CN109870567A - Micro-fluidic chip and its method for separating based on immunization sorting ApoE albumen - Google Patents
Micro-fluidic chip and its method for separating based on immunization sorting ApoE albumen Download PDFInfo
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- CN109870567A CN109870567A CN201910126434.9A CN201910126434A CN109870567A CN 109870567 A CN109870567 A CN 109870567A CN 201910126434 A CN201910126434 A CN 201910126434A CN 109870567 A CN109870567 A CN 109870567A
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- 108010025628 Apolipoproteins E Proteins 0.000 title claims abstract description 21
- 102000013918 Apolipoproteins E Human genes 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000002649 immunization Methods 0.000 title claims abstract description 7
- 230000003053 immunization Effects 0.000 title claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 239000002699 waste material Substances 0.000 claims abstract description 15
- 239000008280 blood Substances 0.000 claims description 9
- 210000004369 blood Anatomy 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000003550 marker Substances 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 5
- 108010060159 Apolipoprotein E4 Proteins 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 108010060219 Apolipoprotein E2 Proteins 0.000 claims description 3
- 108010060215 Apolipoprotein E3 Proteins 0.000 claims description 3
- 102000008128 Apolipoprotein E3 Human genes 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 230000010100 anticoagulation Effects 0.000 claims description 3
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 3
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 3
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical group C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 3
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 8
- 206010039966 Senile dementia Diseases 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 101150037123 APOE gene Proteins 0.000 description 3
- 102100029470 Apolipoprotein E Human genes 0.000 description 3
- 206010012289 Dementia Diseases 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 2
- 102000009091 Amyloidogenic Proteins Human genes 0.000 description 2
- 108010048112 Amyloidogenic Proteins Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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Abstract
Present invention discloses a kind of micro-fluidic chips based on immunization sorting ApoE albumen, constituting independent cavity by substrate and cover plate clamping is respectively sample pool, buffer pool, trapping region and waste liquid pool, independent channel is constituted by substrate and cover plate clamping, wherein three one end of the channel are respectively communicated with one end of sample pool, buffer pool and trapping region, the other end in three channels crosses connection, and the other end of the trapping region is connected to waste liquid pool by channel.The present invention controls the flow velocity and flow of sample to be tested by microflow control technique, to reduce error, increases reaction sensitivity, and it is easy to operate, detection time is short, storage is simple, cost has been saved in easy popularization and use.
Description
Technical field
The present invention relates to microfluidic chip technologies, especially carry out sorting inspection to senile dementia marker protein ApoE albumen
The micro-fluidic chip and its application method of survey.
Background technique
Senile dementia, that is, alzheimer's disease, be a kind of central nervous system degeneration, and the course of disease, which shows, slowly to be aggravated
Process is senile dementia one of the most common type type, is mainly shown as gradual memory obstacle, cognition dysfunction, personality
The mental symptoms such as change and aphasis, seriously affect the daily life of patient.According to statistics, China's old dementia patients at present
About 9,000,000, global a quarter is accounted for, and patient's number can increase sharply with the aggravation of aging of population, every 7 seconds of the whole world
Just there is a people to be diagnosed as old dementia patients, and old dementia patients show the trend of rejuvenation, alzheimer's disease
It is increasingly becoming the biggest obstacle of China senior health and fitness, while also being threatened to the health of countries in the world the elderly.Old age is silly
It stays and has become one of society universal phenomenon, however the pathogenesis about Alzheimer's disease is not yet completely clear, mesh
Before until medical field also not yet find the effective ways of radical cure.Therefore early diagnosis is intervened in time, delays the development of the state of an illness
Key as AD treatment.
A kind of reliable many researchers's note that searching out, spirit have been caused to the early diagnosis of senile dementia in recent years
Quick effective, the biochemistry detection means of non-intrusion type become the common purpose of researcher.Apo E (ApoE) is exactly therein one
The generally acknowledged quasi- risks and assumptions of alzheimer's disease of kind.
Apo E is located at No. 19 chromosomes of people, 3 kinds of allele, respectively 2/3/4 genotype is shared, due to every
Individual has a pair of alleles, shares 6 kinds of phenotypes, and in protein level, finally mainly there are three phenotypes: E2 phenotype, including ε
2/ ε 2,2/ ε 3 of ε, 7%, the APOE2 for accounting for about the mankind have protectiveness, can reduce the accumulation of amyloid protein, such phenotype
Individual is not susceptible to suffer from AD；E3 phenotype, including 3/ ε 3 of ε, 2/ ε 3 of ε, 78%, the APOE3 for accounting for about the mankind plays the part of the idle role without mistake, right
Risk has little effect；E4 phenotype, including 3/ ε 4 of ε, ε 4/ ε 4,15%, the ApoE4 for accounting for about the mankind are greatly promoted β
The deposition of amyloid protein in the brain, individual are susceptible to suffer from AD.Therefore, detect to APOE albumen to sentence to a certain extent
The generation of disconnected AD provides guidance for effectively prevention delayed AD.
Micro-fluidic chip makes multiple functions unit, so that kinds of experiments skill based on micro-processing technology on chip
Art can be realized on the chip of heart size, can be referred to as the laboratory on chip, have analysis speed fast, separative efficiency
The advantages that height, the consumption of reduction kit sample.With the further development of microfluidic chip technology, especially detect sensitive
The raising of degree, the micro-fluidic application to various macromoleculars are increasingly paid attention to, and big point of the detection of micro-fluidic chip combination immunization method
The principle of sub- substance is that corresponding trapping region is arranged in microfluidic channel, and corresponding antibody is embedded in trapping region, object to be detected
When mass flow is through trapping region, the specific reaction due to antigen-antibody is to enable specific substance to capture.
Currently, for the predominantly external progress immune detection of detection of APOE albumen, but this method is complex for operation step,
It expends that the time is longer, and required blood sample is more, therefore finds a kind of simple and quick detected to APOE protein types
Method becomes urgent problem.
Summary of the invention
It is a kind of based on immunology the technical problem to be solved by the present invention is to realize, using microflow control technique to ApoE albumen
The method for carrying out detection and genotyping.
To achieve the goals above, the technical solution adopted by the present invention are as follows: the miniflow based on immunization sorting ApoE albumen
Chip is controlled, constituting independent cavity by substrate and cover plate clamping is respectively sample pool, buffer pool, trapping region and waste liquid pool, by
Substrate and cover plate clamping constitute independent channel, wherein three one end of the channel are respectively communicated with sample pool, buffer pool and trapping region
One end, the other end in three channels crosses connection, and the other end of the trapping region is connected to waste liquid pool by channel, described
Trapping region is successively arranged at least one rectangle capture slot along liquid flow direction, and each rectangle capture trench bottom gathers round
Slot, the antibody of ApoE protein configurations is embedded in the round slot of each rectangle capture slot, and the cover plate is located at sample
Adding mouth, buffer Sample application mouth and observation port are equipped at pond, buffer pool and trapping region.
The trapping region is located at the middle position of sample pool, buffer pool, waste liquid pool, and all channels constitute y-type structure.
The channel and sample pool, buffer pool, waste liquid pool, trapping region are made of the sunk structure on substrate, institute
Stating cover plate is the plate being sealed on substrate, and the material of the substrate and cover plate is PDMS, and the observation port of the cover plate passes through saturating
Bright glass capsulation.
The trapping region both ends pass through the upper delta-shaped region of bell mouth shape structure respectively and lower delta-shaped region is connected to and leads to
Road, the bell mouth shape structure become larger towards trapping region opening, gradually become smaller towards access portal.
The trapping region is successively arranged three rectangle capture slots along liquid flow direction, divides in three rectangle capture slots
It is not embedded with the antibody of three kinds of various configurations of ApoE albumen.
Three rectangle capture slots are embedded with ApoE2, ApoE3, the corresponding antibody of ApoE4 albumen respectively.
The channel and sample pool, buffer pool, waste liquid pool, the sunk structure composition depth of trapping region are identical, described
Capture section length is 5-7mm, and width 2-4mm, the aperture of the circle slot is 8-12 μm, is connected to sample in three channels
This pond is mutually all 6-10mm with two passage lengths of buffer pool, and the length in another channel is 8-12mm.
Method for separating based on the micro-fluidic chip, comprising the following steps:
Step 1, blood sample processing；
Sample pool is added in the blood sample of processing by step 2, and buffer pool is added in buffer later, finally to will delay
The secondary antibody of fliud flushing pond addition fluorescent marker；
Step 3, the type that fluorescence developing region decision ApoE albumen is observed in observation port.
The step 1 will be added EDTA and carry out anticoagulation in blood sample, take out supernatant by centrifugal treating later
As sample.
The step 2, the supernatant that step 1 is collected into the speed of 6-9ul/min from sample pool sample introduction, to supernatant
Incoming seizure area ApoE albumen is combined in trapping region with the antibody of embedding, later from buffer pool with the speed of 6-9ul/min
Buffer is added, rinses trapping region remained on surface substance, then fluorescent marker is added from buffer pool with the speed of 5-7ul/min
In conjunction with first antibody, it is abundant that buffer finally is added from buffer pool in trapping region in secondary antibody, secondary antibody to be marked
Rinse trapping region.
The present invention controls the flow velocity and flow of sample to be tested by microflow control technique, to reduce error, increases reaction spirit
Sensitivity, and it is easy to operate, detection time is short, storage is simple, easily promote the use of, saved cost.
Detailed description of the invention
Below to width attached drawing every in description of the invention expression content and figure in label be briefly described:
Fig. 1 is micro-fluidic chip substrate passageway structural schematic diagram；
Fig. 2 is micro-fluidic chip cover plate structural schematic diagram；
Label in above-mentioned figure is equal are as follows: 1, sample pool；2, buffer pool；3, overcrossing point；4, the undercrossing point；5, upper triangle
Shape region；6, lower delta-shaped region；7, trapping region；8, waste liquid pool.
Specific embodiment
Below against attached drawing, by the description of the embodiment, for example related each component of a specific embodiment of the invention
Shape, construction, the mutual alignment between each section and connection relationship, the effect of each section and working principle, manufacturing process and
Operate with method etc., is described in further detail, to help those skilled in the art to inventive concept of the invention, technology
Scheme has more complete, accurate and deep understanding.
Based on immunization sorting ApoE albumen micro-fluidic chip, including substrate and cover plate, the material of substrate and cover plate is preferred
For PDMS, substrate is equipped with sunk structure, constitutes sample pool 1, buffer pool 2, trapping region 7, waste liquid pool 8, and be connected to these
The channel of sunk structure is integrally in Y type by the sunk structure that channel is connected to, as shown in Figure 1.
Sample pool 1 and buffer pool 2 are located at two independent one end of the channel, the two channels cross at overcrossing point 3
Connection, overcrossing point 3 are the beginning in a channel, and the end in the channel is the undercrossing point 4, and at the undercrossing point 4 and is captured
Area 7 is connected to, and gather 7 bottom of trapping region round slot, and for round slot for embedding ApoE protein antibodies, preferred embodiment is trapping region
Straight flow direction includes that three rectangles capture slot on 7, and round slot, each rectangle capture are densely covered in each rectangle capture slot
Slot embeds and embeds the corresponding antibody of ApoE2 albumen in different antibody, such as the round slot of first rectangle capture slot, and second
The corresponding antibody of ApoE3 albumen is embedded in the round slot of a rectangle capture slot, third rectangle captures in the round slot of slot
Embed the corresponding antibody of ApoE4 albumen.
It is connected between trapping region 7 and waste liquid pool 8 by channel, in use, needing to allow the liquid to substrate slant setting
Trapping region 7 is flowed through by channel using gravity from sample pool 1 and buffer pool 2, finally flow to waste liquid pool 8.
Cover plate includes adding mouth, buffer Sample application mouth, observation port；The position of adding mouth, buffer Sample application mouth, observation port exists
The material of the surface of sample pool 1, buffer pool 2, trapping region 7, trapping region 7 and observation port is glass, and adding mouth, buffer add
Sample mouth is hatch frame, facilitates to sample pool 1, buffer pool 2 and instills liquid.
In addition, 7 rear and front end of trapping region is respectively equipped with delta-shaped region 5 and lower delta-shaped region 6, and pass through triangle
Region and lower 6 communicating passage of delta-shaped region, delta-shaped region and lower delta-shaped region 6 are used to control the purpose of fluid flow rate.
Trapping region 7 is round rectangle structure, length 6mm, width 3mm, depth and the microfluidic channel depth phase of slot
Together, corner is circular arc.The densely covered round slot of trapping region 7 is that sectional dimension is circle, and diameter is 10 μm.3 He of overcrossing point
The distance between undercrossing point 4 is 10mm, and the distance between addition pool, buffer pool 2 and overcrossing point 3 are 8mm.
The method for separating of the micro-fluidic chip, includes the following steps:
(1) blood sample is handled: EDTA being added in blood sample and carries out anticoagulation, then 1500g, 15min are centrifuged, and are taken
Supernatant is detected；
(2) sample is added micro-fluidic chip and is detected: by the supernatant being collected into the speed of 8ul/min from sample pool
11 sample introductions, sample flows decrease when entering inverted triangle region 4, specificity of the ApoE albumen in trapping region 7 and the embedding of its surface
Antibody combines, and buffer is added from buffer pool 2 with the speed of 8ul/min after sample application, rinses surface residue
Matter, then from buffer pool 2 with the secondary antibody of the speed addition fluorescent marker of 6ul/min, the secondary antibody of label is in trapping region 7
In conjunction with first antibody, buffer is added from buffer pool 2 later and sufficiently rinses；
(3) Analysis of test results: after flushing, ApoE albumen is circulated to glass region, observes fluorescence developing regional determination
The type of ApoE albumen.
The present invention is exemplarily described above in conjunction with attached drawing, it is clear that the present invention implements not by aforesaid way
Limitation, as long as the improvement for the various unsubstantialities that the inventive concept and technical scheme of the present invention carry out is used, or without changing
It is within the scope of the present invention into the conception and technical scheme of the invention are directly applied to other occasions.
Claims (10)
1. the micro-fluidic chip based on immunization sorting ApoE albumen, it is characterised in that: constitute independence by substrate and cover plate clamping
Cavity be respectively sample pool, buffer pool, trapping region and waste liquid pool, independent channel is constituted by substrate and cover plate clamping,
In three one end of the channel be respectively communicated with one end of sample pool, buffer pool and trapping region, the other end in three channels crosses
Connection, the other end of the trapping region by channel connection waste liquid pool, the trapping region along liquid flow direction be successively arranged to
A few rectangle captures slot, each densely covered round slot of rectangle capture trench bottom, the circle of each rectangle capture slot
The antibody of ApoE protein configurations is embedded in slot, the cover plate, which is located at sample pool, buffer pool and trapping region, is equipped with sample-adding
Mouthful, buffer Sample application mouth and observation port.
2. micro-fluidic chip according to claim 1, it is characterised in that: the trapping region be located at sample pool, buffer pool,
The middle position of waste liquid pool, all channels constitute y-type structure.
3. micro-fluidic chip according to claim 2, it is characterised in that: the channel and sample pool, give up at buffer pool
Liquid pool, trapping region are made of the sunk structure on substrate, and the cover plate is the plate being sealed on substrate, the substrate and lid
The material of piece is PDMS, and the observation port of the cover plate is sealed by transparent glass.
4. micro-fluidic chip according to claim 1,2 or 3, it is characterised in that: the trapping region both ends pass through loudspeaker respectively
The upper delta-shaped region of mouth shape structure and lower delta-shaped region communicating passage, the bell mouth shape structure are opened towards trapping region
Mouth becomes larger, and gradually becomes smaller towards access portal.
5. micro-fluidic chip according to claim 4, it is characterised in that: the trapping region is successively set along liquid flow direction
There are three rectangles to capture slot, is embedded with the antibody of three kinds of various configurations of ApoE albumen in three rectangle capture slots respectively.
6. micro-fluidic chip according to claim 5, it is characterised in that: three rectangle capture slots are embedded with respectively
The corresponding antibody of ApoE2, ApoE3, ApoE4 albumen.
7. micro-fluidic chip according to claim 1 or 6, it is characterised in that: the channel and sample pool, buffer
Pond, waste liquid pool, trapping region sunk structure to constitute depth identical, the capture section length is 5-7mm, width 2-4mm, described
The aperture of round slot is 8-12 μm, and connection sample pool is mutually all with two passage lengths of buffer pool in three channels
6-10mm, the length in another channel are 8-12mm.
8. the method for separating based on micro-fluidic chip described in any one of claim 1-7, which is characterized in that including following step
It is rapid:
Step 1, blood sample processing；
Sample pool is added in the blood sample of processing by step 2, and buffer pool is added in buffer later, finally to by buffer
The secondary antibody of pond addition fluorescent marker；
Step 3, the type that fluorescence developing region decision ApoE albumen is observed in observation port.
9. method for separating according to claim 8, it is characterised in that: the step 1 will in blood sample be added EDTA into
Row anticoagulation takes out supernatant as sample by centrifugal treating later.
10. method for separating according to claim 8 or claim 9, it is characterised in that: step 1 is collected into upper by the step 2
Clear liquid with the speed of 6-9ul/min from sample pool sample introduction, to supernatant incoming seizure area ApoE albumen in trapping region and embedding
Antibody combines, and buffer is added from buffer pool with the speed of 6-9ul/min later, rinses trapping region remained on surface substance,
The secondary antibody of fluorescent marker is added with the speed of 5-7ul/min from buffer pool again, secondary antibody to be marked is in trapping region
In conjunction with first antibody, buffer finally is added from buffer pool and sufficiently rinses trapping region.
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| CN201910126434.9A CN109870567A (en) | 2019-02-20 | 2019-02-20 | Micro-fluidic chip and its method for separating based on immunization sorting ApoE albumen |
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| CN201910126434.9A CN109870567A (en) | 2019-02-20 | 2019-02-20 | Micro-fluidic chip and its method for separating based on immunization sorting ApoE albumen |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022165751A1 (en) * | 2021-02-05 | 2022-08-11 | 京东方科技集团股份有限公司 | Microfluidic chip |
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