CN107185612A - A kind of micro-fluid chip captured applied to excretion body and preparation method thereof - Google Patents
A kind of micro-fluid chip captured applied to excretion body and preparation method thereof Download PDFInfo
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- CN107185612A CN107185612A CN201710158652.1A CN201710158652A CN107185612A CN 107185612 A CN107185612 A CN 107185612A CN 201710158652 A CN201710158652 A CN 201710158652A CN 107185612 A CN107185612 A CN 107185612A
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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
- 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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Abstract
The present invention relates to a kind of micro-fluidics bio micro-fluid chip captured for excretion body and preparation method thereof.The micro-fluid chip combines microarray post and the affine identification of specificity, realizes the collaboration high efficiency to excretion body in cells and supernatant and Human Fluids and the capture of purity.Micro-fluid chip realizes encapsulation by upper strata cover plate and underlying basal by irreversible bonding.Cover plate is provided with liquid filling hole and fluid hole.It is the biologic specificity molecule for being modified with affine identification, the microarray post arranged with geometry that underlying basal, which is devised in surge chamber and separation chamber, separation chamber,.Excretion body is captured by way of microarray post and antibody affinity purification.The biological micro-fluidic chip of the present invention can efficiently realize the enrichment and extraction of target excretion body, be provided simultaneously with simple in construction, easy to operate, pollution-free, low power consumption and other advantages.
Description
Technical field
The invention belongs to biotechnology and micro-fluidic technologies, and in particular to a kind of capture micro-fluid chip design of excretion body and
Prepare.
Background technology
Excretion body (exosomes) is that a kind of diameter is about that 30-150nm has the vesica of double-deck plasma structure, is led to by cell
Exocytosis is crossed to discharge into extracellular gap or biological body fluid；In recent years, excretion body is received more and more attention, as new
Study hotspot, the various biomolecules such as mRNAs, microRNAs and protein specific to derived cell are included in excretion body,
Played an important role in signal transduction and immune system, more there are some researches show the transfer of excretion body and tumour, the nervous system disease
Etc. closely related, its content is also the marker molecule of a variety of Disease Clinicals detections and diagnosis.
The isolation technics of current excretion body includes supercentrifugation, filter centrifugation method, Density ultracentrifugation, immune
Magnetic bead combination supercentrifugation and chromatography etc.；These technologies require instrument and equipment high, cumbersome time-consuming, it is therefore desirable to have
The excretion body capture technique of effect solves these problems.
Based on described above, the present invention plans microflow control technique, microarray and specific affinity capture technology and combined, and prepares
A kind of micro-pillar array fluid chip based on biologic specificity molecule, and it is applied to the capture of excretion body.
The content of the invention
It is an object of the invention to provide a kind of micro-pillar array fluid chip modified with specific molecular, for excretion
The separation and capture of body.
Technical scheme：Including upper strata cover plate and underlying basal, on the upper strata cover plate provided with sample holes and
Sample outlet hole；
Separation chamber is provided with the underlying basal, is provided with the separation chamber two ends and delaying that separation chamber links together
Rush room,
Described separation chamber is serpentine channel, and the micro-pillar array of cross arrangement is provided with the inside of the serpentine channel,
The surface modification of the micro-pillar array has specific recognition molecules.
The volume size of the micro-fluid chip is 40mm × 30mm × 2mm,
Described surge chamber be square, wide 10-20um, long 1-10mm, high 5-20um,；
The wide 10-20um in described separation chamber, high 5-20um.
It is one kind in circle, triangle, ellipse, water-drop-shaped and polygon at the top of described micro-pillar array；
The micro-pillar array diameter 0.5-5um, high 4-9um, spacing 0.5-2um.
Described specific recognition molecules include the aptamers and specific recognition of specific recognition excretion body surface marker
One kind in the antibody of excretion body surface marker.
The upper strata cover plate is sheet glass, and size is sample holes and sample outlet hole on 40mm × 30mm, the upper strata cover plate
Position it is corresponding with the position of surge chamber on the underlying basal.
The present invention is to live in modified specificity biomolecule by the microarray in micro-fluid chip, so that by sample
Excretion body affinity capture.
Specifically the present invention is made up of the preparation of micro-fluid chip, the surface modification process of microarray post, main logical
Crossing arranging for microarray post increases the contact area of sample and micro-fluid chip micro-fluid chip, and the specificity on post surface is raw
Thing molecule and excretion body affinity interaction, so as to realize the separation and capture of excretion body.
Specific preparation method step：
(1) preparation of micro-fluid chip：The making of micro-structural is realized using the photoetching process of standard；Whole microfluid core
Piece size is about 40mm × 30mm × 2mm, and preparation process is as follows：
1), material pre-preparation：The equal-sized a piece of silicon chip of pre-preparation and a piece of upper strata cover plate,
2), silicon chip is handled：Cleaned silicon chip, drying, spin coating (SU-8 photoresists), whirl coating, front baking, exposure, thickness are dried and aobvious
The steps such as shadow make mould；
PDMS, curing agent is well mixed with 10: 1 mass ratio, remove in mixture and be cast in after bubble on mould, 90
DEG C it is heating and curing 1 hour, mould is carefully peeled off with PDMS solidfied materials, the underlying basal of micro-fluid chip is obtained；
The underlying basal of corona treatment micro-fluid chip,
GPTMS ethanol solutions carry out surface silanization modification to the underlying basal of micro-fluid chip；
The cover plate of micro-fluid chip and substrate are subjected to irreversible bonding,
With perforating needle sample holes and sample outlet hole are beaten on the cover plate of upper strata；Obtain preparing the micro-fluid chip completed.
(2) specific biological molecules are modified：After the making of micro-fluid chip micro-fluid chip is completed, to microfluid core
Piece surface carries out modified biological specific molecular, and preparation process is as follows：
With 0.1mol/l, 2- (N- morpholines) ethyl sulfonic acid (MES), 2mol/l N- ethyls-N '-(3- dimethyl aminopropyls) carbon
Diimine (EDC) and 5mol/l n-hydroxysuccinimide (NHS) mixed solution, which are passed through in microfluid micro-fluid chip, to react
30min, 0.1mol/l phosphate buffer (PBS) rinse micro-fluid chip, are passed through 10ug/ml specific antibody working solution,
After reacting 4 hours at room temperature, with the deionized water rinsing micro-fluid chip serpentine channel of sterilizing, and with the closing of BSA solution,
0.01mol/l PBS bufferings are washed, and are stored for future use under the conditions of 4 degree.
Sample is passed through micro-fluid chip through sample holes when using, adjustment sample flow rate is flow velocity 0-10mL/h, sample stream
The specific molecular modified in excretion body specificity and microarray post in buffered room and the separation chamber of serpentine channel, sample is made
With and combine, so as to capture excretion body.
The advantage of the invention is that：The structure for increasing microarray post in micro-fluid chip significantly increases fluid environment
Area volume ratio, and in micro-pillar array modified specificity combination excretion body biomolecule, so as to substantially increase reaction
Efficiency, reduce the consumption of sample and reagent, reduce cost.
Brief description of the drawings
Fig. 1 is the overall structure diagram of microfluidic arrays micro-fluid chip in the present invention；
Fig. 2 is the structural representation of microfluidic arrays micro-fluid chip cover plate at the middle and upper levels in the present invention；
Fig. 3 is the structural representation of underlying substrate in microfluidic arrays micro-fluid chip in the present invention；
Fig. 4 is microarray post schematic diagram on microfluidic arrays micro-fluid chip serpentine channel in the present invention,
Fig. 5 is microarray post surface modification schematic diagram in the present invention；
1 is upper strata cover plate in figure, and 2 be underlying basal, and 3 be sample holes, and 4 be sample outlet hole, and 5 be separation chamber, and 6 be surge chamber.
Embodiment
Illustrate below in conjunction with the accompanying drawings with regard to embodiment, the present invention is furture elucidated, it should be understood that these embodiments are only
For illustrating the present invention rather than limitation the scope of the present invention, after the present invention has been read, those skilled in the art are to this
The modification of the various equivalent form of values of invention falls within the application appended claims limited range.
In order to understand the present invention, the present invention is described in further detail below.
As illustrated, including upper strata cover plate 1 and underlying basal 2, the upper strata cover plate 1 be preferably sheet glass or
PDMS,
Sample holes 3 and sample outlet hole 4 are provided with the upper strata cover plate 1；
Separation chamber 5 is provided with the underlying basal 2, is provided with and is linked together with separation chamber 5 at the two ends of separation chamber 5
Surge chamber 6,
Described separation chamber 5 is serpentine channel, and the micro-pillar array of cross arrangement is provided with the inside of the serpentine channel,
The surface modification of the micro-pillar array has specific recognition molecules.
The volume size of the micro-fluid chip is 40mm × 30mm × 2mm,
Described surge chamber 6 be square, wide 10-20um, long 1-10mm, high 5-20um,；
Described separation chamber 5 width 10-20um, high 5-20um.
It is one kind in circle, triangle, ellipse, water-drop-shaped and polygon at the top of described micro-pillar array；
The micro-pillar array diameter 0.5-5um, high 4-9um, spacing 0.5-2um.
Described specific recognition molecules include the aptamers and specific recognition of specific recognition excretion body surface marker
One kind in the antibody of excretion body surface marker.
Embodiment 1
The preparation of micro-fluid chip：The schematic diagram of micro-structural is as shown in Figure 1 in micro-fluid chip；Utilize the photoetching work of standard
Skill realizes the making of micro-structural；The volume size of whole micro-fluid chip is about 40mm × 30mm × 2mm, and microtrabeculae is cylinder,
Diameter 0.5um, separation chamber is width 10um, high 5um, micro-pillar array diameter 0.5um, high 4um, spacing 0.5um.
Preparation process is as follows：
1), material pre-preparation：The equal-sized a piece of silicon chip of pre-preparation and a piece of upper strata cover plate 1, silicon chip is base material,
2), silicon chip is handled：Cleaned silicon chip, drying, spin coating (SU-8 photoresists), whirl coating, front baking, exposure, thickness are dried and aobvious
The steps such as shadow make mould；
PDMS, curing agent is well mixed with 10: 1 mass ratio, remove in mixture and be cast in after bubble on mould, 90
DEG C it is heating and curing 1 hour, mould is carefully peeled off with PDMS solidfied materials, the underlying basal 2 of micro-fluid chip is obtained；
The underlying basal 2 of corona treatment micro-fluid chip,
GPTMS ethanol solutions carry out surface silanization modification to the underlying basal 2 of micro-fluid chip；
The upper strata cover plate 1 and underlying basal 2 of micro-fluid chip are subjected to irreversible bonding,
Sample holes 3 and sample outlet hole 4 are beaten on upper strata cover plate 1 with perforating needle；Obtain preparing the micro-fluid chip completed.
Specific biological molecules are modified：After the making of micro-fluid chip is completed, micro-fluid chip surface is modified
Biologic specificity molecule, preparation process is as follows：
With 0.1mol/l, 2- (N- morpholines) ethyl sulfonic acid (MES), 2mol/l N- ethyls-N '-(3- dimethyl aminopropyls) carbon
Diimine (EDC) and 5mol/l n-hydroxysuccinimide (NHS) mixed solution, which are passed through in micro-fluid chip, reacts 30min,
0.1mol/l phosphate buffers (PBS) rinse micro-fluid chip, are passed through 10ug/ml specific antibody working solution, at room temperature
After reaction 4 hours, with the deionized water rinsing micro-fluid chip serpentine channel of sterilizing, and with the closing of BSA solution, 0.01mol/l
PBS bufferings are washed, and are stored for future use under the conditions of 4 degree.
Sample is passed through micro-fluid chip through sample holes 3 when using, adjustment sample flow rate is flow velocity 0-10mL/h, sample stream
Excretion body specificity and the specific molecular modified on microarray post in buffered room 6 and the separation chamber 5 of serpentine channel, sample
Act on and combine, so as to capture excretion body.
Embodiment 2
The preparation of micro-fluid chip：The schematic diagram of micro-structural is as shown in Figure 1 in micro-fluid chip；Utilize the photoetching work of standard
Skill realizes the making of micro-structural；Whole micro-fluid chip size is about 40mm × 30mm × 2mm, and microtrabeculae is cylinder, diameter
2.5um, separation chamber is width 15um, high 13um, micro-pillar array diameter 2.5um, high 6.5um, spacing 1.5um.
Preparation process is as follows：
1), material pre-preparation：The equal-sized a piece of silicon chip of pre-preparation and a piece of upper strata cover plate 1, silicon chip is base material,
2), silicon chip is handled：Cleaned silicon chip, drying, spin coating (SU-8 photoresists), whirl coating, front baking, exposure, thickness are dried and aobvious
The steps such as shadow make mould；
PDMS, curing agent is well mixed with 10: 1 mass ratio, remove in mixture and be cast in after bubble on mould, 90
DEG C it is heating and curing 1 hour, mould is carefully peeled off with PDMS solidfied materials, the underlying basal 2 of micro-fluid chip is obtained；
The underlying basal 2 of corona treatment micro-fluid chip,
GPTMS ethanol solutions carry out surface silanization modification to the underlying basal 2 of micro-fluid chip；
The upper strata cover plate 1 and underlying basal 2 of micro-fluid chip are subjected to irreversible bonding,
Sample holes 3 and sample outlet hole 4 are beaten on upper strata cover plate 1 with perforating needle；Obtain preparing the micro-fluid chip completed.
Specific biological molecules are modified：After the making of micro-fluid chip is completed, micro-fluid chip surface is modified
Biologic specificity molecule, preparation process is as follows：
With 0.1mol/l, 2- (N- morpholines) ethyl sulfonic acid (MES), 2mol/l N- ethyls-N '-(3- dimethyl aminopropyls) carbon
Diimine (EDC) and 5mol/l n-hydroxysuccinimide (NHS) mixed solution, which are passed through in micro-fluid chip, reacts 30min,
0.1mol/l phosphate buffers (PBS) rinse micro-fluid chip, are passed through 10ug/ml specific antibody working solution, at room temperature
After reaction 4 hours, with the deionized water rinsing micro-fluid chip serpentine channel of sterilizing, and with the closing of BSA solution, 0.01mol/l
PBS bufferings are washed, and are stored for future use under the conditions of 4 degree.
Sample is passed through micro-fluid chip through sample holes 3 when using, adjustment sample flow rate is flow velocity 0-10mL/h, sample stream
Excretion body specificity and the specific molecular modified on microarray post in buffered room 6 and the separation chamber 5 of serpentine channel, sample
Act on and combine, so as to capture excretion body.
Embodiment 3
The preparation of micro-fluid chip：The schematic diagram of micro-structural is as shown in Figure 1 in micro-fluid chip；Utilize the photoetching work of standard
Skill realizes the making of micro-structural；Whole micro-fluid chip size is about 40mm × 30mm × 2mm, and microtrabeculae is equilateral triangle, side
Long 5um, separation chamber is width 20um, high 10um, micro-pillar array diameter 5um, high 9um, spacing 2um.
Preparation process is as follows：
1), material pre-preparation：The equal-sized a piece of silicon chip of pre-preparation and a piece of upper strata cover plate 1, silicon chip is base material,
2), silicon chip is handled：Cleaned silicon chip, drying, spin coating (SU-8 photoresists), whirl coating, front baking, exposure, thickness are dried and aobvious
The steps such as shadow make mould；
PDMS, curing agent is well mixed with 10: 1 mass ratio, remove in mixture and be cast in after bubble on mould, 90
DEG C it is heating and curing 1 hour, mould is carefully peeled off with PDMS solidfied materials, the underlying basal 2 of micro-fluid chip is obtained；
The underlying basal 2 of corona treatment micro-fluid chip,
GPTMS ethanol solutions carry out surface silanization modification to the underlying basal of micro-fluid chip；
The upper strata cover plate 1 and underlying basal 2 of micro-fluid chip are subjected to irreversible bonding,
Sample holes 3 and sample outlet hole 4 are beaten on upper strata cover plate 1 with perforating needle；Obtain preparing the micro-fluid chip completed.
Specific biological molecules are modified：After the making of micro-fluid chip is completed, micro-fluid chip surface is modified
Biologic specificity molecule, preparation process is as follows：
With 0.1mol/l, 2- (N- morpholines) ethyl sulfonic acid (MES), 2mol/l N- ethyls-N '-(3- dimethyl aminopropyls) carbon
Diimine (EDC) and 5mol/l n-hydroxysuccinimide (NHS) mixed solution, which are passed through in micro-fluid chip, reacts 30min,
0.1mol/l phosphate buffers (PBS) rinse micro-fluid chip, are passed through 10ug/ml specific antibody working solution, at room temperature
After reaction 4 hours, with the deionized water rinsing micro-fluid chip serpentine channel of sterilizing, and with the closing of BSA solution, 0.01mol/l
PBS bufferings are washed, and are stored for future use under the conditions of 4 degree.
Sample is passed through micro-fluid chip through sample holes 3 when using, adjustment sample flow rate is flow velocity 0-10mL/h, sample stream
Excretion body specificity and the specific molecular modified on microarray post in buffered room 6 and the separation chamber 5 of serpentine channel, sample
Act on and combine, so as to capture excretion body.
Claims (7)
1. a kind of micro-fluid chip captured applied to excretion body, it is characterised in that：Including upper strata cover plate and underlying basal, in institute
Upper strata cover plate is stated provided with sample holes and sample outlet hole；
Separation chamber is provided with the underlying basal, the buffering linked together with separation chamber is provided with the separation chamber two ends
Room,
Described separation chamber is serpentine channel, and the micro-pillar array of cross arrangement is provided with the inside of the serpentine channel,
The surface modification of the micro-pillar array has specific recognition molecules.
2. a kind of micro-fluid chip captured applied to excretion body according to claim 1, it is characterised in that：
The volume size of the micro-fluid chip is 40mm × 30mm × 2mm,
Described surge chamber be square, wide 10-20um, long 1-10mm, high 5-20um,
The wide 10-20um in described separation chamber, high 5-20um.
3. a kind of micro-fluid chip captured applied to excretion body according to claim 1, it is characterised in that：Described is micro-
Post array top is one kind in circle, triangle, ellipse, water-drop-shaped and polygon；
The micro-pillar array diameter 0.5-5um, high 4-9um, spacing 0.5-2um.
4. a kind of micro-fluid chip captured applied to excretion body according to claim 1, it is characterised in that：
Described specific recognition molecules include the aptamers and specific recognition excretion of specific recognition excretion body surface marker
One kind in the antibody of body surface marker.
5. a kind of micro-fluid chip captured applied to excretion body according to claim 1, it is characterised in that：
The upper strata cover plate is sheet glass, and size is 40mm × 30mm, the position of sample holes and sample outlet hole on the upper strata cover plate
Put corresponding with the position of surge chamber on the underlying basal.
6. a kind of a kind of preparation method of the micro-fluid chip captured applied to excretion body as claimed in claim 1, its feature
It is, comprises the following steps：
(1) preparation of micro-fluid chip：
1), material pre-preparation：The equal-sized a piece of silicon chip of pre-preparation and a piece of upper strata cover plate,
2), silicon chip is handled：The steps such as cleaned silicon chip, drying, spin coating, whirl coating, front baking, exposure, thick baking and development make mould：
PDMS, curing agent is well mixed with 10: 1 mass ratio, remove in mixture and be cast in after bubble on mould, 90 DEG C add
Heat cure 1 hour, mould is carefully peeled off with PDMS solidfied materials, obtains the underlying basal of micro-fluid chip；
The underlying basal of corona treatment micro-fluid chip,
GPTMS ethanol solutions carry out surface silanization modification to the underlying basal of micro-fluid chip；
The upper strata cover plate and underlying basal of micro-fluid chip are subjected to irreversible bonding,
With perforating needle sample holes and sample outlet hole are beaten on the cover plate of upper strata；Obtain preparing the micro-fluid chip completed.
(2) specific biological molecules are modified：
Preparation process is as follows：
With 0.1mol/l 2- (N- morpholines) ethyl sulfonic acid, 2mol/l N- ethyls-N '-(3- dimethyl aminopropyls) carbodiimide and
5mol/l n-hydroxysuccinimide mixed solution, which is passed through in micro-fluid chip, reacts 30min, and 0.1mol/l phosphate delays
Fliud flushing rinses micro-fluid chip, is passed through 10ug/ml specific antibody working solution, after reacting 4 hours at room temperature, with going for sterilizing
Ionized water rinses micro-fluid chip serpentine channel, and is closed with BSA solution, and 0.01mol/l phosphate buffers are rinsed, 4 degree of bars
Stored for future use under part.
7. a kind of preparation method of micro-fluid chip captured applied to excretion body according to claim 5, its feature exists
In sample being passed through into micro-fluid chip through sample holes when using, adjustment sample flow rate is flow velocity 0-10mL/h, sample flows through buffering
Excretion body specificity in room and the separation chamber of serpentine channel, sample is acted on and tied with the specific molecular modified on microarray post
Close, so as to capture excretion body.
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| CN110320355B (en) * | 2018-03-30 | 2022-03-01 | 上海市肿瘤研究所 | Micro-fluidic chip and circulating exosome detection method |
| CN110320355A (en) * | 2018-03-30 | 2019-10-11 | 上海市肿瘤研究所 | A kind of micro-fluidic chip and the detection method for recycling excretion body |
| CN111250177A (en) * | 2018-11-30 | 2020-06-09 | 山东大学 | Biomolecule detection method |
| CN111250177B (en) * | 2018-11-30 | 2022-06-24 | 山东大学 | Biomolecule detection method |
| CN111518742A (en) * | 2020-05-07 | 2020-08-11 | 西安交通大学 | Nano-scale single exosome separation method |
| CN111518742B (en) * | 2020-05-07 | 2022-02-11 | 西安交通大学 | Nano-scale single exosome separation method |
| CN111804356A (en) * | 2020-07-16 | 2020-10-23 | 清华大学 | Microfluidic chip and preparation method thereof, microfluidic device and detection method of pathogenic bacteria |
| CN114196503B (en) * | 2020-09-17 | 2024-07-12 | 中国科学院大连化学物理研究所 | Exosome separation enrichment method based on exosome enrichment chip |
| CN114196503A (en) * | 2020-09-17 | 2022-03-18 | 中国科学院大连化学物理研究所 | Exosome separation and enrichment method based on exosome enrichment chip |
| CN114196504A (en) * | 2020-09-17 | 2022-03-18 | 中国科学院大连化学物理研究所 | Exosome enrichment chip based on chitosan positive and negative charge adsorption principle and preparation method thereof |
| CN113005033A (en) * | 2021-02-24 | 2021-06-22 | 清华大学 | Device and method for capturing and separating exosomes and cells in biological sample |
| CN113413933A (en) * | 2021-07-02 | 2021-09-21 | 山东大学第二医院 | Micro-fluidic chip based on glass microspheres and application thereof |
| CN113654953A (en) * | 2021-07-29 | 2021-11-16 | 山东大学深圳研究院 | Method for detecting environmental behaviors and biological effects of nano-particle pollutants |
| CN113576541A (en) * | 2021-08-04 | 2021-11-02 | 张希武 | Capture method of vesicles in tears and contact lens capture chip |
| CN113913367A (en) * | 2021-09-02 | 2022-01-11 | 中国科学院大连化学物理研究所 | Exosome separation and enrichment method based on exosome enrichment device |
| CN114106978A (en) * | 2021-11-22 | 2022-03-01 | 清华大学深圳国际研究生院 | Kit and method for simultaneously detecting internal microRNA and surface protein of exosome |
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