CN109943468A - A kind of micro-fluidic chip and its application for separating intestinal cancer circulating tumor cell - Google Patents
A kind of micro-fluidic chip and its application for separating intestinal cancer circulating tumor cell Download PDFInfo
- Publication number
- CN109943468A CN109943468A CN201910354897.0A CN201910354897A CN109943468A CN 109943468 A CN109943468 A CN 109943468A CN 201910354897 A CN201910354897 A CN 201910354897A CN 109943468 A CN109943468 A CN 109943468A
- Authority
- CN
- China
- Prior art keywords
- cell
- micro
- circulating tumor
- separating
- intestinal cancer
- 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.)
- Pending
Links
Abstract
The invention discloses a kind of for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, which includes the connected micro-fluidic sorting channel composition of six double-spiral structures, including a cell enters channel and three cell flow pass.The present invention provides a kind of micro-fluidic chips of double helix channel design, the path clustering to tumour cell is realized using inertia force and Dien drag force, to reach the sorting purpose of CTC, with independent of antigen-antibody reaction, it can be to the advantage that interstitial type epidermal cell is sorted, with high sensitivity, high specificity, high efficiency and the simple feature of operation.
Description
Technical field
The present invention relates to a kind of micro-fluidic chips more particularly to a kind of for separating the micro-fluidic of intestinal cancer circulating tumor cell
Chip and its application.
Background technique
Currently, in fields such as biomedicines, it is often necessary to from more complicated sample, it is thin to sub-elect specific target
Born of the same parents.Such as detection, sorting simultaneously count circulating tumor cell (CTC, circulating tumor cell) in cancer patient's blood
Number seem ever more important in cancer diagnosis and treatment.The good and bad jumbled together for CTC detection technique at present, is generally exactly to utilize
Do not utilize marker two major classes, based on marker capture generally include antibody coating magnetic bead based on positive concentration method and
Negative concentration method.It is such to belong to first generation CTC capture technique, it is represented as the Cell search system of Johnson & Johnson.But its technological deficiency
It is that antibody capture is limited only to the capture of the epithelial origin CTC to specifically expressing EpCAM epithelium antigen, and cannot achieve work
Cell capture carries out subsequent gene detection.Negative concentration method reversely removes leucocyte in peripheral blood, this method by CD45 magnetic bead
Enrichment detection can not be carried out to the micro CTC in substantially product sample, and there are problems that the non-specific adsorption of magnetic bead.The first generation
Technical filter embrane method can only capture big partial size tumour cell, missing inspection small particle tumour cell, and the cell captured does not have cell
Activity is only used for cell count, morphology and FISH detection.
Micro-fluidic chip is called the support technology of 21 century life science, is the technology core of portable biochemical analysis instrument
The heart.The technology is the channel by constructing minute yardstick, by sample preparation involved in the fields such as biological and chemical, biology and chemistry
The basic operation units such as Reaction Separation and detection are integrated on one piece several square centimeters of chip, to complete different biology or
Chemical reaction process can analyze a large amount of biomolecule in a short time, the accurate bulk information obtained in sample, information content
It is hundreds and thousands of times of traditional detection means.
Before the diagnosis of intestinal cancer primary tumo(u)r, invasion, the process for shifting and sending out are had begun.CTC turns in intestinal cancer early stage
There is central role in the formation of shifting.Sorting existing one is carried out to the circulating tumor cell in blood sample on micro-fluidic chip
The report studied a bit, such as utilize dielectrophoresis, fluid dynamic, be based on immunomagnetic beads and fluorescence separating method.But this method all exists
Different degrees of deficiency is easy to cause cell cracking if the method for direct current dielectrophoresis needs to apply higher electric field strength；Base
It is although improved in immuno magnetic cell separation method and tumour cell separative efficiency is expressed to specific antigen, but still be confined to tumour
The identification of cell antigen epitope, and circulating tumor cell heterogeneity is determined and is carried out using monospecific antibody or several antibody based on anti-
, all there is the missing inspection to non-antigen presentation CTC in the catching method of original identification.And fluorescence sorting method need to cell into
Row fluorescent marker, and need to build complicated Systems for optical inspection.
Summary of the invention
For overcome the deficiencies in the prior art, it is swollen for separating intestinal cancer circulation that one of the objects of the present invention is to provide one kind
The micro-fluidic chip of oncocyte has high sensitivity, high specificity, high efficiency and the simple feature of operation.
The second object of the present invention is the provision of above-mentioned for separating the micro-fluidic chip of intestinal cancer circulating tumor cell
Using.
An object of the present invention adopts the following technical scheme that realization:
It is a kind of for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, the chip including six double-spiral structures be connected
Micro-fluidic sorting channel composition, including a cell enters channel and three cell flow pass.
Further, the length and width of the chip is 45000 μm, with a thickness of 4000 μm.
Further, the radius in the sorting channel is 9350 μm, and internal diameter is 300 μm.
Further, cell inflow entrance and outflux internal diameter are 1000 μm, 300 μm of each channel width, 40 μm high.
Further, the distance between two neighboring helical duct is 450 μm.Length is 334mm.
Further, the internal diameter of three flow pass is respectively 56 μm, 76 μm, 155 μm.
The second object of the present invention adopts the following technical scheme that realization:
The application of the above-mentioned micro-fluidic chip for being used to separate intestinal cancer circulating tumor cell, comprising the following steps:
(1) after by blood sample to be measured using erythrocyte cracked liquid cracking processing, PBS buffer solution is added, is diluted to sample introduction sample；
(2) by mating syringe pump or pressure pump device, sample introduction sample in step (1) is at the uniform velocity pushed away into chip
Note；
(3) the CTC cell after sorting is intercepted through cell filter membrane, remaining cell and clear liquid are thrown aside；
(4) the internal channel outlet filter membrane after finishing CTC separation is cut together with segment chip, is placed in centrifuge tube
It is centrifuged；
(5) above-mentioned steps (4) treated CTC cell is subjected to ring mediated isothermal amplification, setting fluorescence quantitative PCR instrument is every
Minute detection first order fluorescence, record reflection curve, with the separating effect of assessments tumour cell.
Further, blood sample and erythrocyte cracked liquid to be measured are mixed in above-mentioned steps (1) with 1: 5 volume ratio.
Further, the temperature of ring mediated isothermal amplification is 65 DEG C in above-mentioned steps (5).
Compared with prior art, the beneficial effects of the present invention are: the present invention provides a kind of the micro- of double helix channel design
Fluidic chip realizes the path clustering to tumour cell using inertia force and Dien drag force, to reach the sorting mesh of CTC
, have independent of antigen-antibody reaction, there can be high sensitivity to the advantage that interstitial type epidermal cell is sorted, it is high
Specificity, high efficiency and the simple feature of operation.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of micro-fluidic chip of the present invention for separating intestinal cancer circulating tumor cell；
Fig. 2 is LAMP result figure.
Specific embodiment
In the following, being described further in conjunction with attached drawing and specific embodiment to the present invention, it should be noted that not
Under the premise of conflicting, new implementation can be formed between various embodiments described below or between each technical characteristic in any combination
Example.
Embodiment 1
A kind of for separating the micro-fluidic chip of intestinal cancer circulating tumor cell as shown in Figure 1:, which includes six double spiral shells
The connected micro-fluidic sorting channel composition of structure is revolved, including a cell enters channel and three cell flow pass.The core
The length and width of piece is 45000 μm, with a thickness of 4000 μm.The radius in the sorting channel is 9350 μm, and internal diameter is 300 μm.Cell
Inflow entrance and outflux internal diameter are 1000 μm, 300 μm of each channel width, 40 μm high.Between two neighboring helical duct away from
From being 450 μm.Length is 334mm.The internal diameter of three flow pass is respectively 56 μm, 76 μm, 155 μm.
The preparation method of the above-mentioned micro-fluidic chip for being used to separate intestinal cancer circulating tumor cell, comprising the following steps:
(1) floor layout is printed using film printer, and carries out lithography process；
(2) the carry out spin coating processing that above-mentioned photoetching is good, 500 revs/min of first low speed are even with 1500 revs/min after work 20 seconds
Glue 30 seconds, remember the SU8-3025 negative photoresist that uniform spin coating a layer thickness is 100um i.e. on silicon wafer；
(3) film being disposed is placed in 95 DEG C of drying-plate 15min (80 DEG C of drying-plate 30min) cooled to room temperature list
Face photo-etching machine exposal 30 seconds；
(4) film after above-mentioned exposure is again placed in 95 DEG C of drying-plate 15min, 80 DEG C of drying-plate 30min drying and processings, then
It is cooled to room temperature again；
(5) the above-mentioned silicon wafer being disposed is subjected to development treatment；
(6) precursor of dimethyl silicone polymer (PDMS) and curing agent are configured according to 10:1 mass ratio, is poured into template
On, thickness is about 3 millimeters, is placed in vacuum desiccator and is dried；
(7) above-mentioned PDMS is placed in 80 DEG C of hatching 30min, takes the PDMS layer of polymerization after natural cooling off, pattern turns
Shifting finishes, and is cut with blade according to suitably sized；
(8) pattern is smooth into plastic ware downward after punching the figuratum PDMS of above-mentioned band at inlet port；
(9) plastic flexible pipe that outer diameter is 0.96mm is cut into suitable length, and one end is cut into wedge shape, close to wedge-like portion
Smear one layer of silica gel；
(10) above-mentioned plastic flexible pipe plug flat is entered in the PDMS being flattened on plastic ware, imports uncured PDMS to thickness
About 1mm is placed in 80 DEG C of baking oven and toasts 45min；
(11) PDMS after above-mentioned solidification is cut into suitable chip size, is put into plasma oxidation cleaning chamber with slide
In, high frequency cleans 1min, and PDMS and slide are sticked together, and it is light to press, obtain the chip being bonded；
(12) the cell filter membrane purchased is affixed on inner channel outlet, so as to the CTC filtered to isolate.
Embodiment 2
The application of the above-mentioned micro-fluidic chip for being used to separate intestinal cancer circulating tumor cell, comprising the following steps:
(1) median basilic vein blood 4ml (preceding 2ml of the Adult Healthy Volunteers on an empty stomach after 8 hours is taken in 60min before experiment
Blood abandons it, then takes 4ml as research blood sample), blood sample to be measured and erythrocyte cracked liquid are mixed with 1: 5 volume ratio, are placed in 0
In DEG C environment, static 10-15min mixes sample to ensure lysis efficiency every 5min until blood is transparent up and down；
(2) by mating syringe pump or pressure pump device, by sample introduction sample in step (1) with the speed of 25ml/h to chip
Middle carry out constant-speed injection；
(3) the CTC cell after sorting is intercepted through cell filter membrane, remaining cell and clear liquid are thrown aside；
(4) the internal channel outlet filter membrane after finishing CTC separation is cut together with segment chip, is placed in centrifuge tube
It is centrifuged；
(5) PBS rinse chip 3min is used, 1mlTrizol is added after being repeated 3 times, sufficiently piping and druming mixes, static at room temperature
Lysate is transferred in 1.5ml centrifuge tube by 5min, abundant lytic cell, 0.2ml chloroform is added, acutely concussion 15 seconds, in room
Temperature is lower to be incubated for 5min, is centrifuged 15 minutes for 12000 revs/min at 4 DEG C, lower leaf on lysate；
(6) supernatant 0.4ml is drawn, is transferred in 1.5ml centrifuge tube, 0.5ml isopropanol is added, is turned upside down sufficiently mixed
It is even, it is centrifuged after the static 10min of room temperature；
(7) supernatant is taken, 1m175% ethyl alcohol is added, is inhaled after centrifugation and abandons supernatant, is added without RNA enzyme water, dispenses, carry out
LAMP experiment；
(8) LAMP reaction system is mixed according to table 1, LAMP reaction is added in the CTC total serum IgE sample after taking 2 μ l to extract respectively
System, 65 DEG C of isothermal reactions, setting fluorescence quantitative PCR instrument detect first order fluorescence per minute, and record reflects curve, as shown in Fig. 2,
With the separating effect of assessments tumour cell.
Table 1
The successful enrichment and separation of CTC not only can provide its enumerative information, while the expression feelings of its surface marker
The expression of condition and gene all can provide more information for the diagnosis of tumour, achieve the effect that liquid biopsy.Relative to tradition
Aspiration biopsy, the acquisition of CTC will obtain more tumour cells, rather than fibroblast, analysis of molecules and genetic analysis
To all have significant advantage, which especially has important value.
The present invention provides a kind of micro-fluidic chips of double helix channel design, are realized using inertia force and Dien drag force
To the path clustering of tumour cell, to reach the sorting purpose of CTC, have independent of antigen-antibody reaction, it can be to interstitial
The advantage that type epidermal cell is sorted has high sensitivity, high specificity, high efficiency and the simple feature of operation.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Claims (9)
1. a kind of for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that the chip includes six double spiral shells
The connected micro-fluidic sorting channel composition of structure is revolved, including a cell enters channel and three cell flow pass.
2. according to claim 1 for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that the core
The length and width of piece is 45000 μm, with a thickness of 4000 μm.
3. according to claim 1 for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that described point
The radius for gating road is 9350 μm, and internal diameter is 300 μm.
4. according to claim 1 for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that cell stream
Entrance and outflux internal diameter are 1000 μm, 300 μm of each channel width, 40 μm high.
5. according to claim 1 for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that adjacent two
The distance between a helical duct is 450 μm.Length is 334mm.
6. according to claim 1 for separating the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that three streams
The internal diameter in channel is respectively 56 μm, 76 μm, 155 μm out.
7. a kind of application for the micro-fluidic chip for being used to separate intestinal cancer circulating tumor cell as described in claim 1 to 6, feature
It is, comprising the following steps:
(1) after by blood sample to be measured using erythrocyte cracked liquid cracking processing, PBS buffer solution is added, is diluted to sample introduction sample；
(2) by mating syringe pump or pressure pump device, sample introduction sample in step (1) is subjected to constant-speed injection into chip；
(3) the CTC cell after sorting is intercepted through cell filter membrane, remaining cell and clear liquid are thrown aside；
(4) the internal channel outlet filter membrane after finishing CTC separation is cut together with segment chip, is placed in centrifuge tube and is carried out
Centrifugation；
(5) above-mentioned steps (4) treated CTC cell is subjected to ring mediated isothermal amplification, setting fluorescence quantitative PCR instrument is per minute
Detect first order fluorescence, record reflection curve, with the separating effect of assessments tumour cell.
8. according to claim 7 for separating the application of the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that
Blood sample and erythrocyte cracked liquid to be measured are mixed in above-mentioned steps (1) with 1: 5 volume ratio.
9. according to claim 7 for separating the application of the micro-fluidic chip of intestinal cancer circulating tumor cell, which is characterized in that
The temperature of ring mediated isothermal amplification is 65 DEG C in above-mentioned steps (5).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910354897.0A CN109943468A (en) | 2019-04-30 | 2019-04-30 | A kind of micro-fluidic chip and its application for separating intestinal cancer circulating tumor cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910354897.0A CN109943468A (en) | 2019-04-30 | 2019-04-30 | A kind of micro-fluidic chip and its application for separating intestinal cancer circulating tumor cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109943468A true CN109943468A (en) | 2019-06-28 |
Family
ID=67016588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910354897.0A Pending CN109943468A (en) | 2019-04-30 | 2019-04-30 | A kind of micro-fluidic chip and its application for separating intestinal cancer circulating tumor cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109943468A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102879377A (en) * | 2012-10-29 | 2013-01-16 | 重庆科技学院 | Microfluidic chip for detecting early leukemia |
| KR20160064768A (en) * | 2014-11-28 | 2016-06-08 | 한국과학기술연구원 | Apparatus and method for microfluidic chip filtration using spiral branch channel |
| WO2017131580A1 (en) * | 2016-01-28 | 2017-08-03 | Clearbridge Biomedics Pte Ltd | Multi-stage target cell enrichment using a microfluidic device |
| CN107084916A (en) * | 2017-03-29 | 2017-08-22 | 无锡纳奥生物医药有限公司 | A kind of circulating tumor cell separating micro-fluidic chip device and its application method |
| CN108795869A (en) * | 2018-06-28 | 2018-11-13 | 亚能生物技术（深圳）有限公司 | A kind of circulating tumor cell positive enrichment method |
| CN109082368A (en) * | 2018-10-29 | 2018-12-25 | 上海理工大学 | The multistage micro flow control chip device of circulating tumor cell sorting, enrichment and detection |
-
2019
- 2019-04-30 CN CN201910354897.0A patent/CN109943468A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102879377A (en) * | 2012-10-29 | 2013-01-16 | 重庆科技学院 | Microfluidic chip for detecting early leukemia |
| KR20160064768A (en) * | 2014-11-28 | 2016-06-08 | 한국과학기술연구원 | Apparatus and method for microfluidic chip filtration using spiral branch channel |
| WO2017131580A1 (en) * | 2016-01-28 | 2017-08-03 | Clearbridge Biomedics Pte Ltd | Multi-stage target cell enrichment using a microfluidic device |
| CN107084916A (en) * | 2017-03-29 | 2017-08-22 | 无锡纳奥生物医药有限公司 | A kind of circulating tumor cell separating micro-fluidic chip device and its application method |
| CN108795869A (en) * | 2018-06-28 | 2018-11-13 | 亚能生物技术（深圳）有限公司 | A kind of circulating tumor cell positive enrichment method |
| CN109082368A (en) * | 2018-10-29 | 2018-12-25 | 上海理工大学 | The multistage micro flow control chip device of circulating tumor cell sorting, enrichment and detection |
Non-Patent Citations (4)
| Title |
|---|
| JIASHU SUN ET AL.: "Double spiral microchannel for label-free tumor cell separation and enrichment", 《LAB ON A CHIP》 * |
| JIDONG WANG ET AL.: "Label-Free Isolation and mRNA Detection of Circulating Tumor Cells from Patients with Metastatic Lung Cancer for Disease Diagnosis and Monitoring Therapeutic Efficacy", 《ANALYTICAL CHEMISTRY》 * |
| LASZLO HAJBA ET AL.: "Circulating tumor-cell detection and capture using microfluidic devices", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
| 唐文博: "微流控芯片分离胰腺癌患者循环肿瘤细胞的基础实验及其临床应用", 《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10677708B2 (en) | Microfluidic device and method for detecting rare cells | |
| CN106076441B (en) | A kind of micro fluidic device and method based on size detection circulating tumor cell | |
| CN107402295B (en) | Circulating tumor cell is automatically separated purifying micro-fluidic chip and its isolation and purification method | |
| CN106635995B (en) | Negative enrichment method for circulating tumor cells | |
| CN105062866B (en) | For disposable separating chips module and the using method thereof of Peripheral Circulation tumour cell | |
| CN111733056B (en) | Micro-fluidic chip integrating circulating tumor cell separation and single-cell immunoblotting | |
| CN105689028B (en) | Micro-fluid chip, method and its application for the single distribution of immune microsphere | |
| CN107085107A (en) | A kind of microfluid system for detecting esophageal squamous cell carcinoma circulating tumor cell and its application | |
| CN110496655A (en) | A kind of tumour cell detection chip based on microflow control technique | |
| CN104073428A (en) | Cell separating micro-structural system | |
| CN105087493B (en) | Three kinds of monoclonal antibody coupling immunomagnetic beads of combination are applied in tumour cell sorting | |
| CN108753571A (en) | A kind of cell typing capture chip and method | |
| CN104062428B (en) | A kind of test kit detecting circulating tumor cell | |
| CN111909823A (en) | Inertial micro-fluidic chip for enriching circulating tumor cells | |
| CN111826349B (en) | Erythrocyte cluster based on size filtration method for enriching circulating tumor cells | |
| WO2018214623A1 (en) | Microfluidic chip for circulating tumor cell separation, circulating tumor cell separation method and counting method | |
| CN106970225A (en) | A kind of kit of application CD45 immunofluorescences joint probe identification circulating tumor cells of CEP 8 and its application | |
| CN106970224A (en) | A kind of kit of application CD45 immunofluorescences joint CEP probe identification circulating tumor cells and its application | |
| CN106980018A (en) | A kind of kit of application CD45 immunofluorescences joint CEP17 probe identification circulating tumor cells and its application | |
| CN108795693A (en) | A kind of micro-fluidic chip of capture blood rare cell | |
| CN107400623A (en) | Circulating tumor cell automatic capture micro-fluidic chip and its automatic capture method | |
| CN207130273U (en) | A kind of magnetic force micro-fluidic chip for capturing bacterium | |
| CN106190770B (en) | A kind of double layer micro fluidic chip for tumour cell sorting | |
| CN111939992A (en) | Preparation method of micro-fluidic system for capturing and enriching circulating tumor cells and micro-fluidic device | |
| CN108546676A (en) | The method that tunnel magnetic field technology detaches and is enriched with rare cell in peripheral blood |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190628 |
|
| RJ01 | Rejection of invention patent application after publication |