CN108956558A - A kind of micro-fluidic chip and immunofluorescence analysis instrument - Google Patents
A kind of micro-fluidic chip and immunofluorescence analysis instrument Download PDFInfo
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- CN108956558A CN108956558A CN201810509672.3A CN201810509672A CN108956558A CN 108956558 A CN108956558 A CN 108956558A CN 201810509672 A CN201810509672 A CN 201810509672A CN 108956558 A CN108956558 A CN 108956558A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
This application discloses a kind of micro-fluidic chip and immunofluorescence analysis instrument, the micro-fluidic chip includes: substrate, is provided with protrusion；Cover board is provided with groove that is corresponding with the position of the protrusion and matching, and the protrusion on the substrate can be separated with the groove on the cover board, so that the substrate of the micro-fluidic chip is detachably connected with the cover board.By the above-mentioned means, the application can be such that the substrate of micro-fluidic chip is detachably connected with cover board, and then technical support is provided for micro-fluidic chip is reusable.
Description
Technical field
This application involves technical field of medical equipment, more particularly to a kind of micro-fluidic chip and immunofluorescence analysis instrument.
Background technique
Microfluidic chip technology is a kind of micro (10 to be handled in the structure of micron dimension-9L-10-18L) the reaction of sample
System is now widely used for cell screening, immune detection, Cytometric Analysis etc.；Traditional micro-fluidic chip is with micro-pipe
Road network microstructure features, by micro-processing technology by Functional Units such as microchannel, Micropump, micro-valve, micro- liquid storage device, micro- detecting elements
Device is integrated on chip material as integrated circuit, to complete the processing and detection of sample.
Present inventor has found that existing most of micro-fluidic chips cannot repeat to make in long-term research process
With difficulty of processing is larger, and processing cost is generally 50-200 member/piece, and the testing cost of the relevant detection project of hospital is 30
Member or so, the cost-range that the cost of micro-fluidic chip can bear considerably beyond hospital.
Summary of the invention
The application can make micro- mainly solving the technical problems that provide a kind of micro-fluidic chip and immunofluorescence analysis instrument
The substrate of fluidic chip is detachably connected with cover board, and then provides technical support for micro-fluidic chip is reusable.
In order to solve the above technical problems, the technical solution that the application uses is: a kind of micro-fluidic chip is provided, it is described
Micro-fluidic chip includes: substrate, is provided with protrusion；Cover board is provided with and phase corresponding with the position of the protrusion
The groove matched, the protrusion on the substrate can be separated with the groove on the cover board, so that the micro-fluidic core
The substrate of piece is detachably connected with the cover board.
In order to solve the above technical problems, another technical solution that the application uses is: providing a kind of immunofluorescence analysis
Instrument, the immunofluorescence analysis instrument include the micro-fluidic chip in any of the above-described embodiment.
The beneficial effect of the application is: being in contrast to the prior art, is provided on substrate provided herein convex
It rises, groove that is corresponding with the position of protrusion and matching, the protrusion when cover plate lid is on substrate, on substrate is provided on cover board
Position substrate and the cover board in groove on insertion cover board, and the protrusion on substrate can be separated with the groove on cover board,
So that the substrate of the micro-fluidic chip is detachably connected with the cover board, and then reusable for micro-fluidic chip
Technical support is provided.
In an application scenarios, after micro-fluidic chip use, substrate is separated with detachable cover plate, and by substrate
It is placed in the cleaning solution containing effective components such as sodium hypochlorite and is cleaned with cover board, in case weight next time after cleaning is completed and dried
It is multiple to use.
Detailed description of the invention
In order to more clearly explain the technical solutions in the embodiments of the present application, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, the drawings in the following description are only some examples of the present application, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.Wherein:
Fig. 1 is the structural schematic diagram of one embodiment of the application micro-fluidic chip；
Fig. 2 is the structural schematic diagram of one embodiment of substrate in Fig. 1；
Fig. 3 is the structural schematic diagram of one embodiment of Fig. 1 cover plate；
Fig. 4 is the structural schematic diagram of another embodiment of substrate in Fig. 1；
Fig. 5 is the structural schematic diagram of another embodiment of the application micro-fluidic chip；
Fig. 6 is the structural schematic diagram of one embodiment of substrate in Fig. 5；
Fig. 7 is the structural schematic diagram of one embodiment of Fig. 5 cover plate；
Fig. 8 is the structural schematic diagram of one embodiment of the application immunofluorescence analysis instrument.
Specific embodiment
Below in conjunction with the attached drawing in the embodiment of the present application, technical solutions in the embodiments of the present application carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of embodiments of the present application, rather than whole embodiments.Based on this
Embodiment in application, those of ordinary skill in the art are obtained every other under the premise of not making creative labor
Embodiment shall fall in the protection scope of this application.
Currently, existing most of micro-fluidic chips cannot be reused, difficulty of processing is larger, and processing cost is generally
50-200 member/piece, and the testing cost of the relevant detection project of hospital is at 30 yuan or so, the cost of micro-fluidic chip considerably beyond
The cost-range that hospital can bear, therefore, developing the reusable micro-fluidic chip of one kind is particularly important.
Fig. 1-Fig. 3 is please referred to, Fig. 1 is the structural schematic diagram of one embodiment of the application micro-fluidic chip, and Fig. 2 is in Fig. 1
The structural schematic diagram of one embodiment of substrate, Fig. 3 are the structural schematic diagram of one embodiment of Fig. 1 cover plate.It is provided herein
Micro-fluidic chip 1 include substrate 10 and cover board 12；In an application scenarios, the material of above-mentioned micro-fluidic chip 1 is surface
Modified dimethyl silicone polymer (PDMS), wherein the modified mode in the surface PDMS includes plasma, surfactant, purple
Any in external exposure, ozone treatment, the application is not construed as limiting this.PDMS itself is a kind of strong hydrophobic material, in the material
Upper building microchannel 102, if modified without surface, entirety after the assembly is completed, i.e., covers cover board on the substrate 10
After 12, due to the strong-hydrophobicity on the surface PDMS, flowing resistance of the polar liquid of similar aqueous solution in microchannel 102 can be made
Power is larger, and therefore, the surface modification for the PDMS material is very necessary.It, can be with by above-mentioned modified to the surface PDMS
Make inert PDMS surface active, enhance the reciprocation at interface, is easier sample in its surface flow.In other application field
The material of Jing Zhong, above-mentioned micro-fluidic chip 1 can also be other, for example, silicon, glass, quartz, plastics etc., the application does not make this
It limits.
Specifically, protrusion 100 is provided on substrate 10；The number of protrusion 100 can be 1,2,3 etc., work as protrusion
When 100 number is even number, protrusion 100 can be symmetrically or non-symmetrically arranged two-by-two, and the application is not construed as limiting this；For convenient for dress
Match, in the present embodiment, the cross section of protrusion 100 is circle, and in other embodiments, the cross section of protrusion 100 can also be it
He (for example, triangle, rectangle etc.), the application is not construed as limiting this.In an application scenarios, miniflow is provided on substrate 10
Channel 102, microchannel 102 are the channel formed in a side surface of substrate 10 using techniques such as photoetching, and microchannel 102 exists
Shared region is defined as functional areas 104 on substrate 10, and the region on substrate 10 in addition to microchannel 102 is defined as nonfunctional area
106, it is the realization for not influencing the function of functional areas 104 on substrate 10, protrusion 100 can be set in the nonfunctional area of substrate 10
On 106.
Specifically, cover board 12 cover the microchannel 102 on substrate 10, are provided with corresponding with the position of protrusion 100
And the groove 120 to match, when cover board 12 covers on the substrate 10, the protrusion 100 on substrate 10 is embedded in the groove on cover board 12
Position substrate 10 and cover board 12 in 120, and the protrusion 100 on substrate 10 can be separated with the groove 120 on cover board 12, so that
The substrate 10 for obtaining micro-fluidic chip 1 is detachably connected with cover board 12.Groove 120 can penetrate through or not penetrate through cover board 12；When recessed
When slot 120 does not penetrate through cover board 12, the height h1 of protrusion 100 can be equal to or the depth d1 of slightly below groove 120；When groove 120 passes through
When logical cover board 12, the height of protrusion 100 can be greater than or equal to or lower than groove 120 depth, the application is not construed as limiting this.
Certainly, in other embodiments, it may also set up groove on aforesaid substrate 10, may also set up on cover board 12 and groove
Position it is corresponding and the protrusion that matches, the application are not construed as limiting this.1 knot of micro-fluidic chip provided in above-described embodiment
Structure is simple, and detachable；After the completion of above-mentioned 1 use of micro-fluidic chip, it is put into after substrate 10 and cover board 12 are separated equipped with secondary
It is cleaned in the ultrasonic cleaning equipment of the cleaning solution of the effective components such as sodium chlorate, cleaning repeats after completing drying in case of next
It utilizes.By the above-mentioned means, 1 repetitive cycling of micro-fluidic chip can be made to use, and then achieve the purpose that reduce testing cost.
In one embodiment, please continue to refer to Fig. 1-Fig. 3, the microchannel 102 being arranged on substrate 10 includes sample
Injection region 108, the position that sample injection region 108 is corresponded on cover board 12 are provided with through-hole 122, through-hole 122 and sample injection region 108
Connection, so that sample enters sample injection region 108 by through-hole 122.In an application scenarios, sample injection region 108
Contact surface with through-hole 122 is respectively the first plane A and the second plane B, and the first plane A covers the second plane B, so that sample
This is all flowed into sample injection region 108 by through-hole 122；The cross section of through-hole 122 can be round, rectangular, oval etc.,
Through-hole 122 can for it is isometrical or not isometrical hole (for example, the diameter of the cross section of through-hole 122 along towards the direction of substrate 10 by
It gradually reduces or gradually increases)；The cross section of sample injection region 108 can be rectangular, round, oval, pentagon etc., to make
Subsequent detection zone can be entered by obtaining the sample in sample injection region 108, and sample injection region 108 can be at edge (for example, working as sample
When this injection region 108 is pentagon, in pentagonal corner) open up one or more outlet C.
In another embodiment, the microchannel 102 on substrate 10 goes back shape after above-mentioned sample injection region 108
At having successively sample filtering area 101 interconnected, trapping region 103, sample collection area 105.
Specifically, the first end C of sample injection region 108 is connect with the first end D of sample filtering area 101.Sample filtering area
101 for filtering red blood cell and other impurities in sample, and sample filtering area 101 is provided with the multiple of 10 surface of self-reference substrate extension
The radius of pillar E (for example, 5,10,50 etc.), pillar E are 15um-20um (for example, 15um, 18um, 20um etc.), phase
Spacing between two pillar E of neighbour is 5um-10um (for example, 5um, 8um, 10um etc.).The half of pillar E in sample filtering area 101
Spacing between diameter and two neighboring pillar E can be adjusted according to specific requirements, and the application is not construed as limiting this.
Specifically, the bottom of trapping region 103 is provided with multiple recess portion F of microarray arrangement (for example, 5,10,50
Deng), recess portion F is used to capture the microparticle substance into the sample of trapping region 103, and the space size of recess portion F may only accommodate
One or two microparticle substance.In an application scenarios, microparticle substance can be magnetic bead, microballon (for example, polystyrene
Microballoon etc.) or cell etc..When microparticle substance is magnetic bead or microballon, can also be coated on magnetic bead or microballon antibody etc. its
His substance, the application are not construed as limiting this.Microparticle substance flowing to guarantee in sample is not blocked, the miniflow on substrate 10
Minimum range between the bottom and cover board 12 in channel 102 is first distance d2, and first distance d2 is greater than the straight of microparticle substance
Diameter.In another application scenarios, the shape of the cross section of recess portion F includes circle, triangle, rectangle, diamond shape etc..As recess portion F
Circular in cross-section when, circular diameter is greater than or equal to the diameter of microparticle substance, and is less than or equal to microparticle substance
Twice of diameter.In one embodiment, circular diameter is 10um-30um, for example, 10um, 20um, 30um etc..
Specifically, sample collection area 105 is used to collect the sample in non-incoming seizure area 103.
In an application scenarios, sample be in advance with the magnetic of specifying protein bound fluorescent marker good in whole blood/serum
Pearl sample, after magnetic bead sample via through holes 122 enter sample injection region 108, under the driving of capillary force, to sample filtering area
101 flowings；Enter trapping region 103 under the driving of capillary force by the magnetic bead sample of sample filtering area 101；In trapping region
103 apply external magnetic field, and magnetic bead sample is fallen under the magneticaction of external magnetic field in multiple recess portion F of trapping region 103, and one
It is no more than two magnetic bead samples in recess portion F.Apply external magnetic field and remove external magnetic field after a certain period of time, does not fall within trapping region 103
Multiple recess portion F in remaining sample under the driving of capillary force enter sample collection area 105.Provided by the present embodiment
Sample in micro-fluidic chip 1 flows under the driving of capillarity, without by extraneous electro-mechanical drive component, operation letter
It is single, it is practical.By reasonable light path design, with the single recess portion F of the laser irradiation trapping region 103 of specific wavelength, and lead to
The recess portion F for crossing fluorescence microscope irradiation current goes through all recess portion F in trapping region 103 with the presence or absence of fluorescence, and then sentences
Whether contain specified albumen in random sample sheet.The fluorescence detection method of above-mentioned trapping region 103 and magnetic bead sample is utilized in the application,
The quantitative detection that single magnetic bead may be implemented, is detected relative to traditional chemoluminescence method, and sensitivity is higher.Work as micro-fluidic chip
When 1 material is transparent material, laser can be with the trapping region 103 of direct irradiation micro-fluidic chip 1；When the material of micro-fluidic chip 1
When matter is nontransparent material, via hole (not shown) can be opened up in the position of the corresponding trapping region 103 of cover board 12, laser passes through the via hole
It is irradiated to the trapping region 103 of micro-fluidic chip 1.
In another embodiment, referring to Fig. 4, Fig. 4 is the structural schematic diagram of another embodiment of substrate.The substrate
20 include the sample injection region 108 interconnected, sample filtering area 101, sample reaction zone 200, trapping region 103, sample collection
Area 105；In the present embodiment, the structure in sample injection region 108, sample filtering area 101, trapping region 103, sample collection area 105
Details are not described herein for part same with the above-mentioned embodiment.It is first anti-to be embedded with fluorescent marker in advance for sample reaction zone 200
Body is embedded with the secondary antibody of marked by magnetic bead in the recess portion (not indicating) of trapping region 103 in advance, wherein first antibody can be with
Specified protein binding in sample, secondary antibody can be with the specified protein bindings in sample, alternatively, secondary antibody can be with
One antibody combines.
In an application scenarios, the corresponding sample of substrate 20 provided in above-described embodiment is whole blood/serum sample.
After whole blood/serum sample via through holes 122 enter sample injection region 108, under the driving of capillary force, to sample filtering area
101 flowings；Enter sample reaction zone under the driving of capillary force by whole blood/serum sample of sample filtering area 101
200, the first antibody of the fluorescent marker embedded in the specified albumen and sample reaction zone 200 in whole blood/serum sample in advance is anti-
It answers；Whole blood/serum sample after reaction enters trapping region 103, trapping region 103 apply external magnetic field, trapping region 103 it is multiple
The secondary antibody for being embedded with marked by magnetic bead in recess portion in advance is further reacted with the sample after above-mentioned react.Apply external magnetic field one
External magnetic field is removed after fixing time, and does not fall within remaining sample in multiple recess portions of trapping region 103 in the driving of capillary force
It is lower to enter sample collection area 105.It is single recessed with the laser irradiation trapping region 103 of specific wavelength by reasonable light path design
Portion, and fluorescence whether there is by the recess portion of fluorescence microscope irradiation current, all recess portions in trapping region 103 are gone through,
And then whether determine in sample containing specified albumen.When the material of micro-fluidic chip 1 is transparent material, laser can directly shine
Penetrate the trapping region 103 of micro-fluidic chip 1；It, can be in the corresponding capture of cover board 12 when the material of micro-fluidic chip 1 is nontransparent material
The position in area 103 opens up via hole, and laser is irradiated to the trapping region 103 of micro-fluidic chip 1 by the via hole.
It is micro- for flow velocity of control sample (magnetic bead sample, or, whole blood sample or serum sample etc.) in microchannel 102
Circulation road 102 further includes serpentine bend portion, and the flow velocity of sample and the length in serpentine bend portion are inversely proportional；As shown in Fig. 2, the first snake
Shape bending section 107 between sample injection region 108 and sample filtering area 101, and/or, the second serpentine bend portion 109 is located at sample
Between this filtering area 101 and trapping region 103；As shown in figure 4, the first serpentine bend portion 107 is located at sample injection region 108 and sample
Between filtering area 101, and/or, third serpentine bend portion 202 between sample filtering area 101 and sample reaction zone 200, and/
Or, the 4th serpentine bend portion 204 is between sample reaction zone 200 and trapping region 103.
Currently, traditional micro-fluidic chip can only be detected for unitem, when to the multiple items of same sample progress
When visual inspection is surveyed, it is necessary first to sample to be detected are divided into more parts, then by more parts of samples after distribution respectively in different miniflows
Corresponding item detection is carried out on control chip, which not only increases the sample size of test, but also makes micro-fluidic chip cost
Increase.Therefore, develop it is a kind of can the micro-fluidic chip of entry joint inspection be particularly important.
Referring to Fig. 5, Fig. 5 is the structural schematic diagram of another embodiment of the application micro-fluidic chip, Fig. 6 is base in Fig. 5
The structural schematic diagram of one embodiment of plate.In the present embodiment, micro-fluidic chip 3 is detachable structure, and detachable structure is set
Count same with the above-mentioned embodiment, details are not described herein.
In the present embodiment, microchannel 300 includes sample shunting zone 302 on substrate 30, and sample shunting zone 302 includes dry
Road runner 3000 and multiple branch flow channels 3002 (for example, 2,4,9 etc.), main line runner 3000 are used to that sample to be made to flow out
First end H of the first end G respectively with multiple branch flow channels 3002 for flowing into sample connect；Wherein, multiple branch streams
The cross-sectional area sum in road 3002 is the cross-sectional area of main line runner 3000.
In an application scenarios, the number N of branch flow channel 3002, the radius R1 of branch flow channel 3002, main line runner
3000 radius R2 meets following relationship: R2=R1*N1/2.In one embodiment, the radius of main line runner is 200um-
500um, for example, 200um, 300um, 400um, 500um etc..
In another application scenarios, the number N of branch flow channel 3002, the length L1 of branch flow channel 3002, main line runner
3000 length L2 meets following relationship: L2=L1*N1/2.In one embodiment, the length L2 of main line runner 3000 and branch
The number N of road runner 3002 is proportional；The length L2 of main line runner 3000 is 3cm-8cm, for example, 3cm, 5cmc, 8cm
Deng.
For example, the cross-sectional area sum of 4 branch flow channels 3002 is equal to main line when the number of branch flow channel 3002 is 4
The cross-sectional area of runner 3000；The radius R2 of main line runner 3000 is 2 times of the radius R1 of branch flow channel 3002；It meanwhile being true
The sample that guarantor flows through main line runner 3000 is sufficiently paved with branch flow channel 3002, and the length of branch flow channel 3002 is as short as possible, does
The length L2 of road runner 3000 is 2 times of the length L1 of branch flow channel 3002.It should be noted that main line runner 3000 and branch
The length of runner 3002 includes and is not limited to straight length, and main line runner 3000, branch flow channel 3002 may be designed as curve, into
And increase sample storage volume.In addition, being that can pass through so that sample is as uniform as possible to branch to each branch flow channel 3002
The mode of sample flow is reduced, for example, designing serpentine bend portion (not shown) on main line runner 3000, passes through serpentine bend portion
Warp architecture and length reduce the flow velocity of sample.
In one embodiment, the microchannel 300 being located in the present embodiment on substrate 30 further includes sample injection region
304, the second end I for flowing into sample of one end and main line runner 3000 that sample injection region 304 is used to that sample to be made to flow out
Connection.The position that sample injection region 304 is corresponded on cover board 32 is formed with through-hole 320, and through-hole 320 is connected to sample injection region 304,
So that sample enters sample injection region 304 by the through-hole 320.
In another embodiment, the sample injected from sample injection region 304 is whole blood/serum sample, to avoid
The corresponding detection project of the subsequent each branch flow channel 3002 of red blood cell or impurity effect therein, miniflow provided herein are logical
Road 300 further includes sample filtering area 306, between sample injection region 304 and main line runner 3000, sample filtering area 306
Structure is same with the above-mentioned embodiment, and details are not described herein.
In another embodiment, the microchannel 300 on substrate 30 further include it is multiple be sequentially communicated and be arranged side by side
Sample reaction zone 308, trapping region 301 and sample collection area 303, wherein each branch flow channel 3002 for making sample flow
Second end J out is respectively with corresponding each sample reaction zone 308 for making one end K connection of sample inflow.Each branch
The corresponding sample reaction zone 308 of runner 3002 and trapping region 301, multiple branch flow channels 3002 can share the same sample collection
Area 303, alternatively, each branch flow channel 3002 corresponds to a sample collecting region 303, the application is not construed as limiting this.Each sample is anti-
Area 308 is answered to be embedded with the first antibody of fluorescent marker in advance, it is second anti-to be embedded with marked by magnetic bead in advance for each trapping region 301
Body, wherein first antibody can be with the specified protein binding in sample with the specified protein binding in sample, secondary antibody
Or the secondary antibody can be in conjunction with the first antibody.By reasonable light path design, shone with the laser of specific wavelength
The single recess portion of trapping region 301 is penetrated, and fluorescence whether there is by the recess portion of fluorescence microscope irradiation current.When micro-fluidic
When the material of chip 3 is transparent material, laser can be with the trapping region 301 of direct irradiation micro-fluidic chip 3；When micro-fluidic chip 3
Material when being nontransparent material, via hole can be opened up in the position of the trapping region 301 of the corresponding each branch flow channel 3002 of cover board 32
322, laser is irradiated to the trapping region 301 of micro-fluidic chip 3 by the via hole 322.
Referring to Fig. 8, Fig. 8 is the structural schematic diagram of one embodiment of the application immunofluorescence analysis instrument.The application is mentioned
The immunofluorescence analysis instrument 4 of confession includes the micro-fluidic chip (not showing in Fig. 8) in any of the above-described embodiment.In an application scenarios
In, as shown in figure 8, the immunofluorescence analysis instrument 4 includes entrance 40, micro-fluidic chip is placed into fluorescence by the entrance 40
In the detection platform (not shown) of analyzer 4.When in use, shown in Fig. 1 for micro-fluidic chip 1, first by miniflow
The substrate 10 and cover board 12 for controlling chip 1 assemble, and complete sample injection；Then, micro-fluidic chip 1 is put by entrance 40
In the detection platform for setting fluorescence analyser 4；After the completion of detection, micro-fluidic chip 1 is taken out from entrance 40, in immunofluorescence
The substrate 10 of micro-fluidic chip 1 is separated with the disassembly of cover board 12 outside analyzer 4.
In the following, being described further with several specific application scenarios to micro-fluidic chip provided herein.
Embodiment one: Procalcitonin detection；
Procalcitonin (PCT) is a kind of protein, when serious bacterial, fungi, parasitic infection and pyemia and how dirty
Its level in blood plasma can increase when device functional failure.And PCT will not be increased when autoimmunity, allergy and virus infection.Office
The limited bacterium infection in portion, slight infection and chronic inflammation not will lead to its raising.PCT reflects the work of systemic inflammatory response
Jump degree, for the test of PCT, clinically meaning is significant.Detection for PCT, the application of double antibodies sandwich immunoluminescence method are wide
General, principle is with bispecific monoclonal antibody, and one of antibody is calcitonin antibody (first antibody), with magnetic bead and PCT points
The calcitonin position of son combines；Another is anticalcium element antibody (secondary antibody), with the anticalcium of mark fluorescent element and PCT molecule element
Position.Two antibody form sandwich complex in conjunction with PCT molecule, issue under the laser excitation of specific wavelength later glimmering
Light can determine whether corresponding PCT value height according to fluorescence power, and this method is easy to operate, high specificity, and sensibility is high.
The sample that 1 pair of micro-fluidic chip as shown in Figure 1 has been coated with can be used to the detection of above-mentioned PCT and carry out screening, tool
Body operating process is as follows:
A, the magnetic bead and first antibody that diameter is 5um are added into reaction cup, is incubated for coating 20- at a suitable temperature
30min.The sample completed will be incubated for be put into magnetic field, under magnetic fields, magnetic bead can be adsorbed on reaction cup wall of cup, use cleaning solution
2-3 first antibody not being coated on magnetic bead with removing of cleaning, obtains the magnetic bead that theoretically first antibody has been coated with；
B, sample is added into the magnetic bead reaction cup that first antibody has been coated with, is incubated for coating 20- at a suitable temperature
30min.The sample completed will be incubated for be put into magnetic field, under magnetic fields, magnetic bead can be adsorbed on reaction cup wall of cup, use cleaning solution
2-3 sample not being coated on magnetic bead with removing of cleaning, obtains the magnetic bead that theoretically first antibody and sample have been coated with；
C, the good secondary antibody of fluorescent marker is added into the magnetic bead reaction cup that first antibody and sample have been coated with, suitable
At a temperature of be incubated for coating 20-30min.The sample completed will be incubated for be put into magnetic field, under magnetic fields, magnetic bead can be adsorbed on
Reaction cup wall of cup cleans 2-3 times with cleaning solution to remove the secondary antibody not being coated on magnetic bead, obtains theoretically interlayer structure
Magnetic bead-first antibody-sample-fluorescent second antibody sample；
D, certain buffer is added into theoretic magnetic bead-first antibody-sample-fluorescent second antibody sample, obtains
The sample detected for micro-fluidic chip 1；
E, the substrate 10 of micro-fluidic chip 1 and the combination of cover board 12 are assembled, takes 10-15uL sample, Xiang Wei with sampling pipe
The sample injection region 108 of fluidic chip 1 is loaded, and after waiting 5-10min, sample is driven in capillarity downstream to trapping region 103；
F, apply externally-applied magnetic field in trapping region 103, wait 5-10min, remove external magnetic field, extra sample is through trapping region
103 flow into sample collection area 105；
G, the laser of specific wavelength successively irradiates the single recess portion F on micro-fluidic chip 1, detects whether to be collected into fluorescence letter
Number, yin and yang attribute can determine whether according to the presence or absence of fluorescence signal and power, be positive if being collected into fluorescence signal, otherwise be negative
Property；
H, the substrate 10 of micro-fluidic chip 1 and cover board 12 are separated, utilizes the cleaning solution ultrasound for pressing ingredient containing hypochlorous acid
Wave cleans micro-fluidic chip 1, is put into drying box dry in case reusing again.
Embodiment two, brain natriuretic peptide and troponin joint-detection；
The clinical meaning of joint-detection brain natriuretic peptide (BNP) and troponin I (cTn I) to auxiliary diagnosis cardiac function.Usually
In the case of, the detection of brain natriuretic peptide (BNP) and troponin I (cTn I) will be carried out in two times, on the one hand increase testing cost, separately
On the one hand detection is more cumbersome and complicated.Detection for brain natriuretic peptide (BNP) and troponin I (cTn I) is adopted in the present embodiment
With double antibodies sandwich immuno-fluorescence assay, principle is with bispecific monoclonal antibody, and wherein first antibody is brain natriuretic peptide primary antibody, with
Brain natriuretic peptide (BNP) molecule combines, and a resistance to surface is modified with fluorescein；Secondary antibody is brain natriuretic peptide secondary antibody, with magnetic bead and brain natriuretic peptide
(BNP) another site of molecule combines.The testing principle of troponin I (cTn I) is consistent with brain natriuretic peptide (BNP) testing principle, herein
It repeats no more.
Detection speed can be promoted using the micro-fluidic chip 3 of support entry joint inspection as shown in Figure 5, simplify operation step
Suddenly, specific operation process is as follows:
A, the first branch runner for the micro-fluidic chip 3 surveyed in entry joint inspection and the corresponding sample of second branch runner are anti-
Answer the antibody in area and trapping region embedded corresponding brain natriuretic peptide (BNP) and troponin I (cTn I)；
B, the substrate 30 of micro-fluidic chip 3 and the combination of cover board 32 are assembled, takes 10-15uL whole blood/serum with sampling pipe
Sample is loaded to the sample injection region 304 of micro-fluidic chip 3, includes brain natriuretic peptide (BNP) and troponin I (cTn I) in sample
Antigen, after waiting 5-6min, sample flows through sample filtering area 306 under capillarity driving；
C, first branch runner and second are separately flowed under capillarity driving by the sample of sample filtering area 306
The corresponding sample reaction zone 308 of road runner, the brain natriuretic peptide (BNP) or troponin I (cTn I) in sample are corresponding to being embedded in
The antibody (primary antibody) in channel is immunoreacted；
It flows down in capillarity power drive through catching in conjunction with the antigen (brain natriuretic peptide (BNP) or troponin I (cTn I)) of primary antibody
Area 301 is obtained, it is anti-anti- to form magnetic bead-two for two anti-bindings of the magnetic bead surfaces embedded in advance in the antigen and trapping region 301 of primary antibody
Original-primary antibody interlayer structure；
Apply external magnetic field in trapping region 301, unbonded extra sample flows into sample collection area 303；
D, the laser of specific wavelength successively irradiates the magnetic bead sample of multiple recess portions in the trapping region 301 of different branch runner
This, detects whether to be collected into fluorescence signal, can determine whether yin and yang attribute according to the presence or absence of fluorescence signal and power, if be collected into glimmering
Optical signal is then positive, otherwise is feminine gender；
E, the substrate 30 of micro-fluidic chip 3 and cover board 32 are separated, utilizes the cleaning solution ultrasound for pressing ingredient containing hypochlorous acid
Wave cleans micro-fluidic chip 3, is put into drying box dry in case reusing again.
The foregoing is merely presently filed embodiments, are not intended to limit the scope of the patents of the application, all to utilize this
Equivalent structure or equivalent flow shift made by application specification and accompanying drawing content, it is relevant to be applied directly or indirectly in other
Technical field similarly includes in the scope of patent protection of the application.
Claims (13)
1. a kind of micro-fluidic chip, which is characterized in that the micro-fluidic chip includes:
Substrate is provided with protrusion；
Cover board is provided with groove that is corresponding with the position of the protrusion and matching, and the protrusion on the substrate can
It is separated with the groove on the cover board, so that the substrate of the micro-fluidic chip detachably connects with the cover board
It connects.
2. micro-fluidic chip according to claim 1, which is characterized in that
When the cover plate lid on the substrate when, the protrusion on the substrate is embedded in the groove on the cover board
And position the substrate and the cover board.
3. micro-fluidic chip according to claim 1, which is characterized in that
Microchannel is additionally provided on the substrate, the microchannel includes sample shunting zone, and the sample shunting zone includes dry
Road runner and multiple branch flow channels, the main line runner for make sample flow out first end respectively with multiple branch streams
The first end connection for flowing into sample in road；Wherein, the cross-sectional area sum of the multiple branch flow channel is the main line
The cross-sectional area of runner.
4. micro-fluidic chip according to claim 3, which is characterized in that the number N of the branch flow channel, the branch stream
The radius R1 in road, the main line runner radius R2 meet following relationship: R2=R1*N1/2；The radius of the main line runner is
200 microns -500 microns.
5. micro-fluidic chip according to claim 3, which is characterized in that
The number N of the branch flow channel, the length L1 of the branch flow channel, the main line runner length L2 meet with ShiShimonoseki
System: L2=L1*N1/2；The length of the main line runner is 3 centimetres -8 centimetres.
6. micro-fluidic chip according to claim 3, which is characterized in that
The main line runner includes serpentine bend portion, the serpentine bend portion for reducing sample flow velocity.
7. micro-fluidic chip according to claim 3, which is characterized in that
The microchannel further includes sample injection region, wherein the sample injection region for make sample flow out one end with
The second end connection for flowing into sample of the main line runner.
8. micro-fluidic chip according to claim 7, which is characterized in that the microchannel further includes sample filtering area,
The sample filtering area is arranged between the sample injection region and the main line runner；The sample filtering area is provided with from institute
State multiple pillars of substrate surface extension.
9. micro-fluidic chip according to claim 8, which is characterized in that the radius of the pillar is 15 microns -20 microns,
Spacing between the two neighboring pillar is 5 microns -10 microns.
10. micro-fluidic chip according to claim 7, which is characterized in that
The position that the sample injection region is corresponded on the cover board is provided with through-hole, and the through-hole and the sample injection region connect
It is logical, so that sample enters the sample injection region by the through-hole.
11. micro-fluidic chip according to claim 1, which is characterized in that
Microchannel is additionally provided on the substrate, the microchannel includes trapping region, and the bottom of the trapping region is provided with micro-
The recess portion of array arrangement, the recess portion are used to capture the microparticle substance into the sample of the trapping region, the recess portion
Space size may only accommodate one or two microparticle substance.
12. micro-fluidic chip according to claim 11, which is characterized in that circular in cross-section, the triangle of the recess portion
Shape, rectangle or diamond shape；The circular diameter is greater than or equal to the diameter of the microparticle substance, and is less than or equal to described micro-
Twice of the diameter of particulate matter；The circular diameter is 10 microns -30 microns；The microparticle substance be magnetic bead, microballon,
Or cell.
13. a kind of immunofluorescence analysis instrument, which is characterized in that the immunofluorescence analysis instrument includes any one of claim 1-12
The micro-fluidic chip.
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