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 PDF

Info

Publication number
CN109628277A
CN109628277A CN201910064022.7A CN201910064022A CN109628277A CN 109628277 A CN109628277 A CN 109628277A CN 201910064022 A CN201910064022 A CN 201910064022A CN 109628277 A CN109628277 A CN 109628277A
Authority
CN
China
Prior art keywords
mirna
excretion
tumor
separation
detection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910064022.7A
Other languages
Chinese (zh)
Other versions
CN109628277B (en
Inventor
章寅
赵佳斌
董隽
陈云飞
司伟
沙菁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southeast University
Original Assignee
Southeast University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Southeast University filed Critical Southeast University
Priority to CN201910064022.7A priority Critical patent/CN109628277B/en
Publication of CN109628277A publication Critical patent/CN109628277A/en
Application granted granted Critical
Publication of CN109628277B publication Critical patent/CN109628277B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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

The separation of excretion in-vivo tumour mark miRNA a kind of and detection system and method
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.
CN201910064022.7A 2019-01-23 2019-01-23 System and method for separating and detecting tumor marker miRNA in exosome Active CN109628277B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910064022.7A CN109628277B (en) 2019-01-23 2019-01-23 System and method for separating and detecting tumor marker miRNA in exosome

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910064022.7A CN109628277B (en) 2019-01-23 2019-01-23 System and method for separating and detecting tumor marker miRNA in exosome

Publications (2)

Publication Number Publication Date
CN109628277A true CN109628277A (en) 2019-04-16
CN109628277B CN109628277B (en) 2022-02-01

Family

ID=66063246

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910064022.7A Active CN109628277B (en) 2019-01-23 2019-01-23 System and method for separating and detecting tumor marker miRNA in exosome

Country Status (1)

Country Link
CN (1) CN109628277B (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111286456A (en) * 2020-03-05 2020-06-16 清华大学 Microfluidic channel, microfluidic chip and method for preparing vesicle
CN111440697A (en) * 2020-03-05 2020-07-24 清华大学 Microfluidic channel, microfluidic chip and method for processing cells
CN111961584A (en) * 2020-08-24 2020-11-20 山东大学齐鲁医院 Cerebrospinal fluid exosome RNA detection device, system and method based on microfluidic technology
CN112048502A (en) * 2019-06-06 2020-12-08 承启医学(深圳)科技有限公司 Method for separating exosome in body fluid by electric field capture mechanism and microfluidic chip
CN112557670A (en) * 2020-12-07 2021-03-26 东南大学 Method and kit for detecting IMN based on urine exosome PLA2R and application of method and kit

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140356877A1 (en) * 2012-04-16 2014-12-04 Biological Dynamics, Inc. Nucleic acid sample preparation
US20150368635A1 (en) * 2013-01-16 2015-12-24 The Regents Of The University Of California Microfluidic devices to extract, concentrate and isolate molecules
CN105388055A (en) * 2015-12-11 2016-03-09 浙江省肿瘤医院 Method for separating tumor cell derived-exosomes from urine
CN105505772A (en) * 2015-12-07 2016-04-20 中国科学院苏州生物医学工程技术研究所 Pyrolysis device for single exosomes
US20160215328A1 (en) * 2013-03-01 2016-07-28 Somagenics, Inc. Methods, compositions and systems for the analysis of nucleic acid molecules
US20160230237A1 (en) * 2015-04-20 2016-08-11 The Research Foundation For The State University Of New York Molecular Analysis using a Magnetic Sifter and Nanowell System
US20160275239A1 (en) * 2013-11-08 2016-09-22 Cartagenia N.V. Genetic analysis method
CN106093392A (en) * 2016-06-05 2016-11-09 浙江大学 The integrated testing method secrete body separation outside a kind of urine, being enriched with and detecting and detection chip
CN106124282A (en) * 2016-07-26 2016-11-16 广州海力特生物科技有限公司 A kind of method secreting body outside lamination centrifugal filtration separation and Extraction
CN106222126A (en) * 2016-07-27 2016-12-14 郑州点石生物技术有限公司 The extracting method of body is secreted by human body fluid China and foreign countries
CN106282107A (en) * 2016-08-30 2017-01-04 章毅 Human plactnta mescenchymal stem cell source separates outer method and the application thereof secreting body
US20170065978A1 (en) * 2014-03-14 2017-03-09 University Of Kansas Non-invasive monitoring cancer using integrated microfluidic profiling of circulating microvesicles
CN206082559U (en) * 2016-06-05 2017-04-12 浙江大学 A microfluid chip that outside being used for, secretes body separation, enrichment and detection
US20170108503A1 (en) * 2008-11-12 2017-04-20 Caris Science, Inc. Methods and systems of using exosomes for determining phenotypes
CN107151661A (en) * 2016-03-02 2017-09-12 上海润腾生物科技有限公司 A kind of people's excretion body protein, kit and its application
CN107271655A (en) * 2017-05-18 2017-10-20 成都中医药大学附属医院 A kind of kit and method for detecting urine excretion body load miRNAs
CN107267440A (en) * 2017-06-09 2017-10-20 李刚 The extracts reagent and application and its extracting method extracted suitable for excretion body
CN107442186A (en) * 2016-05-31 2017-12-08 陈欲超 Micro-fluidic chip, the analytical equipment based on micro-fluidic chip and analysis method
CN107449631A (en) * 2016-05-31 2017-12-08 陈欲超 Sampler, analytical equipment and analysis method
CN107576555A (en) * 2017-06-02 2018-01-12 温州汇芯生物科技有限公司 Separate and collect the device and method of target particles in liquid sample
CN207012992U (en) * 2017-07-26 2018-02-16 深圳宣泽生物医药有限公司 A kind of microfluidic devices of Trace bio-element, detection excretion body
CN108126522A (en) * 2017-12-21 2018-06-08 深圳汇芯生物医疗科技有限公司 The method of target particles in separating chips, separator and separation liquid sample
CN108192951A (en) * 2017-12-26 2018-06-22 东南大学 Observe tumour cell excretion body and the method for miRNA DYNAMIC DISTRIBUTIONs in recipient cell inside excretion body
CN108593916A (en) * 2018-04-08 2018-09-28 国家纳米科学中心 Cancer detection system and method based on excretion body
CN108802374A (en) * 2018-06-25 2018-11-13 中山大学附属第五医院 Excretion body nucleic acid detection technique based on magnetic enrichment electrochemical luminescence
CN108872564A (en) * 2018-09-12 2018-11-23 杭州多泰科技有限公司 A kind of external instant detection platform and its detection method based on excretion body

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170108503A1 (en) * 2008-11-12 2017-04-20 Caris Science, Inc. Methods and systems of using exosomes for determining phenotypes
US20140356877A1 (en) * 2012-04-16 2014-12-04 Biological Dynamics, Inc. Nucleic acid sample preparation
US20150368635A1 (en) * 2013-01-16 2015-12-24 The Regents Of The University Of California Microfluidic devices to extract, concentrate and isolate molecules
US20160215328A1 (en) * 2013-03-01 2016-07-28 Somagenics, Inc. Methods, compositions and systems for the analysis of nucleic acid molecules
US20160275239A1 (en) * 2013-11-08 2016-09-22 Cartagenia N.V. Genetic analysis method
US20170065978A1 (en) * 2014-03-14 2017-03-09 University Of Kansas Non-invasive monitoring cancer using integrated microfluidic profiling of circulating microvesicles
US20160230237A1 (en) * 2015-04-20 2016-08-11 The Research Foundation For The State University Of New York Molecular Analysis using a Magnetic Sifter and Nanowell System
CN105505772A (en) * 2015-12-07 2016-04-20 中国科学院苏州生物医学工程技术研究所 Pyrolysis device for single exosomes
CN105388055A (en) * 2015-12-11 2016-03-09 浙江省肿瘤医院 Method for separating tumor cell derived-exosomes from urine
CN107151661A (en) * 2016-03-02 2017-09-12 上海润腾生物科技有限公司 A kind of people's excretion body protein, kit and its application
CN107442186A (en) * 2016-05-31 2017-12-08 陈欲超 Micro-fluidic chip, the analytical equipment based on micro-fluidic chip and analysis method
CN107449631A (en) * 2016-05-31 2017-12-08 陈欲超 Sampler, analytical equipment and analysis method
CN206082559U (en) * 2016-06-05 2017-04-12 浙江大学 A microfluid chip that outside being used for, secretes body separation, enrichment and detection
CN106093392A (en) * 2016-06-05 2016-11-09 浙江大学 The integrated testing method secrete body separation outside a kind of urine, being enriched with and detecting and detection chip
CN106124282A (en) * 2016-07-26 2016-11-16 广州海力特生物科技有限公司 A kind of method secreting body outside lamination centrifugal filtration separation and Extraction
CN106222126A (en) * 2016-07-27 2016-12-14 郑州点石生物技术有限公司 The extracting method of body is secreted by human body fluid China and foreign countries
CN106282107A (en) * 2016-08-30 2017-01-04 章毅 Human plactnta mescenchymal stem cell source separates outer method and the application thereof secreting body
CN107271655A (en) * 2017-05-18 2017-10-20 成都中医药大学附属医院 A kind of kit and method for detecting urine excretion body load miRNAs
CN107576555A (en) * 2017-06-02 2018-01-12 温州汇芯生物科技有限公司 Separate and collect the device and method of target particles in liquid sample
CN107267440A (en) * 2017-06-09 2017-10-20 李刚 The extracts reagent and application and its extracting method extracted suitable for excretion body
CN207012992U (en) * 2017-07-26 2018-02-16 深圳宣泽生物医药有限公司 A kind of microfluidic devices of Trace bio-element, detection excretion body
CN108126522A (en) * 2017-12-21 2018-06-08 深圳汇芯生物医疗科技有限公司 The method of target particles in separating chips, separator and separation liquid sample
CN108192951A (en) * 2017-12-26 2018-06-22 东南大学 Observe tumour cell excretion body and the method for miRNA DYNAMIC DISTRIBUTIONs in recipient cell inside excretion body
CN108593916A (en) * 2018-04-08 2018-09-28 国家纳米科学中心 Cancer detection system and method based on excretion body
CN108802374A (en) * 2018-06-25 2018-11-13 中山大学附属第五医院 Excretion body nucleic acid detection technique based on magnetic enrichment electrochemical luminescence
CN108872564A (en) * 2018-09-12 2018-11-23 杭州多泰科技有限公司 A kind of external instant detection platform and its detection method based on excretion body

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CONTRERAS-NARANJO JC, ET AL.: "Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine.", 《LAB CHIP》 *
GU LQ, ET AL.: "Detection of miRNAs with a nanopore single-molecule counter.", 《EXPERT REV MOL DIAGN.》 *
HANNAFON BN, ET AL.: "Plasma exosome microRNAs are indicative of breast cancer.", 《BREAST CANCER RES.》 *
JOYCE DP, ET AL.: "Exosome-encapsulated microRNAs as circulating biomarkers for breast cancer.", 《INT J CANCER.》 *
KANWAR SS, ET AL.: "Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes.", 《LAB CHIP》 *
KO J, ET AL.: "miRNA Profiling of Magnetic Nanopore-Isolated Extracellular Vesicles for the Diagnosis of Pancreatic Cancer.", 《CANCER RES.》 *
TIAN K, ET AL.: "Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection.", 《METHODS MOL BIOL.》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112048502A (en) * 2019-06-06 2020-12-08 承启医学(深圳)科技有限公司 Method for separating exosome in body fluid by electric field capture mechanism and microfluidic chip
CN111286456A (en) * 2020-03-05 2020-06-16 清华大学 Microfluidic channel, microfluidic chip and method for preparing vesicle
CN111440697A (en) * 2020-03-05 2020-07-24 清华大学 Microfluidic channel, microfluidic chip and method for processing cells
CN111286456B (en) * 2020-03-05 2021-08-10 清华大学 Microfluidic channel, microfluidic chip and method for preparing vesicle
CN111440697B (en) * 2020-03-05 2022-03-29 清华大学 Microfluidic channel, microfluidic chip and method for processing cells
CN111961584A (en) * 2020-08-24 2020-11-20 山东大学齐鲁医院 Cerebrospinal fluid exosome RNA detection device, system and method based on microfluidic technology
CN112557670A (en) * 2020-12-07 2021-03-26 东南大学 Method and kit for detecting IMN based on urine exosome PLA2R and application of method and kit

Also Published As

Publication number Publication date
CN109628277B (en) 2022-02-01

Similar Documents

Publication Publication Date Title
CN109628277A (en) The separation of excretion in-vivo tumour mark miRNA a kind of and detection system and method
CN106093392B (en) The integrated testing method and detection chip of a kind of urine excretion body separation, enrichment and detection
Pinzani et al. Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase–polymerase chain reaction results and feasibility of molecular analysis by laser microdissection
JP6912064B2 (en) Use of circulating cell biomarkers in blood for disease detection and diagnosis and methods of isolating them
CN109946446A (en) Using electric difference counter to grain count
JP2018009993A (en) Method and apparatus for detection of biomarker by alternating current electrokinetics
JP2001258868A (en) Method and device for blood analysis
US20080050769A1 (en) Method for detecting bioparticles
CN114127562A (en) Detection method of tumor cell surface marker molecule PD-L1
JP6936984B2 (en) How to Predict the Prognosis of Cancer Patients Using Rare Cells
CN110907416A (en) Circulating tumor cell detection device based on hollow nano needle tube electroporation system and detection method thereof
TWI616534B (en) Method and device for purifying and separating blood circulation tumor cells using non-contact and automatic identification
JP2007139556A (en) New analysis method and kit
CN109457032A (en) Thyroid cancer molecule diagnosis kit
JP2018141775A (en) Method of detecting biomarker by alternating current electrokinetics
CN206082559U (en) A microfluid chip that outside being used for, secretes body separation, enrichment and detection
Li et al. Microfluidic chip for cancer cell detection and diagnosis
CN104004824B (en) A kind of blood serum of patients with human breast carcinoma dissociative DNA immue quantitative detection reagent box and application method thereof
Naoe et al. Detection of circulating tumor cells and the importance of their measurement in urological cancers
CN108504736A (en) Detect application of the system of ORM1 gene expression amounts in diagnosis of tuberculosis
CN210506297U (en) Micro flow channel chip and micro flow channel structure
CN109911425B (en) Pulmonary tuberculosis detection kit and application method thereof
CN205398640U (en) Detecting system is united to single biological sample multi -parameter
CN104569100A (en) Functionalized graphene based electrochemical sensor array
CN105116152B (en) A kind of ELISA detection kit and detection method of humanized's urine Endocan albumen

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant