CN106513062B - The micro-fluidic chip and its pretreatment and detection method that enzyme linked immunological quickly detects - Google Patents
The micro-fluidic chip and its pretreatment and detection method that enzyme linked immunological quickly detects Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 32
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 32
- 230000001900 immune effect Effects 0.000 title claims abstract description 28
- 238000002203 pretreatment Methods 0.000 title abstract description 4
- 238000002347 injection Methods 0.000 claims abstract description 121
- 239000007924 injection Substances 0.000 claims abstract description 121
- 238000011534 incubation Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000012545 processing Methods 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 8
- 239000000427 antigen Substances 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 229920002873 Polyethylenimine Polymers 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000007790 solid phase Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000000799 fluorescence microscopy Methods 0.000 claims description 4
- 235000020183 skimmed milk Nutrition 0.000 claims description 4
- 108010090804 Streptavidin Proteins 0.000 claims description 3
- 241000446313 Lamella Species 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 8
- 238000002965 ELISA Methods 0.000 description 11
- 239000000758 substrate Substances 0.000 description 6
- 230000008901 benefit Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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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
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- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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Abstract
This application discloses a kind of micro-fluidic chip that enzyme linked immunological quickly detects and its pretreatment and detection methods, the micro-fluidic chip includes: the first chip layer, at least one sense channel is distributed with thereon, the sense channel has the first injection port, the second injection port and outlet, and second injection port is between the outlet and the first injection port；Second chip layer, relatively described first chip layer is removable, and offer third injection port and the 4th injection port thereon, and meet: second chip layer is at first position, the third injection port is connected to first injection port, and the 4th injection port is not connected to the second injection port；At the second position, the third injection port is not connected to second chip layer with first injection port, and the 4th injection port is connected to the second injection port.The present invention detects sample used amount and greatly reduces, and after pretreatment, after sample to be tested incubation is added, result can quickly be analyzed by adding detection reagent.
Description
Technical field
The application belongs to enzyme linked immunological rapid detection technical field, quickly detects more particularly to a kind of enzyme linked immunological micro-
Fluidic chip and its pretreatment and detection method.
Background technique
Traditional enzyme linked immunoassay analysis method (ELISA) is operated on 96 orifice plates, firstly, by primary antibody adding hole
It is incubated in plate, antibody is adsorbed on orifice surface after a certain period of time.After washing away the antibody not adsorbed, consolidating without absorption primary antibody is closed
Body surface face is to reduce the non-specific adsorption of albumen.Then, standard items or sample to be tested containing known antigens are added, incubate
The secondary antibody for adding specific enzymes label after a certain period of time is educated, is incubated for after a certain period of time, secondary antibody and antigen binding are washed out and remove
Remove the extra secondary antibody not combined.Finally, substrate is added, by enzymic catalytic reaction chromogenic reaction occurs for substrate, generates detection letter
Number (color) is detected.Detection signal is generated since each enzyme molecule can be catalyzed a large amount of substrate reactions,
ELISA method detection sensitivity is high.However, routine ELISA analysis carries out on 96 orifice plates, grasped by hand for technology-intensive type
Make, complex for operation step, time-consuming, the even longer time general a few hours, the operation antibody and reagent consumption are big, and result
Error is big, is also easy to produce cross reaction, poor reproducibility, causes ELISA Expenses of laboratory examination high.
Micro-fluidic chip is by the process integration of a variety of chemistry and biologies by a variety of microchannel network structures to together
The analytical technology platform of one chip fast and automatically changed.Existing market is to ELISA reagent consumptive material and automates enzyme-linked exempt from
Epidemic disease detection is in great demand, and existing ELISA orifice plate is not only expensive, and cannot detect plurality of target albumen simultaneously
Content.Therefore, using the advantage of microfluidic chip technology, it is fast that easy to operate, inexpensive, low sample size, plurality of target albumen are developed
The enzyme linked immunological micro-fluidic chip of speed detection is developing direction important at present.
Summary of the invention
It is an object of the invention to by micro-fluidic chip microchannel advantage, design is a to may be implemented same sample to be tested
The chip of the detection of interior one or more target proteins.
To achieve the above object, the invention provides the following technical scheme:
The embodiment of the present application discloses a kind of micro-fluidic chip that enzyme linked immunological quickly detects, comprising:
At least one sense channel is distributed in first chip layer thereon, the sense channel have the first injection port, second into
Sample mouth and outlet, second injection port is between the outlet and the first injection port；
Second chip layer, relatively described first chip layer is removable, offers third injection port and the 4th injection port thereon,
And meet:
At first position, the third injection port is connected to second chip layer with first injection port, and described
4th injection port is not connected to the second injection port；
At the second position, the third injection port is not connected to second chip layer with first injection port, and institute
The 4th injection port is stated to be connected to the second injection port.
Preferably, in the micro-fluidic chip that above-mentioned enzyme linked immunological quickly detects, second chip layer is bonded and leads to
It crosses guiding device and slides on the first chip layer surface.
Preferably, in the micro-fluidic chip that above-mentioned enzyme linked immunological quickly detects, the guiding device includes matching
Sliding groove and sliding tenon.
Preferably, in the micro-fluidic chip that above-mentioned enzyme linked immunological quickly detects, the first chip layer surface is protruded out
There are two sliding tenons disposed in parallel, the recessed surface in second chip layer of the sliding groove.
It preferably, further include third chip layer in the micro-fluidic chip that above-mentioned enzyme linked immunological quickly detects, described
Two chip layers, the first chip layer and third chip layer are sequentially overlapped up and down, and the sense channel is recessed in first chip layer
Lower surface.
Preferably, it in the micro-fluidic chip that above-mentioned enzyme linked immunological quickly detects, is distributed in first chip layer
A plurality of sense channel, one end of a plurality of sense channel are connected to same first injection port, a plurality of sense channel it is another
End is connected to the same outlet.
Preferably, in the micro-fluidic chip that above-mentioned enzyme linked immunological quickly detects, on sense channel described in each also
It is communicated with a detection channel, the width of the detection channel is greater than the sense channel.
Preferably, in the micro-fluidic chip that above-mentioned enzyme linked immunological quickly detects, it is located at first injection port and the
The width of sense channel between two injection ports is less than the width of the sense channel between second injection port and outlet.
Correspondingly, disclosed herein as well is a kind of enzyme linked immunological rapid detection methods, comprising:
(1), it pre-processes:
S11, the second chip layer slide into first position, and NaOH is added from third injection port and handles certain time, rinse dry
Only, polyethyleneimine room temperature is added and handles certain time, glutaraldehyde room temperature is added and handles certain time；Distilled water is rinsed well,
Then it is dried；
S12, the second chip layer slide into the second position, and primary antibody is added from the 4th injection port, so that primary antibody is logical full of detection
Then road is incubated for, so that primary antibody is sufficiently combined with chip solid phase carrier, complete the coating processing of different primary antibodies；
After s13, incubation, the second chip layer is slid into first position, PBS buffer solution is added from third injection port,
Wash away no coated primary antibody, be then added closed protein or skimmed milk power, closing without absorption primary antibody the surface of solids with
Reduce the non-specific adsorption of albumen；
(2), it detects:
S21, it takes pretreatment good and has been coated with the chip of primary antibody, sample to be tested is added from third injection port or containing
Know the standard items of antigen, is incubated for after a certain period of time, it is clean with PBS rinse；
S22, biotinylated detection antibody is added from third injection port as secondary antibody, is incubated for after a certain period of time, is moistened with PBS
Wash clean；
S23, the Streptavidin albumen that fluorescent molecule label is added are incubated for after a certain period of time, clean with PBS rinse；
Under the microscope, analysis of fluorescence intensity makes fluorescence intensity and antigen concentration by standard items for s24, fluorescence microscopy
Standard curve corresponds to the fluorescence intensity on standard curve, analyzes the concentration of sample to be tested.
A kind of preprocess method of micro-fluidic chip that enzyme linked immunological quickly detects is also disclosed in the application, comprising:
S11, the second chip layer slide into first position, and NaOH is added from third injection port and handles certain time, rinse dry
Only, polyethyleneimine room temperature is added and handles certain time, glutaraldehyde room temperature is added and handles certain time；Distilled water is rinsed well,
Then it is dried；
S12, the second chip layer slide into the second position, and primary antibody is added from the 4th injection port, so that primary antibody is logical full of detection
Then road is incubated for, so that primary antibody is sufficiently combined with chip solid phase carrier, complete the coating processing of different primary antibodies；
After s13, incubation, the second chip layer is slid into first position, PBS buffer solution is added from third injection port,
Wash away no coated primary antibody, be then added closed protein or skimmed milk power, closing without absorption primary antibody the surface of solids with
Reduce the non-specific adsorption of albumen；
S14, chip have been coated with primary antibody, and after Seal treatment, chip is sealed up for safekeeping.
Compared with the prior art, the advantages of the present invention are as follows:
1, ingenious to complete different reagent sample introductions, the first injection port and second by the joinery and its construction of setting sliding (slip)
Injection port can arbitrarily switch between opening and closing, and structure is simple, easy to operate, avoid the complexity of valve mechanism.
2, in chip preprocessing process, surface modification is easy to operate, is almost not necessarily to any instrument and equipment, at low cost, modified
Surface is evenly distributed, and in batch and differences between batches are smaller, and test reproducibility is more preferable.
3, quickly, after chip pretreatment, chip is directly used in sample detection for detection.
4, reagent and sample usage amount are reduced, the total usage amount of sample within 10 μ L, reagent totality usage amount 50 μ L with
It is interior.50 μ L of sample is added in the every hole of 96 orifice plates of traditional ELISA detection.
5, detection time is reduced, and traditional ELISA detection time 3 hours or so, the chip detection time most 1 hours.
6, multiple targets detect simultaneously in same sample, and in traditional ELISA detection, a kind of kit can only detect a kind of mesh
Albumen is marked, which may be implemented multiple targets and in parallel, quickly detect, when further shortening the unit of target protein detection
Between.ELISA kit is expensive in market simultaneously, which reduces the testing cost of target protein.
Detailed description of the invention
In order to illustrate the technical solutions in the embodiments of the present application or in the prior art more clearly, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The some embodiments recorded in application, for those of ordinary skill in the art, without creative efforts,
It is also possible to obtain other drawings based on these drawings.
Fig. 1 show the stereochemical structure decomposing schematic representation of micro-fluidic chip in the specific embodiment of the invention；
Fig. 2 show the top view of the first chip layer in the specific embodiment of the invention；
Fig. 3 show the side view of micro-fluidic chip in the specific embodiment of the invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out detailed retouch
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, those of ordinary skill in the art's every other implementation obtained without making creative work
Example, shall fall within the protection scope of the present invention.
Referring to figs. 1 and 2, the micro-fluidic chip that enzyme linked immunological quickly detects, including being sequentially overlapped setting up and down
Second chip layer 1, the first chip layer 2 and third chip layer 3.
At least one sense channel 21 is distributed in first chip layer 2 thereon, which has the first injection port
22, the second injection port 23 and outlet 24, the second injection port 23 are located between outlet 24 and the first injection port 22.
Second chip layer 1, opposite first chip layer 2 is removable, offers third injection port 11 and the 4th injection port thereon
12, and meet:
At first position, third injection port 11 is connected to second chip layer 1 with the first injection port 22, and the 4th injection port 12
It is not connected to the second injection port 23；
At the second position, third injection port 11 is not connected to second chip layer 1 with the first injection port 22, and the 4th injection port
12 are connected to the second injection port 23.
Third injection port and the 4th injection port are the through-hole being opened in the second chip layer, are used at the top end opening of the through-hole
It is injected in sample etc., the bottom end of the through-hole is realized after being aligned with the first injection port or the second injection port and is connected to.
As shown in connection with fig. 3, the second chip layer 1 patch merga pass guiding device slides on 2 surface of the first chip layer.It is preferred that
, guiding device includes the sliding groove 25 matched and sliding tenon 13.
In a preferred embodiment, 2 surface of the first chip layer has protruded out two sliding tenons disposed in parallel, and sliding groove is recessed
In the surface of the second chip layer.
In another embodiment, sliding tenon convexedly stretches in the lower surface of the second chip layer, correspondingly, sliding groove is recessed in first
The upper surface of chip layer.
The section for sliding tenon and sliding groove is preferably trapezoidal, and the limit of up and down direction is realized by trapezoid cross section, so that the
One chip layer and the second chip layer remain fitting.
In one embodiment, guiding device can also be formed in the first chip layer and the second chip layer side there is side
To the guide rail of recess, the edge of the second chip layer is limited and is slided in the recess.
The recessed lower surface in the first chip layer 2 of sense channel 21.Third chip layer is a plate, with the first chip layer
Between surround closed sense channel.
Preferably, a plurality of sense channel 21 (the present embodiment is for 4), a plurality of detection are distributed in the first chip layer
The one end in channel 21 is connected to same first injection port 22, and the other end of a plurality of sense channel is connected to the same outlet
24。
In other embodiments, sense channel can be by parallel channels using injection port as the center of circle, radial arrangement, are in disk
Formula array structure is completed to detect while more target proteins.
A detection channel 211 is also communicated on each sense channel, the width of the detection channel is greater than sense channel.Position
The width of sense channel between the first injection port and the second injection port is less than the detection between the second injection port and outlet
The width in channel.
In a preferred embodiment, the sense channel width between the first injection port and the second injection port is 100 μ
M, the width of the sense channel between the second injection port and outlet are 200 μm, and the length of detection channel 211 is 5mm, and width is
400μm。
Realize that enzyme linked immunological rapid detection method includes: using said chip
1. the incipient stage, upper layer injection port 11 and middle layer injection port 22 are interconnected, and injection port 12 and middle layer into
Sample mouth 23 is not connected to, and injection port 23 is in closed state.Pretreating reagent is sequentially added from injection port 11: 1mol/L NaOH,
55 DEG C of processing 30min；It rinses well, handles 30min polyethyleneimine (PEI) (0.2% PH=7) room temperature is added；Glutaraldehyde
(1%, w/v) room temperature handles 30min；Distilled water is rinsed well, is dried or is dried with nitrogen.(test the specific time, concentration range can
To expand)
2. upper layer chip is slided to the right relative to middle layer, so that 4 23 phases of injection port of 4 injection ports 12 and middle layer
It is intercommunicated, and injection port 11 is disconnected with middle layer injection port 22, injection port 22 is closed.The injection ports 23 different from 4 divide
Different pre-coated antibody (primary antibody) is not added, so that pre-coated antibody is full of sense channel.Chip is carefully placed into 4 DEG C, mistake
Night is incubated for, or chip is put into 37 DEG C, is incubated for 1 hour, so that primary antibody is sufficiently combined with chip solid phase carrier, completes not same
Anti- coating processing.
4. upper layer chip is slid back into origin-location, upper layer injection port 11 and middle layer to the left after primary antibody is incubated for
Injection port 22 is interconnected, and injection port 12 is not connected to middle layer injection port 23, and injection port 23 is in closed state.From sample introduction
PBS buffer solution are added in mouth 11, wash away no coated primary antibody.Then it is added closed protein (bovine serum albumin(BSA) BSA) or de-
Rouge milk powder closes the surface of solids without adsorbing primary antibody to reduce the non-specific adsorption of albumen.
5. chip has been coated with primary antibody, and after Seal treatment, chip is sealed up for safekeeping, it is put into 4 DEG C of preservations, target egg to be detected
It can be used directly when white.
Two, it detects
1. taking and handling well and be coated with the chip of primary antibody.Sample to be tested is added from injection port 11 or containing known
The standard items of antigen are clean with PBS rinse after being incubated for 20min.
2. biotinylated detection antibody (secondary antibody) is added from injection port 11, it is clean with PBS rinse after being incubated for 20min.
In the step, the detection antibody (secondary antibody) for being directly added into fluorescent marker can be, after being incubated for 20min, with PBS rinse
Completely, then in fluorescence microscopy microscopic observation.
3. the Streptavidin albumen of fluorescent molecule label is added, it is clean with PBS rinse after being incubated for 20min.
In the step, it can be addition and substrate be added, by enzymic catalytic reaction chromogenic reaction occurs for substrate, generates detection letter
Number (color) is detected.Such detecting step and traditional ELISA detection, substrate and enzyme target secondary antibody is needed to send out
Raw chromogenic reaction, needs certain incubation time.
4. fluorescence microscopy is under the microscope, analysis of fluorescence intensity.The mark of fluorescence intensity and antigen concentration is made by standard items
Directrix curve corresponds to the fluorescence intensity on standard curve, analyzes the concentration of sample to be tested.
In above-mentioned detection method, the practical incubation time in detection process can be changed.
Based on said chip and method, the invention has the advantages that:
1, it is provided with sliding shutter, is mutually slided by upper layer with middle layer, at the sample-adding for completing different pre-coated antibody
Reason.
Upper layer injection port 11 and middle layer injection port 22 are interconnected, for reagent treatment, cleaning solution and detection to be added
Sample.Upper layer injection port 12 is not connected to middle layer injection port 23 in beginning link, and injection port 23 is in closed state.Work as upper layer
After chip slides to the right relative to middle layer chip, injection port 12 and middle layer injection port 23 are interconnected, and injection port 11 with
Middle layer injection port 22 disconnects, and injection port 22 is closed, to smoothly complete the sample-adding operation of different pre-coated antibody.
2, after 4 pre-coated different antibodies of different sense channels, it can be used for detecting different target proteins.
It should be noted that, in this document, relational terms such as first and second and the like are used merely to a reality
Body or operation are distinguished with another entity or operation, are deposited without necessarily requiring or implying between these entities or operation
In any actual relationship or order or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to
Non-exclusive inclusion, so that the process, method, article or equipment including a series of elements is not only wanted including those
Element, but also including other elements that are not explicitly listed, or further include for this process, method, article or equipment
Intrinsic element.In the absence of more restrictions, the element limited by sentence "including a ...", it is not excluded that
There is also other identical elements in process, method, article or equipment including the element.
The above is only the specific embodiment of the application, it is noted that for the ordinary skill people of the art
For member, under the premise of not departing from the application principle, several improvements and modifications can also be made, these improvements and modifications are also answered
It is considered as the protection scope of the application.
Claims (10)
1. a kind of micro-fluidic chip that enzyme linked immunological quickly detects characterized by comprising
At least one sense channel is distributed in first chip layer thereon, which has the first injection port, the second injection port
And outlet, second injection port is between the outlet and the first injection port；
Second chip layer, relatively described first chip layer is removable, offers third injection port and the 4th injection port thereon, and full
Foot:
At first position, the third injection port is connected to second chip layer with first injection port, and the described 4th
Injection port is not connected to the second injection port；
At the second position, the third injection port is not connected to second chip layer with first injection port, and described
Four injection ports are connected to the second injection port.
2. the micro-fluidic chip that enzyme linked immunological according to claim 1 quickly detects, it is characterised in that: second chip
Layer patch merga pass guiding device slides on the first chip layer surface.
3. the micro-fluidic chip that enzyme linked immunological according to claim 2 quickly detects, it is characterised in that: the guiding device
Including the sliding groove and sliding tenon matched.
4. the micro-fluidic chip that enzyme linked immunological according to claim 3 quickly detects, it is characterised in that: first chip
Layer surface has protruded out two sliding tenons disposed in parallel, the recessed surface in second chip layer of the sliding groove.
5. the micro-fluidic chip that enzyme linked immunological according to claim 1 quickly detects, it is characterised in that: further include third core
Lamella, second chip layer, the first chip layer and third chip layer are sequentially overlapped up and down, and the sense channel is recessed in described
The lower surface of first chip layer.
6. the micro-fluidic chip that enzyme linked immunological according to claim 1 quickly detects, it is characterised in that: first chip
A plurality of sense channel is distributed on layer, one end of a plurality of sense channel is connected to same first injection port, a plurality of detection
The other end in channel is connected to the same outlet.
7. the micro-fluidic chip that enzyme linked immunological according to claim 1 quickly detects, it is characterised in that: examined described in each
It surveys on channel and is also communicated with a detection channel, the width of the detection channel is greater than the sense channel.
8. the micro-fluidic chip that enzyme linked immunological according to claim 1 quickly detects, it is characterised in that: be located at described first
It is logical that the width of sense channel between injection port and the second injection port is less than the detection between second injection port and outlet
The width in road.
9. the detection method for the micro-fluidic chip that any enzyme linked immunological of claim 1 to 8 quickly detects, feature exist
In, comprising:
(1), it pre-processes:
S11, the second chip layer slide into first position, and NaOH is added from third injection port and handles certain time, rinses well, adds
Enter polyethyleneimine room temperature processing certain time, glutaraldehyde room temperature is added and handles certain time；Distilled water is rinsed well, is then done
Dry processing；
S12, the second chip layer slide into the second position, and primary antibody is added from the 4th injection port, so that primary antibody is full of sense channel, so
After be incubated for so that primary antibody is sufficiently combined with chip solid phase carrier, complete the coating processing of different primary antibodies；
After s13, incubation, the second chip layer is slid into first position, PBS buffer solution is added from third injection port, washes away
There is no coated primary antibody, closed protein or skimmed milk power is then added, closes the surface of solids without adsorbing primary antibody to reduce
The non-specific adsorption of albumen；
(2), it detects:
S21, it takes pretreatment good and has been coated with the chip of primary antibody, sample to be tested is added from third injection port or containing known anti-
Former standard items are incubated for after a certain period of time, clean with PBS rinse；
S22, biotinylated detection antibody is added from third injection port as secondary antibody, is incubated for after a certain period of time, it is dry with PBS rinse
Only；
S23, the Streptavidin albumen that fluorescent molecule label is added are incubated for after a certain period of time, clean with PBS rinse；
Under the microscope, analysis of fluorescence intensity makes the standard of fluorescence intensity and antigen concentration by standard items for s24, fluorescence microscopy
Curve corresponds to the fluorescence intensity on standard curve, analyzes the concentration of sample to be tested.
10. the preprocess method for the micro-fluidic chip that any enzyme linked immunological of claim 1 to 8 quickly detects, feature
It is, comprising:
S11, the second chip layer slide into first position, and NaOH is added from third injection port and handles certain time, rinses well, adds
Enter polyethyleneimine room temperature processing certain time, glutaraldehyde room temperature is added and handles certain time；Distilled water is rinsed well, is then done
Dry processing；
S12, the second chip layer slide into the second position, and primary antibody is added from the 4th injection port, so that primary antibody is full of sense channel, so
After be incubated for so that primary antibody is sufficiently combined with chip solid phase carrier, complete the coating processing of different primary antibodies；
After s13, incubation, the second chip layer is slid into first position, PBS buffer solution is added from third injection port, washes away
There is no coated primary antibody, closed protein or skimmed milk power is then added, closes the surface of solids without adsorbing primary antibody to reduce
The non-specific adsorption of albumen；
S14, chip have been coated with primary antibody, and after Seal treatment, chip is sealed up for safekeeping.
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| CN108375559B (en) * | 2018-02-08 | 2021-01-15 | 南京岚煜生物科技有限公司 | Myocardial troponin kit based on micro-fluidic chip and preparation and detection methods thereof |
| CN108387564B (en) * | 2018-02-08 | 2020-08-21 | 南京岚煜生物科技有限公司 | Procalcitonin detection kit based on micro-fluidic chip and preparation and detection methods thereof |
| CN111044735A (en) * | 2019-12-31 | 2020-04-21 | 武汉大学 | A kind of method for measuring follicle-stimulating hormone |
| CN113406338B (en) * | 2021-07-21 | 2025-03-25 | 陕西省人民医院 | A microfluidic control cartridge |
| CN115283026B (en) * | 2022-07-06 | 2024-09-20 | 上海交通大学 | Integrated sliding chip |
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