet, chassis 2 is arranged
In the bottom of the axis of rotation away from the bottom surface of upper strata chip 1；When the magnet on chassis 2 is electromagnet, chassis 2 is attached to upper strata chip
1 bottom surface, electromagnet is correspondingly positioned at the lower section of magnetic particle coating area 112 of upper strata chip 1, electromagnet no power；With reference to shown in Fig. 4,
Whole blood sample is added from sample application zone 10, starts instrument, the rotation of the axis of rotation 3, under the influence of centrifugal force, whole blood is configured to be loaded
The anti-erythrocyte hemofiltration membrane filtration in area 10, into sample Disengagement zone 110；
2）With reference to shown in Fig. 5, after sample flow dynamic stability, make sample by increasing the rotation rotating speed of the axis of rotation 3 to increase centrifugal action
The blood sample sample of this Disengagement zone 110 breaks through the capillary micro-valve effect of capillary microchannels 114 and flows to antibody coating area 111,
One strain antibody of the determinand that the determinand in blood sample sample marks with luminescent substance, have in a pair of materials of pathoklisis
Another strain antibody of determinand of a mark combine to form immune complex；
3）With reference to shown in Fig. 6, by increase again centrifugal force make immune complex break through capillary microchannels 115 capillary it is micro-
Valve action enters magnetic particle coating area 112, marks another in a pair of materials with pathoklisis on magnetic particle surface
One with one in a pair of materials with pathoklisis in immune complex it is quick react to be formed it is micro- with reference to magnetic
The immune complex of grain, then, the chassis 2 with permanent magnet is moved upward to the bottom surface of fitting upper strata chip 1, permanent magnet is corresponding
Positioned at the lower section of magnetic particle coating area 112 of upper strata chip 1；Or the electromagnet energization to the chassis 2 with electromagnet makes it rich in magnetic
Property；Due to the magnetic attraction of magnet, the bottom in magnetic particle coating area is enriched under the influence of a magnetic field with reference to the immune complex of magnetic particle
End, then makes the sample for not participating in reaction flow to waste collection area 113 through capillary microchannels 116 by increasing centrifugal force again；
4）With reference to shown in Fig. 7, immune complex of the cleaning fluid washing with reference to magnetic particle is added from sample application zone 10, cleaning fluid is moved to
When magnetic particle is coated with area 112, the chassis 2 with permanent magnet is moved down away from into upper strata chip 1, or disconnect power device to make electricity
Magnet loses magnetism, sonic oscillation, is fully washed with reference to the immune complex of magnetic particle, by the chassis 2 with permanent magnet to
Moved at upper strata chip 1, or the electromagnet energization to the chassis 2 with electromagnet makes it to combine exempting from for magnetic particle rich in magnetic
Epidemic disease compound is enriched to the bottom in magnetic particle coating area 112, cleaning fluid is flowed into waste collection area 113 by increasing centrifugal force；
5）With reference to shown in Fig. 8, luminous substrate liquid is added from sample application zone 10, luminous substrate liquid is transferred to magnetic particle bag by centrifuging
By area 112, the chassis 2 with permanent magnet is moved down away from into upper strata chip 1, or disconnecting power device makes electromagnet lose magnetic
Property, after sonic oscillation, instrument detecting system detects the intensity of luminous signal, so as to realize the quantitative detection of determinand.
The centrifugal force that the detection process of the micro-fluidic chemiluminescence detection system based on magnetic particle of the present invention needs comes from
In supporting detecting instrument.The axis of rotation is not belonging to a part for micro-fluidic chemiluminescence detection system, but with micro-fluidic chemistry
A part in the matching used instrument of luminescent detection system, bearing are connected to a fixed with chip tray；Chip can be clipped in core
On tablet tray；Chassis does not depend on the axis of rotation, and it is set on the axis of rotation by through hole freely to move up and down.
The magnetic field of magnetic field generation device is provided by magnet, and permanent magnet can pass through the mobile relative position with chip（Electromagnetism
Energization or power-off are crossed by Tie Tong）, magnetic particle is in or depart from the magnetic field of magnet, magnetic particle technique effect collected to realize.
Hereinafter, to detect anti-gyneduct hormone（AMH）Exemplified by the present invention is further described.
Step 1 is from sample application zone（There is anti-erythrocyte hemofiltration film herein）15 ~ 150 μ L whole blood samples are added, cover blood lid,
Micro-fluidic chemiluminescence detection system is put into supporting instrument, starts instrument, under the influence of centrifugal force, whole blood is through anti-red
Cell hemofiltration membrane filtration, then fill up three sample Disengagement zone successively by Loading channel；
Step 2 makes the sample of sample Disengagement zone break through capillary microchannels after sample flow dynamic stability by increasing centrifugal action
The effect of capillary micro-valve flow to antibody coating area, sample is anti-by the powdered alkali phosphatase enzyme mark formed after drying
The anti-AMH antibody of another strain of AMH antibody and biotin labeling is redissolved, and the antigen and alkali phosphatase enzyme mark in sample resist
AMH antibody, the anti-AMH antibody bindings of another strain of biotin labeling form immune complex；
Step 3 makes immune complex enter magnetic particle coating area by capillary microchannels by increasing centrifugal force, redissolves
The magnetic particle of the marked by streptavidin in the region（1 μm of average grain diameter）, the biotin in Streptavidin and immune complex
The quick immune complex to be formed with reference to magnetic particle that reacts, after 1 ~ 5 min, by the chassis with permanent magnet at the chip of upper strata
Motion, the bottom in magnetic particle coating area is enriched under the influence of a magnetic field with reference to the immune complex of magnetic particle, then by again
Secondary increasing centrifugal force makes the sample for not participating in reaction flow to waste collection area through capillary microchannels；
Step 4 adds immune complex of the cleaning fluid washing twice with reference to magnetic particle from adding mouth, and cleaning fluid is moved to magnetic particle
When being coated with area, the chassis with permanent magnet is moved down away from into upper strata chip, sonic oscillation, with reference to the immune complex of magnetic particle
Fully washed, by the chassis with permanent magnet to motion at the chip of upper strata, the immune complex for combining magnetic particle is enriched to
Magnetic particle is coated with the bottom in area, cleaning fluid is flowed into waste collection area by increasing centrifugal force；
The alkaline phosphatase luminous substrate that step 5 adds from adding mouth, luminous substrate liquid is transferred to magnetic particle bag by centrifuging
By area, the chassis with permanent magnet is moved down away from into upper strata chip, after sonic oscillation, instrument detecting system detection luminous signal
Intensity, so as to realize the quantitative detection of analyte.
AMH result is as shown in table 1 below in whole blood sample, and detection sensitivity scope is 0 ~ 20 ng/mL, and herein
The detection CV values of scope are less than 10%.
| Concentration ng/mL | Relative light unit（RLU） | The coefficient of variation（CV） |
| 0 | 1545 | 2.3% |
| 0.1 | 4429 | 0.9% |
| 0.4 | 19520 | 0.7% |
| 1.5 | 67218 | 3.2% |
| 5 | 290853 | 1.4% |
| 20 | 847238 | 0.8% |
1. one plant of anti-AMH antibody of alkali phosphatase enzyme mark
By 2.5mg alkaline phosphatase（50IU/mg）It is added to 200 μ L 100mM PBS cushioning liquid（pH=6.8）In, wherein
Containing 1.25% glutaraldehyde, stir and evenly mix, activate 24 hours, dialysed to 50mM at 4 DEG C（pH=7.2）, 18 hours, change liquid 3 times；
The anti-AMH monoclonal antibodies of one plant of 1.8mg mouse are dissolved in 120 μ L 1M carbonate solution（pH=9）In；By the alkaline phosphorus of activation
Sour enzyme is added in the solution of the anti-AMH monoclonal antibodies of mouse of configuration, is well mixed, 24h is reacted at 4 DEG C, then adds 20 μ
The mM of L 100 lysine solution, it is well mixed, reacts 4h at 20 DEG C；Dialyse 12h to 50 mM PBS at 4 DEG C（pH=7.2）,
Change liquid 3 times；Supernatant liquor is removed in centrifugation, with the NaN of 50mM TB7.4+0.6% BSA+0.05%3Dilution, is preserved at -20 DEG C.
2. the anti-AMH antibody of another strain of biotin labeling
The anti-AMH monoclonal antibodies of another plant of mouse are first diluted to 1mg/mL with sodium carbonate buffer, and with sodium carbonate buffer room
Temperature（25℃±5℃）Lucifuge is stirred 4 hours and dialysed；Then use N, N- dimethylformamides（DMF）By 6-aminocaprolc acid-N hydroxyl ambers
Amber acid imide-biotin（BCNHS）It is configured to 1mg/mL；Added in the anti-AMH monoclonal antibody solutions of another plant of mouse of 1mL above-mentioned
The μ L of μ L of DMF solution 125 ~ 66.7, mix in vial, room temperature（25℃±5℃）Lucifuge stirs 2 hours；Add 1mol/L chlorinations
The μ L of ammonium salt solution 9.6, room temperature（25℃±5℃）Lucifuge stirs 10 minutes；Then mixed solution is transferred to bag filter, uses phosphate buffer
4 DEG C of dialysed overnights.Finally take out plus -20 DEG C of equivalent glycerine preserves.
3. the processing of anti-erythrocyte hemofiltration film at well
Selected materials are glass fibre element film or polyester fiber film, and it is single to be soaked in the mouse anti-human RBC that concentration is 30 mg/L
Clonal antibody solution, a length of 1 ~ 6 h during immersion；It is subsequently placed at humidity<Drained away the water in 35% environment, duration 8 ~ 16 is small
When；Anti-erythrocyte hemofiltration film is finally cut into dimension, is affixed on instrument at well.
4. antibody is coated with the processing in area
The antibody that alkaline phosphatase luminescent substance is marked：Another strain antibody=4 ~ 1 of biotin labeling：1（Mol ratio）Phase is pressed in mixing
Answer specification（The amount of the antibody of biotin labeling is 5 ~ 50 μ g）Antibody coating area in chip, is placed in humidity<Done in 35% environment
Dry 8 ~ 16 hours.
5. magnetic particle is coated with the processing in area
The configuration of magnetic particle solution：There are the magnetic bionanoparticles with superparamagnetism of Streptavidin from surface markers,
1 μm of diameter, it is dissolved into 1 ~ 20 mg/mL with magnetic particle dilution.
Magnetic particle is coated with the drying in area：The magnetic particle coating that the magnetic particle solution for taking 5 ~ 15 μ L to configure is placed on chip
Area, it is placed in humidity<Dried 0.5 hour in 35% environment.
6. detection
Taking 15-150 μ L droplets of whole blood, in the presence of capillary or centrifugal force, sample flows through chip in sample area.5-20 minutes
Afterwards, the luminous intensity in magnetic particle coating area is detected under chemical illumination immunity analysis instrument, you can inverse goes out the dense of AMH in sample
Degree.
It is attached：Required solution is prepared
（1）Mouse anti-human RBC's monoclonal antibody soak
Sodium dihydrogen phosphate 0.99g
Disodium hydrogen phosphate 5.16g
Bovine serum albumin 1g
Sodium chloride 0.9g
Mouse anti-human RBC's monoclonal antibody 34mg
Sodium azide 1g
Purified water is settled to 1000mL.
（2）Magnetic particle dilution
Sodium dihydrogen phosphate 0.99g
Disodium hydrogen phosphate 5.16g
Sodium chloride 0.9g
Bovine serum albumin 5g
Hexadecyltrimethylammonium chloride 0.224g
Sodium azide 0.5g
Proclin300 1mL
Roche cleans antibody（HBR-3） 50mg
Purified water is settled to 1000mL.
（3）Cleaning fluid
Sodium dihydrogen phosphate 0.99g
Disodium hydrogen phosphate 5.16g
Tween-20 1ml
Proclin300 1mL。
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
Enclose not limited to this.The equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
Claims (10)
1. a kind of micro-fluidic chemiluminescence detection system based on magnetic particle, it is characterised in that including chassis and upper strata chip；
The upper strata chip is included positioned at the sample application zone of upper strata chip center and at least one miniflow connected with the sample application zone
Control reaction detection passage；
The micro-fluidic reaction detection passage includes：
Sample Disengagement zone, it is connected with sample application zone；Antibody is coated with area, and it is connected by capillary microchannels and the sample Disengagement zone
It is logical；Magnetic particle is coated with area, and it is coated with area with the antibody by capillary microchannels and connected；Waste collection area, it passes through capillary
Pipe microchannel connects with magnetic particle coating area；Wherein, the antibody coating area is coated with the determinand of luminescent substance mark
A strain antibody and with pathoklisis a pair of materials in one mark determinand another strain antibody；The magnetic is micro-
Grain coating area is coated with the magnetic particle of another mark in a pair of materials with pathoklisis；
Using when, the chassis is arranged at the lower section of upper strata chip, and the position that the chassis corresponds to the magnetic particle coating area is set
There is magnet, the magnet is permanent magnet or electromagnet.
2. the micro-fluidic chemiluminescence detection system according to claim 1 based on magnetic particle, it is characterised in that described to add
Sample area is connected by Loading channel with the sample Disengagement zone；The Loading channel is the circular passage formed around sample application zone,
One end of Loading channel connects with the sample application zone, and the side wall of Loading channel connects the sample Disengagement zone.
3. the micro-fluidic chemiluminescence detection system according to claim 1 based on magnetic particle, it is characterised in that the hair
Stimulative substance is horseradish peroxidase, alkaline phosphatase, glucose oxidase or acridinium ester；It is described with pathoklisis one
It is biotin and Streptavidin to material, biotin and Avidin, or be fluorescein and anti-fluorescein.
4. the micro-fluidic chemiluminescence detection system according to claim 1 based on magnetic particle, it is characterised in that magnetic particle
Average grain diameter at 0.5 ~ 2 μm.
5. the micro-fluidic chemiluminescence detection system according to claim 1 based on magnetic particle, it is characterised in that the hair
The determinand of one strain antibody of the determinand of stimulative substance mark and a mark in a pair of materials with pathoklisis
The mol ratio of another strain antibody is 4 ~ 1:1.
6. the micro-fluidic chemiluminescence detection system according to claim 1 based on magnetic particle, it is characterised in that described anti-
The inlet diameter that body is coated with the capillary microchannels between area and the sample Disengagement zone is less than outlet diameter；The magnetic particle bag
Outlet diameter is less than by the inlet diameter of the capillary microchannels between area and antibody coating area；The waste collection area with
The inlet diameter of capillary microchannels between the magnetic particle coating area is less than outlet diameter；Antibody coating area with it is described
Between the inlet diameter of capillary microchannels between sample Disengagement zone, magnetic particle coating area and antibody coating area
The inlet diameter of capillary microchannels and the waste collection area and the magnetic particle are coated with the capillary microchannels between area
Inlet diameter reduces successively；
Or the inlet diameter of the capillary microchannels between the antibody coating area and the sample Disengagement zone is more than outlet directly
Footpath；The inlet diameter of capillary microchannels between the magnetic particle coating area and antibody coating area is more than outlet diameter；
The inlet diameter of capillary microchannels between the waste collection area and magnetic particle coating area is more than outlet diameter；It is described
Antibody be coated with the outlet diameter of the capillary microchannels between area and the sample Disengagement zone, magnetic particle coating area with it is described
Between the outlet diameter of capillary microchannels between antibody coating area and the waste collection area and magnetic particle coating area
The outlet diameters of capillary microchannels reduce successively.
7. the micro-fluidic chemiluminescence detection system according to claim 1 based on magnetic particle, it is characterised in that on described
Layer chip is that shape identical is circular with the chassis, and the sample application zone is located at the center of circle of upper strata chip, the micro-fluidic reaction
Radial direction of the sense channel along the upper strata chip is formed；The center on chassis is provided with through hole.
8. the micro-fluidic chemiluminescence detection system according to claim 7 based on magnetic particle, it is characterised in that described micro-
Stream control reaction detection passage is multiple；Magnet on the chassis is annular, corresponding positioned at the lower section in magnetic particle coating area.
9. the preparation method of the micro-fluidic chemiluminescence detection system based on magnetic particle described in any one of claim 1 ~ 8, its
It is characterised by, comprises the following steps：
1）The sample application zone and the micro-fluidic reaction detection passage are opened up on chip substrate；
2）By a strain antibody of the determinand of luminescent substance mark and there is a mark in a pair of materials of pathoklisis
The solution of another strain antibody of determinand be overlying on antibody coating area, dry；
3）The solution that surface markers are had to another the magnetic particle in a pair of materials with pathoklisis is overlying on magnetic particle
Area is coated with, is dried；
4）The position that chassis corresponds to the magnetic particle coating area sets magnet.
10. the application process of the micro-fluidic chemiluminescence detection system based on magnetic particle described in any one of claim 1 ~ 8, its
It is characterised by, comprises the following steps：
1）Upper strata chip is fixed on the axis of rotation top of detecting instrument；When the magnet on chassis is permanent magnet, chassis is arranged
In the bottom of the axis of rotation away from upper strata die bottom surface；When the magnet on chassis is electromagnet, chassis is attached to upper strata chip bottom
Face, electromagnet is correspondingly positioned at the magnetic particle coating area lower section of upper strata chip, electromagnet no power；Whole blood is added from sample application zone
This, startup instrument, axis of rotation rotation, under the influence of centrifugal force, the configured anti-erythrocyte hemofiltration film mistake in sample application zone of whole blood
Filter, into sample Disengagement zone；
2）After whole blood sample flowing is stable, make sample Disengagement zone by increasing axis of rotation rotation rotating speed to increase centrifugal action
Blood sample sample breaks through the capillary micro-valve effect of capillary microchannels and flows to antibody coating area, the determinand in blood sample sample with
The determinand of a mark in one strain antibody of the determinand that luminescent substance marks, a pair of materials with pathoklisis
Another strain antibody combines to form immune complex；
3）By increase again centrifugal force make immune complex break through capillary microchannels capillary micro-valve effect enter magnetic
Particulate be coated with area, mark in a pair of materials with pathoklisis on magnetic particle surface another with immune complex
A pair of materials with pathoklisis in a quick immune complex to be formed with reference to magnetic particle that reacts, so
Afterwards, the chassis with permanent magnet is moved upward to the bottom surface of fitting upper strata chip, magnetic of the permanent magnet correspondingly positioned at upper strata chip is micro-
Below grain coating area；Or the electromagnet energization to the chassis with electromagnet makes it rich in magnetic；Due to the magnetic attraction of magnet, knot
Close magnetic particle immune complex be enriched under the influence of a magnetic field magnetic particle coating area bottom, then by increase again from
Mental and physical efforts make the sample for not participating in reaction flow to waste collection area through capillary microchannels；
4）Immune complex of the cleaning fluid washing with reference to magnetic particle is added from sample application zone, cleaning fluid is moved to magnetic particle coating area
When, the chassis with permanent magnet is moved down away from into upper strata chip, or disconnecting power device makes electromagnet lose magnetism, ultrasound
Vibration, is fully washed with reference to the immune complex of magnetic particle, then moves on the chassis with permanent magnet at the chip of upper strata,
Or the electromagnet energization to the chassis with electromagnet makes it that the immune complex for combining magnetic particle are enriched into magnetic rich in magnetic
Particulate is coated with the bottom in area, cleaning fluid is flowed into waste collection area by increasing centrifugal force；
5）Luminous substrate liquid is added from sample application zone, luminous substrate liquid is transferred into magnetic particle by centrifugation is coated with area, by carry magnet
Chassis move down away from upper strata chip, or disconnecting power device makes electromagnet lose magnetism, after sonic oscillation, instrument inspection
Examining system detects the intensity of luminous signal, so as to realize the quantitative detection of determinand.
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Address after: 101111 Four Floor F4E of Building No. 2, Kechuang East Five Street, Tongzhou District, Beijing Patentee after: Beijing Huaketai Biotechnology Co., Ltd. Address before: 100070 Room 501, 2 building, 8 Haiying Road, Science City, Fengtai District, Beijing (Park) Patentee before: Beijing Elcoteq Bio-Technology Co., Ltd. |