CN109628277A - The separation of excretion in-vivo tumour mark miRNA a kind of and detection system and method - Google Patents
The separation of excretion in-vivo tumour mark miRNA a kind of and detection system and method Download PDFInfo
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Abstract
The separation of excretion in-vivo tumour mark miRNA of the present invention a kind of and detection system and method, the system include excretion body separation module, tumor-marker miRNA separation module and nano-pore detection module；The excretion body separation module is connected on nano-pore detection module by tumor-marker miRNA separation module, and the excretion body separation module is equipped with sample inlet, and the nano-pore detection module connection is on computers；The present invention uses microflow control technique, and the separation of excretion body can be realized on the basis of few sample, while by nano-pore detection technique, on the basis of not needing amplification and fluorescent marker detected material, can effectively improve the efficiency of tumor-marker miRNA detection.
Description
Technical field
The present invention relates to medical treatment and micromechanics electronic technology field more particularly to a kind of excretion in-vivo tumour mark miRNA
Separation and detection system and method.
Background technique
In recent years, our national Malignant Tumor Cases are gradually increasing, and already become and endanger our national people's healths
Principal disease, and in the following more than ten years, Cancer Mortality can also Continued, prevention and treatment of malignant tumors allows of no optimist.Reply
The high-incidence the only way of malignant tumour is " early discovery, early diagnosis, early treatment ".Therefore, the early diagnosis of malignant tumour has weight
The research significance wanted.
But diagnosing tumor is mainly carried out by iconography and histopathological analysis on traditional clinical.It is clinical generally to use electricity
Sub- computed tomography (CT) and Magnetic resonance imaging (MRI) carry out the diagnosis of tumour, but its discovery in tumour early stage is deposited
In difficulty, and it is highly dependent on the experience level of doctor, while inspection fee is high；On the other hand, though organizing biopsy
It is so the goldstandard of diagnosing tumor, but still premised on the positive discovery of iconography, it may be according to the position of tumour, it is also necessary to
Biopsy specimen can be obtained by carrying out operation, and there are tumour spread risks.So being diagnosed as using these two types of conventional methods pernicious swollen
When tumor, the state of an illness of patient has often been developed to middle and advanced stage, so that patient vitals are difficult to be continued.So one kind can early stage into
The detection of row malignant tumour is very necessary.
Research find cancer early stage, tumour can release cycle tumour cell (CTC), Circulating tumor DNA (ctDNA) and
Excretion body enters in blood, by the detection changed to these markers, can realize Precise Diagnosis at tumour initial stage.But
Malignant tumour early stage, CTC and both tumor markers contents of ctDNA are seldom, are difficult to carry out the enrichment of both markers.Together
When research it has also been found that miRNA content can change in the excretion body of tumor cell secretion, at present in breast cancer, ovary
The expression quantity and healthy population that a variety of excretion body miRNA are found in the Malignant Tumor Cases such as cancer, lung cancer, nasopharyngeal carcinoma, cancer of pancreas are not
Together.Additionally due to the phospholipid molecule layer of excretion body can effective protection nucleic acid material with prevent its cell envelope degrade, outside
It is more stable to secrete the miRNA molecule isolated in body.So excretion in-vivo tumour mark miRNA molecule have become at present most have it is latent
One of early diagnosis of tumor marker of power.
Currently, realizing that the detection means of excretion in-vivo tumour mark miRNA molecule needs to undergo three steps: 1) dividing from body fluid
From excretion body；2) it cracks the external film of excretion and is enriched with miRNA molecule；3) table of excretion in-vivo tumour mark miRNA molecule is detected
It reaches.Wherein each step is required to the equipment or kit of profession, there are samples easy to pollute, complicated for operation, somewhat expensive, detection
The problems such as low efficiency, limits clinical application of the technology in terms of diagnosing early malignant tumor.
Summary of the invention
In view of the above problems, it is an object of that present invention to provide one kind to pass through, separating rate few with sample consumption
The microflow control technique of the advantages that fast and at low cost realizes excretion body and the fast and effective separation of miRNA；On the other hand pass through combination
Highly sensitive nanopore sensor carries out the separation and detection of the excretion in-vivo tumour mark miRNA of the detection of tumor-marker miRNA
System and method.
In order to achieve the above object, The technical solution adopted by the invention is as follows: a kind of excretion in-vivo tumour mark miRNA
Separation and detection system, system include excretion body separation module, tumor-marker miRNA separation module and nano-pore detection module；
The excretion body separation module is connected on nano-pore detection module by tumor-marker miRNA separation module, and described is outer
Body separation module is secreted equipped with sample inlet, and the nano-pore detection module connection is on computers.
Nano-pore institute detection module of the invention includes tumor-marker miRNA testing agency and patch-clamp, the tumour
Indicate that miRNA testing agency connects patch-clamp by electrode, the patch-clamp connects computer by data line；Detection
The signal that mechanism obtains is amplified by patch-clamp, then patch-clamp is connected with computer by data line, is finally existed
Detection signal is observed on computer.
Tumor-marker miRNA testing agency of the invention by two organic glass liquid pools being mutually matched, rubber washer and
Nano-pore chip is formed, and the rubber washer and nano-pore chip are arranged between two organic glass liquid pools, and two have
Machine glass liquid pool passes through electrode and is connected in patch-clamp；One of organic glass liquid pool connects tumor-marker by fluid channel
MiRNA separation module.
Excretion body separation module of the invention includes filter structure, fluid channel, micro-valve, enrichment pond and waste liquid pool；Described
Filter structure is made of by-pass filtration structure and secondary filtration structure, is set respectively in by-pass filtration structure and secondary filtration structure
Having aperture is the porous membrane of 200nm and 20nm；The sample inlet is sequentially connected by-pass filtration structure, two by fluid channel
Grade filter structure and enrichment pond, waste liquid pool is connected in the secondary filtration structure, is enriched with pond by fluid channel and is connected tumour
Indicate miRNA separation module.
Cleaning solution and few nucleosides are connected in fluid channel between by-pass filtration structure and secondary filtration structure of the invention
Acid probe import；Cleaning solution and oligonucleotide probe import are connected in fluid channel by micro-valve；The by-pass filtration structure
With the porous membrane for being equipped with different pore size in secondary filtration structure；Between the sample inlet and by-pass filtration structure, second level
Between filter structure and enrichment pond, micro-valve is equipped between secondary filtration structure and waste liquid pool.
Tumor-marker miRNA separation module of the invention includes fluid channel, micro-valve, magnetic bead fixed area, miRNA separation module
It is enriched with pond and miRNA separation module waste liquid pool；The magnetic bead fixed area is equipped with inlet and outlet, and the magnetic bead in module is fixed
Region is fixed with magnetic bead under extraneous magnetic fields, wherein there is p19 albumen on magnetic bead.The import connects excretion body splitting die
Block, the outlet is separately connected miRNA separation module by fluid channel and is enriched with pond and miRNA separation module waste liquid pool, described
MiRNA separation module enrichment pond by fluid channel connection nano-pore detection module.
Be also connected with cleaning solution and eluent import on magnetic bead fixed area of the invention, it is described connect with magnetic bead fixed area it is micro-
Micro-valve is equipped on runner.Cleaning solution is passed first into, will not rinsed with the protein bound substance of p19 to waste liquid pool in sample；Punching
After washing three times, it is passed through eluent, has the tumor-marker miRNA of oligonucleotide probe to elute from magnetic bead hydridization, completes tumour
Indicate the separation and enrichment of miRNA.
Excretion body separation module, tumor-marker miRNA separation module and tumor-marker miRNA detection machine of the present invention
Structure is mounted on an integrated carrier.
The present invention provides a kind of methods of separation and the detection of excretion in-vivo tumour mark miRNA, and the method is such as
Under:
1) humoral sample to be measured is passed through excretion body separation module, by multistage filtering, isolated outer in humoral sample
Secrete body；Lysate is added in excretion body separation module, excretion body is cracked, while oligonucleotide probe is added, with excretion body
The target tumor mark miRNA molecule hydridization included；
2) sample will be obtained in step 1) is passed through tumor-marker miRNA separation module, the tumor-marker of hydridization in sample
MiRNA molecule specifically binds with the p19 albumen on magnetic bead when by magnetic bead fixed area, isolates tumor-marker
miRNA；Eluent is added after the completion of separation, the tumor-marker miRNA of hydridization is eluted from magnetic bead；
3) the tumor-marker miRNA of hydridization obtained in step 2) is passed into nano-pore detection module, to tumor-marker
MiRNA is detected and is transmitted the result to computer.
The present invention has the advantages that integrated excretion in-vivo tumour mark miRNA separation proposed by the present invention and detection
System and method, compared with prior art, the method achieve the extraction of excretion body to tumor-marker miRNA is separated from body fluid
With detection integrated design and integrated, the efficiency of tumor-marker miRNA detection is improved, testing cost and technology door are reduced
Sill.And this method can detect the intracorporal kinds of tumors mark miRNA of excretion simultaneously, improve the technology and be applied to pernicious swell
The accuracy of tumor early diagnosis.
At the same time, this method is in the isolation technics of excretion body, using microflow control technique, few with sample consumption,
The fast and at low cost advantage of separating rate；In the detection of tumor-marker miRNA, using nano-pore detection technique, compared to
Existing miRNA detection technique, does not both need to expand, and does not need fluorescent marker detected material yet, has low cost high-throughput
Advantage.
Detailed description of the invention
Fig. 1 and mounting structure schematic diagram for integrated carrier of the present invention；
Fig. 2 is the connection schematic diagram of nano-pore detection module and computer of the invention；；
Fig. 3 A to 3D is excretion body separation module structure and separation process schematic diagram；
Fig. 3 A is excretion body separation module schematic diagram；
Fig. 3 B is that humoral sample enters separation module schematic diagram；
Fig. 3 C is excretion body separation process schematic diagram；
Fig. 3 D is the cracking of excretion body and tumor-marker miRNA and oligonucleotide probe hydridization schematic diagram；
Fig. 4 A to 4F is tumor-marker miRNA separation module structure and separation process schematic diagram；
Wherein Fig. 4 A is tumor-marker miRNA separation module schematic diagram；
Fig. 4 B is to be combined with P19 albumen magnetic bead to enter schematic diagram；
Fig. 4 C is that the tumor-marker miRNA of the excretion body and hydridization after cracking enters schematic diagram；
Fig. 4 D is that the tumor-marker miRNA of probe hydridization is adsorbed in the schematic diagram of magnetic bead surfaces p19；
Fig. 4 E is that the small molecules such as protein, nucleic acid enter tumor-marker miRNA separation module waste liquid pool schematic diagram；
Fig. 4 F is the tumor-marker miRNA process schematic that hydridization is eluted from magnetic bead；
Fig. 5 A and 5B are miRNA testing agency schematic diagram；
Fig. 5 A is each part schematic diagram of testing agency；
Fig. 5 B is testing agency's assembly schematic diagram.
Wherein, 1- integrated carrier, 2- excretion body separation module, 3- tumor-marker miRNA separation module, 4- tumor-marker
MiRNA testing agency, 5- electrode, 6- patch-clamp, 7- data line, 8- computer, 9- sample inlet, 10,12,14,17,
25,28,29,31,33 be micro-valve, 11- by-pass filtration structure, 13- cleaning solution and oligonucleotide probe import, 15- waste liquid pool,
16- be enriched with pond, 18- secondary filtration structure, small molecules such as bulky grain, 20- excretion body, 21- protein, nucleic acid in 19- sample,
The aperture 22- 200nm filter membrane, the aperture 23- 20nm filter membrane, 24- and oligonucleotide probe hydridization tumor-marker miRNA, 26-
Magnetic bead fixed area, 27- cleaning solution and eluent import, 30-miRNA separation module are enriched with pond, 31-miRNA separation module waste liquid
Pond, 34-miRNA separation module collecting pit, 35- magnetic bead, 36-p19 albumen, 37,40- organic glass liquid pool, 38- silicone rubber pad
Circle, 39- nano-pore chip.
Specific embodiment
The present invention is described in further detail with specific embodiment for explanation with reference to the accompanying drawing.
Embodiment 1: as illustrated in fig. 1 and 2, a kind of separation and detection system of excretion in-vivo tumour mark miRNA, system packet
Include excretion body separation module 2, tumor-marker miRNA separation module 3 and nano-pore detection module；The excretion body separation module
2 are connected on nano-pore detection module by tumor-marker miRNA separation module 3, and the excretion body separation module 2 is equipped with
Sample inlet 9, the nano-pore detection module are connected on computer 8；Nano-pore institute detection module includes tumor-marker
MiRNA testing agency 4 and patch-clamp 6,4 structure of tumor-marker miRNA detection machine connects patch-clamp 6 by electrode 5, described
Patch-clamp 6 pass through data line connect computer 8；The signal that testing agency 4 obtains is amplified by patch-clamp 6, then
Patch-clamp 7 is connected with computer 8 by data line, detection signal is finally observed on computer 8；It is of the present invention
Excretion body separation module 2, tumor-marker miRNA separation module 3 and tumor-marker miRNA testing agency 4 be mounted on a collection
At on carrier 1.
Embodiment 2: as shown in Fig. 3 A to 3D, excretion body separation module 2 includes filter structure 11 and 18, fluid channel, micro-valve
10,12,14,17, pond 16 and waste liquid pool 15 are enriched with；The filter structure is by by-pass filtration structure 11 and secondary filtration structure 18
It is formed, the porous membrane that aperture is 200nm and 20nm is respectively equipped in by-pass filtration structure 11 and secondary filtration structure 18；Institute
The sample inlet 9 stated is sequentially connected by-pass filtration structure 11, secondary filtration structure 18 and enrichment pond 16 by fluid channel, described
Waste liquid pool 15 is connected in secondary filtration structure 18, enrichment pond 16 connects tumor-marker miRNA separation module 3 by fluid channel.
Its workflow and principle are as follows: humoral sample to be measured enters in excretion body separation module 2, wherein in humoral sample
Contain the small molecules such as middle bulky grain, excretion body, protein, nucleic acid；The module is divided into double filtration design, wherein by-pass filtration knot
The aperture of structure 11 is 200nm, so middle bulky grain can not pass through, and the small molecules such as excretion body, protein, nucleic acid can pass through；Together
When secondary filtration structure 18 aperture be 20nm, excretion body can not pass through, and the small molecules such as protein, nucleic acid can pass through；It is logical
The filter structure 11 and 18 for crossing two-stage different pore size can filter out that diameter is less than 20nm greater than 200nm and diameter respectively
Grain, therefore excretion body is trapped between I and II filter structure to achieve the purpose that separation.
Embodiment 3: as shown in Fig. 3 A to 3D, connect in the fluid channel between by-pass filtration structure 11 and secondary filtration structure 18
It is connected to cleaning solution and oligonucleotide probe import 13；Cleaning solution and 13 import of oligonucleotide probe pass through micro-valve 12 and are connected to miniflow
On road.
Its workflow and principle are as follows: when humoral sample by the isolated excretion body of double filtration design 11 and 18 it
Afterwards, but secondary filtration structure 18 may not enter into the useless of excretion body separation module 2 there are also small molecules such as protein, nucleic acid
In liquid pool 15, it is passed through cleaning solution from 13 import of cleaning solution at this time, cleaning solution will not influence excretion body, not by secondary filtration structure 18
The small molecules such as protein, nucleic acid into waste liquid pool 15 are flushed into waste liquid pool 15.
After rinsing three times, it is passed through lysate, excretion body is cracked, inclusion can pass through secondary filtration structure in excretion body at this time
18 enter in enrichment pond 16, then are passed through oligonucleotide probe, and oligonucleotide probe can occur miscellaneous with target tumor mark miRNA
Change, forms duplex structure.
Embodiment 4: as shown in Fig. 4 A to 4F, tumor-marker miRNA separation module 3 includes fluid channel, micro-valve 25,28,29,
32,33, magnetic bead fixed area 26, miRNA separation module enrichment pond 30 and miRNA separation module waste liquid pool 31；The magnetic bead is solid
Determine area 26 and be equipped with inlet and outlet, the magnetic bead fixed area 26 in module is fixed with magnetic bead under extraneous magnetic fields, wherein magnetic
There is p19 albumen on pearl；The import connects excretion body separation module 2, and the outlet is separately connected miRNA by fluid channel
Separation module is enriched with pond 30 and miRNA separation module waste liquid pool 31, and the miRNA separation module enrichment pond 30 passes through fluid channel
Connect nano-pore detection module.
Its workflow and principle are as follows: the sample in excretion body separation module 2 enters tumor-marker miRNA separation module 3,
In addition to the tumor-marker miRNA with oligonucleotide probe hydridization in sample, there are also original protein, nucleic acid in excretion body etc. are small
Molecule；The p19 albumen on magnetic bead in tumor-marker miRNA separation module 3 can only specifically bind length 20-23nt's
Double-stranded RNA；Sample is by the way that when magnetic bead fixed area 26, the p19 albumen on magnetic bead can capture the mesh in sample after hydridization in module
Tumor-marker miRNA is marked, to isolate tumor-marker miRNA.
Embodiment 5: as shown in Fig. 4 A to 4F, being also connected with cleaning solution and eluent import 27 on magnetic bead fixed area 26, described
Micro-valve is equipped in the fluid channel connecting with magnetic bead fixed area 26；
Its workflow is as follows: pass first into cleaning solution, will not be rinsed with the protein bound substance of p19 in sample to
MiRNA separation module waste liquid pool 31；After rinsing three times, it is passed through eluent, hydridization is had to the tumor-marker of oligonucleotide probe
MiRNA is eluted from magnetic bead, completes the separation and enrichment of tumor-marker miRNA.
Embodiment 6: as shown in Fig. 2,5A and 5B, organic glass that tumor-marker miRNA testing agency 4 is mutually matched by two
Glass liquid pool 37 and 40, rubber washer 38 and nano-pore chip 39 are formed, and the rubber washer 38 and nano-pore chip 39 are set
It sets between two organic glass liquid pools 40, two organic glass liquid pools 40 are connected in patch-clamp 6 by electrode 5；Wherein
One organic glass liquid pool 37 connects tumor-marker miRNA separation module by fluid channel；Organic glass liquid pool 37 is equipped with more
A circular through-hole；
Its workflow and principle are as follows: full of certain density in two organic glass liquid pools 37 and 40 in testing agency
Salting liquid, and pass through the nano-pore connection on nano-pore chip 39；Two Ag/AgCl electrodes being connected with 6 amplifier of patch-clamp
It is inserted into two liquid pools respectively, applies transmembrane voltage, while detecting the ionic conductance by nano-pore；As tumor-marker miRNA
Behind the liquid pool end that tumor-marker miRNA separation module enters that added electrode is cathode, since miRNA molecule is negatively charged,
Under the driving of electric field, miRNA can pass through nano-pore；When via hole, due to the occupation time process of miRNA molecule, cause nano-pore from
Sub- conductance decline can observe that the conductance signal changes by 6 amplifier of patch-clamp, to complete tumor-marker in real time
The detection of miRNA.
Embodiment 7: as illustrated in fig. 1 and 2, the separation of excretion in-vivo tumour mark miRNA of the invention and detection system
Operating method is as follows:
1) micro-valve 10 and 14 of excretion body separation module is opened, remaining micro-valve is all in closed state, and humoral sample is from sample
Product import 9 enters, containing small molecules 21 such as excretion body 20, middle bulky grain 19, protein, nucleic acid molecules in sample, due to filtering
22 aperture 200nm of filter membrane in structure 11, so middle bulky grain 19 can not pass through, but excretion body 20, protein, nucleic acid etc.
Small molecule 21 can pass through, and enter filter structure 18 by fluid channel, due to 23 hole of filter membrane in filter structure 18
Diameter is 20nm, so excretion body 20 can not pass through, but the small molecules such as protein, nucleic acid 21 can pass through, thus by fluid channel
It enters in waste liquid pool 15, process is as shown in Figure 3B.
2) micro-valve 10 is closed, micro-valve 12 is opened, micro-valve 14 is still in open state, remaining micro-valve is still in closing shape
State is passed through cleaning solution from import 13, the small molecules such as remaining protein, nucleic acid 21 is punched into waste liquid pool 15, and process is as schemed
Shown in 3C.
3) after cleaning three times, micro-valve 14 is closed, opens micro-valve 17, micro-valve 12 is still in open state, logical from import 13
Entering lysate, excretion body 20 is sufficiently cracked, 20 content of excretion body enters in excretion body separation module enrichment pond 16 at this time,
Including miRNA；It is passed through oligonucleotide probe from import 13 at this time, and is entered in excretion body enrichment pond 16, with target
Hydridization occurs for miRNA, and process is as shown in Figure 3D.
4) miRNA separation module sets that there are four mechanisms in parallel in embodiment, since 4 body functions are the same, here with
It is described in detail for one of them:
5) sample by excretion body separation module 2 crack and with after oligonucleotide probe hydridization, in excretion body inclusion by
Fluid channel enters in tumor-marker miRNA separation module 3, but at this time since micro-valve 25 is closed, so the tumor-marker of hydridization
MiRNA rests on fluid channel, as shown in Figure 4 A.
6) micro-valve 28 and micro-valve 33 are first opened, the magnetic bead 35 of surface modification p19 protein 36 is injected from import 27, magnetic bead is outside
Under boundary's magnetic fields, magnetic bead fixed area 26 can be rested on, process is as shown in Figure 4 B.
7) it is then turned on micro-valve 25, closes micro-valve 28, then inclusion can enter magnetic bead in the excretion body that fluid channel stops
Fixed area 26, in excretion body the tumor-marker miRNA molecule 24 of inclusion hydridization can specific adsorption on p19 protein 36,
To be trapped in magnetic bead fixed area 26, the magnetic bead for being adsorbed with the tumor-marker miRNA molecule of hydridization is as shown in Figure 4 D, process
As shown in Figure 4 C.
8) it is then shut off micro-valve 25, micro-valve 28 is opened, is passed through flushing liquor from import 27, it will be unadsorbed in inclusion in excretion body
Substance be flushed into tumor-marker miRNA separation module waste liquid pool, process is as shown in Figure 4 E.
9) micro-valve 32 is finally closed, micro-valve 29 is opened, eluent is passed through from import 27, by the tumor-marker miRNA of hydridization
Molecule 24 is eluted from p19 protein 36, is entered in tumor-marker miRNA separation module enrichment pond 30, process such as Fig. 4 F
It is shown.
10) the hydridization tumor-marker miRNA molecule 24 in tumor-marker miRNA separation module enrichment pond 30 is by fluid channel
It enters in liquid pool 37.Contain identical salting liquid in liquid pool 37 and liquid pool 40, while added with bias field, the tumour of hydridization
Indicate that miRNA molecule, can be by the nano-pore in nano-pore chip 39, to generate the change of current signal under electric field action
Change, amplifies through patch clamp apparatus 6, computer 8 is transferred to by data line 7.
11) current signal of variation is finally observed on computer 8, to complete the tumor-marker miRNA to hydridization
The detection of molecule 24.
It should be noted that above-mentioned is only presently preferred embodiments of the present invention, protection model not for the purpose of limiting the invention
It encloses, any combination or equivalents made on the basis of the above embodiments all belong to the scope of protection of the present invention.
Claims (10)
1. a kind of separation and detection system of excretion in-vivo tumour mark miRNA, which is characterized in that the separation and detection system
System includes excretion body separation module, tumor-marker miRNA separation module and nano-pore detection module；The excretion body splitting die
Block is connected on nano-pore detection module by tumor-marker miRNA separation module, and the excretion body separation module is equipped with
Sample inlet, the nano-pore detection module connection is on computers.
2. the separation and detection system of excretion in-vivo tumour mark miRNA as described in claim 1, which is characterized in that described
Nano-pore detection module include tumor-marker miRNA testing agency and patch-clamp, the tumor-marker miRNA testing agency
Patch-clamp is connected by electrode, the patch-clamp connects computer by data line.
3. the separation and detection system of excretion in-vivo tumour mark miRNA as claimed in claim 2, which is characterized in that described
Tumor-marker miRNA testing agency by two organic glass liquid pools being mutually matched, rubber washer and nano-pore chip institute group
At the rubber washer and nano-pore chip are arranged between two organic glass liquid pools, and two organic glass liquid pools are logical
Electrode is crossed to be connected in patch-clamp；One of organic glass liquid pool connects tumor-marker miRNA separation module by fluid channel.
4. the separation and detection system of excretion in-vivo tumour mark miRNA as described in claim 1, which is characterized in that described
Excretion body separation module include filter structure, fluid channel, micro-valve, enrichment pond and waste liquid pool；The filter structure is by level-one
Filter structure and secondary filtration structure are formed, and the sample inlet is sequentially connected by-pass filtration structure, two by fluid channel
Grade filter structure and enrichment pond, waste liquid pool is connected in the secondary filtration structure, is enriched with pond by fluid channel and is connected tumour
Indicate miRNA separation module.
5. the separation and detection system of excretion in-vivo tumour mark miRNA as claimed in claim 4, which is characterized in that described
By-pass filtration structure and secondary filtration structure between fluid channel on be connected with cleaning solution and oligonucleotide probe import；Cleaning
Liquid and oligonucleotide probe import are connected in fluid channel by micro-valve.
6. the separation and detection system of excretion in-vivo tumour mark miRNA as claimed in claim 4, which is characterized in that described
Sample inlet and by-pass filtration structure between, between secondary filtration structure and enrichment pond, secondary filtration structure and waste liquid pool it
Between be equipped with micro-valve.
7. the separation and detection system of excretion in-vivo tumour mark miRNA as described in claim 1, which is characterized in that described
Tumor-marker miRNA separation module include fluid channel, micro-valve, magnetic bead fixed area, miRNA separation module enrichment pond and miRNA
Separation module waste liquid pool；The magnetic bead fixed area is equipped with inlet and outlet, and the import connects excretion body separation module,
The outlet is separately connected miRNA separation module by fluid channel and is enriched with pond and miRNA separation module waste liquid pool, described
MiRNA separation module is enriched with pond and connects nano-pore detection module by fluid channel.
8. the separation and detection system of excretion in-vivo tumour mark miRNA as claimed in claim 7, which is characterized in that described
Magnetic bead fixed area on be also respectively connected miRNA separation module collecting pit and magnetic bead and eluent import, it is described solid with magnetic bead
Determine to be equipped with micro-valve in the fluid channel of area's connection.
9. the separation and detection system of excretion in-vivo tumour mark miRNA as claimed in claim 2, which is characterized in that described
Excretion body separation module, tumor-marker miRNA separation module and tumor-marker miRNA testing agency be mounted on one and integrated carry
On body.
10. the side that a kind of system external using described in claim 1 secretes separation and the detection of in-vivo tumour mark miRNA
Method, which is characterized in that the method is as follows:
1) humoral sample to be measured is passed through excretion body separation module, by multistage filtering, isolates the excretion body in humoral sample；
Lysate is added in excretion body separation module, excretion body is cracked, while oligonucleotide probe is added, in excretion body
The target tumor mark miRNA molecule hydridization contained；
2) sample will be obtained in step 1) and is passed through tumor-marker miRNA separation module, the tumor-marker miRNA of hydridization points in sample
Son specifically binds with the p19 albumen on magnetic bead when by magnetic bead fixed area, isolates tumor-marker miRNA；Separation
Eluent is added after the completion, the tumor-marker miRNA of hydridization is eluted from magnetic bead；
3) the tumor-marker miRNA of hydridization obtained in step 2) is passed into nano-pore detection module, to tumor-marker miRNA
It is detected and transmits the result to computer.
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| CN201910064022.7A CN109628277B (en) | 2019-01-23 | 2019-01-23 | System and method for separating and detecting tumor marker miRNA in exosome |
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| CN111286456A (en) * | 2020-03-05 | 2020-06-16 | 清华大学 | Microfluidic channel, microfluidic chip and method for preparing vesicle |
| CN111518668A (en) * | 2020-05-06 | 2020-08-11 | 上海思路迪生物医学科技有限公司 | Microfluidic system for exosome extraction and detection |
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| CN113005021A (en) * | 2020-06-17 | 2021-06-22 | 山东大学 | Microfluidic chip and method for exosome cracking and detection |
| CN113005021B (en) * | 2020-06-17 | 2023-12-08 | 山东大学 | Microfluidic chip and method for exosome cracking and detection |
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