CN208791620U - It is a kind of enrichment, purification cycle tumour cell micro-fluidic chip - Google Patents

Abstract

The utility model discloses a kind of enrichments, the micro-fluidic chip of purification cycle tumour cell, location hole including being located at chip body quadrangle, well, example reaction chamber, physically screening runner, waste liquid runner, waste collection chamber, upper surface is coated with the magnetic-adsorption cavity of magnet, product runner, collection of products chamber and product draw hole, it is connected between example reaction chamber and magnetic-adsorption cavity by physically screening runner, it is connected between magnetic-adsorption cavity and collection of products chamber by product runner, the lower wall of physically screening runner is connected by opening up notch with waste liquid runner；Using the enrichment of above-mentioned micro-fluidic chip, purification cycle tumour cell, it is only necessary to which experiment can be completed in 1 desk centrifuge, and operation is quick, easy, without the use of expensive EpCAM antibody, relative inexpensiveness；Gained circulating tumour cell is not limited only to the type of expression EpCAM antibody, isolates and purifies that effect is more preferable, and without immunomagnetic beads, biological activity is more preferable on surface.

Description

It is a kind of enrichment, purification cycle tumour cell micro-fluidic chip

Technical field

The utility model relates to molecular biosciences medicine technology field, specifically a kind of enrichment, purification cycle tumour cell Micro-fluidic chip.

Background technique

In recent years, the disease incidence of tumour just rises year by year, and malignant tumour has become the primary cause of disease of China's death. One main feature of malignant tumour is transfer, refers to that tumour cell falls off after a large amount of proliferation from primary tumor, and then invade Blood vessel, lymphatic vessel or body cavity, a small number of tumour cells finally survived move to another remote part with blood flow, lymph stream Or orga- nogenesis transfer stove.Circulating tumor cell (Circulating Tumor Cells, CTCs) is departing from the primary portion of tumour The general designation of position and metastasis site and all kinds of tumour cells in entrance blood circulation.CTCs is as between primary tumor and transfer stove Bridge, carries the information of a large amount of tumour progression and transfer, experienced epithelial-mesenchymal conversion in transfer process, makes its tool There is stronger metastatic potential, particularly important role is play during tumour metastasis and recurrence.

Detection CTCs can help us to early diagnose malignant tumour, and this diagnosis index has height A small amount of CTCs also can be detected in sensitivity and specificity in the asymptomatic Peripheral Blood from Patients with Malignant of early stage, and CTCs was not detected in benign disease and physical examination of healthy population blood.By the quantity for the CTCs that gauge check arrives, more may be used To judge neoplasm staging, from the state of development of another side reaction tumour.The quantity variation for monitoring CTCs, can also judge medicine Object therapeutic effect monitors disease progression.

Other than counting, also play an important role for the genetic test of CTCs.Studies have shown that tumor in CTCs and primary tumor Cell has similar gene genetics feature.It also means that by testing and analyzing the gene information in CTCs, can substitute Biopsy is organized, sample, relatively inexpensive mode is obtained by more minimally invasive, sustainable, obtains tumor cell gene information, refer to Its medication treatment is led, realizes the target of precisely medical treatment.

However, due to the CTCs quantity being present in peripheral blood be it is very small amount of, there are 1 in about 1,000,000,000 haemocytes CTCs.And the heterogeneity of CTCs itself and lack effective wide usage Specific marker, leading to the research of CTCs, there is huge Big challenge.System is currently the only one product for obtaining FDA approval listing, and principle is based on immune magnetic Pearl concentration method is coated with tumour cell epithelium marker EpCAM antibody in magnetic bead surfaces, from circulating by way of forward direction enrichment Middle separation CTCs.But there are still some drawbacks for this method, EpCAM antibody consumed first is expensive, and this method can not The cell that the cell of EpCAM is not expressed in capture or EpCAM epitope is lost, cell after separation may because of magnetic bead can not with it is thin Born of the same parents are kept completely separate clean and cell integrity and activity are caused to be destroyed, and cause the recycling for capturing cell by a fixing It rings.

Application No. is the Chinese patents of CN201711446591.5 to disclose a kind of micro-fluidic chip, including chip body, The chip body includes channel, and the channel is equipped with several turbulent structures；The turbulent structure is to be in the side of channel edge 10~20 degree of microchannel or discontinuity set circular configuration or rectangular configuration on a passage, the circular configuration or rectangle knot The internal diameter of structure level cross-sectionn is greater than the width of channel level cross-sectionn.The patent application also disclose above-mentioned micro-fluidic chip into The method of circulating tumor cell in row separating trap blood comprising following steps: step S1, the blood for being blended with cancer cell are thin Born of the same parents' mixed liquor injects in chip, and the piezoelectric ceramic piece by being pasted onto chip bottom, which generates actively to be vortexed, to be shaken, and passes through microchannel When generate air bubble so that cell solution generates vortex, big cell is trapped at the microchannel of channel edge；Step PBS irrigation channel and cell solution are added into channel with the speed of 0.03~0.4mL/min, and keeps piezoelectric ceramic piece by S2 In the state that continues working, leucocyte and red blood cell are gradually washed out channel by PBS, finally only cancer cell it is captured be enriched in it is logical In road.Method described in Chinese patent application No. is CN201711446591.5 mainly utilizes oscillator analog to be centrifuged situation, And utilize the location of different size cell when centrifugation difference, the isolated biggish circulating tumour cell of volume；And it should Method is not screened further using magnetic bead, can not the biggish leucocyte of separated volume.In addition to this it is made pottery using piezoelectricity Tile generates the mode of concussion simulation centrifugation for the more demanding of equipment, operates also more cumbersome.

A kind of circulating tumor cell, which is disclosed, application No. is the Chinese patent of CN201610344918.7 is automatically separated purifying Micro-fluidic chip, including isolate and purify room, shunt column and microtrabeculae, the room of isolating and purifying has sample inlet, for for target The first outlet and be distributed in the first outlet two sides for going out for the second of non-target cell outflow that mark cell flows out Mouthful；Described to isolate and purify indoor tool there are two micro-pillar array, each micro-pillar array is made of multiple microtrabeculae rows, each described There are multiple microtrabeculaes on microtrabeculae row, have between two adjacent microtrabeculaes on each microtrabeculae row gap and should between Gap forms the non-target cell passage for passing through for non-target cell, between having gap between two micro-pillar arrays and being somebody's turn to do Gap forms target cell channel, and the width in the target cell channel is greater than the width of the non-target cell passage；The microtrabeculae It is capable to accompany acute angle with the target cell channel, and the acute angle is toward the sample inlet；The shunting column is arranged described Isolate and purify interior and close at the sample inlet, the shunting column is used to the sample branching to the described micro- of two sides In column array.The patent application also discloses a kind of circulating tumor cell and is automatically separated purification process, includes the following steps: sample Product are isolated and purified the indoor sample and branch to two sides by shunting column by isolating and purifying the entrance sample introduction of room into described In two micro-pillar arrays；The interval and two microtrabeculae battle arrays that the sample passes through the micro-pillar array between the microtrabeculae The channel having between column is separated, and radius is not sent out after colliding less than the non-target cell and micro-pillar array of microtrabeculae critical radius Raw lateral displacement is flowed out by the second outlet for isolating and purifying room described in the original interval warp flowed through between the microtrabeculae； After colliding with micro-pillar array lateral displacement occurs for the target cell that radius is greater than the cell of microtrabeculae critical radius, gradually collects entrance The first outlet outflow of room is isolated and purified in the channel having between two micro-pillar arrays, and described in warp, it is thin into target Born of the same parents' collecting chamber.The structure of microarray column used in method described in Chinese patent application No. is CN201610344918.7 compared with Complexity requires very fine, higher cost for the production of micro-fluidic chip.And it needs when this method feed liquor using additional Pump, for sample apply fixed flow rate, it is higher to equipment requirement, whole flow process cannot a step complete, operate it is more complicated.

Application No. is the Chinese patents of CN201610344920.4 to disclose a kind of circulating tumor cell automatic capture miniflow Control chip, including sample feeding module, sample sorting module and buffer sample introduction module；The sample feeding module include into Sample unit and sample introduction microchannel；The sample sorting module includes that sample separation unit, target cell collector unit and non-target are thin Born of the same parents' collector unit；The buffer sample introduction module includes oscillatory flow unit and continuous flow unit；The sample separation unit has Accommodating cavity, the accommodating cavity is interior to have the micro-pillar array being made of multiple microtrabeculae rows, and each microtrabeculae row is by multiple microtrabeculae groups At the microtrabeculae is about to the accommodating cavity and is separated into multiple collecting regions, has between the adjacent microtrabeculae on each microtrabeculae row There is equal interval and the interval forms sample channel, the width of the sample channel differs on the different microtrabeculae rows； By first microtrabeculae row of the micro-pillar array to the last one microtrabeculae row, the width of the sample channel is gradually reduced；It is described Sample injection unit, the sample introduction microchannel and the accommodating cavity are sequentially communicated, and the sample introduction microchannel is connected to the microtrabeculae First collecting region of array, the sample injection unit are used to sample to be tested being sent into the accommodating by the sample introduction microchannel In chamber, into the sample to be tested in the accommodating cavity by the micro-pillar array and according to the thin of the sample to be tested Born of the same parents' size sorts；The target cell collector unit is connected to sample channel described in the micro-pillar array not less than target cell Collecting region where the microtrabeculae row of critical size is for collecting sorted obtained target cell；The non-target cell collector unit Sample channel described in the micro-pillar array is connected to less than the collecting region where the microtrabeculae row of target cell critical size to be used for Collect it is sorted after obtained non-target cell；The oscillatory flow unit and the continuous flow unit are respectively communicated in the accommodating Chamber, for the oscillatory flow unit for providing the buffering liquid stream for oscillation into the accommodating cavity, the oscillatory flow unit can Pressure is alternately provided into the accommodating cavity and suction is provided；The continuous flow unit into the accommodating cavity for mentioning For continuously buffering liquid stream, the continuous flow unit continuously can provide pressure into the accommodating cavity.The patent application is also Disclose a kind of circulating tumor cell automatic capture method, include the following steps: through the sample injection unit of sample feeding module and In sample introduction microchannel sample introduction to the accommodating cavity of sample separation unit；Sample in the accommodating cavity passes through micro- in the accommodating cavity The microtrabeculae row sample channel that width is gradually reduced on column array is sorted, and the biggish target cell of cell size is trapped in described micro- For sample channel not less than in the collecting region where all microtrabeculae rows of target cell critical size, cell size is lesser in column array Non-target cell enters the receipts that sample channel described in the micro-pillar array is less than where all microtrabeculae rows of target cell critical size Collect in area；The continuous flow unit of buffer sample introduction module provides continuous buffering liquid stream, and the company into the accommodating cavity Afterflow unit continuously provides pressure into the accommodating cavity, so that the target cell and non-target cell in the collecting region are in Flow regime, the oscillatory flow unit of the buffer sample introduction module provide the buffering liquid stream for oscillation into the accommodating cavity, And the oscillatory flow units alternately pressure is provided into the accommodating cavity and suction is provided, the oscillatory flow unit is to described The pressure provided in accommodating cavity promotes target cell and non-target cell longitudinally through the sample channel of each microtrabeculae row, the oscillatory flow The suction that unit is provided into the accommodating cavity promotes target cell and the non-target cell being stuck in the sample channel of each microtrabeculae row It sucks back to the collecting region；Target cell in the target cell collecting region is under the pressure effect that the continuous flow unit provides It flows in lateral flow state and towards target cell collector unit, exists into the non-target cell in the non-target cell collecting region The pressure effect that the continuous flow unit provides is lower to be flowed in lateral flow state and towards non-target cell collector unit.Application Method and Chinese patent application No. is CN201711446591.5 described in Chinese patent number for CN201610344920.4 Described in method it is similar, structure is complicated for microarray column used in this method, the production of micro-fluidic chip is required very smart Carefully, at high cost.And this method lacks the process that magnetic bead is further screened, can not the biggish leucocyte of separated volume, into Liquid is also needed using additional pump, applies fixed flow rate for sample, higher to equipment requirement.

Utility model content

The utility model aim is overcome the deficiencies in the prior art, adapts to realistic development, provide it is a kind of rapidly and efficiently, at The micro-fluidic chip of this cheap circulating tumour cell separation, enrichment, purifying.

A kind of enrichment described in the utility model, purification cycle tumour cell micro-fluidic chip, including chip body, institute Chip body is stated to include the location hole positioned at the chip body quadrangle, well, example reaction chamber, physically screening runner, give up Magnetic-adsorption cavity, product runner, collection of products chamber and the product that liquid stream road, waste collection chamber, upper surface are coated with magnet are drawn Hole, the well are located on the example reaction chamber, and the product is drawn hole and is located on the collection of products chamber, the physics It screens runner and the product runner is pipeline configuration, by described between the example reaction chamber and the magnetic-adsorption cavity Physically screening runner is connected, and is connected between the magnetic-adsorption cavity and the collection of products chamber by the product runner Logical, the physically screening runner is connected with the waste liquid runner by being located at the notch of the physically screening runner lower wall.

Further, the width of the notch is 8~12 μm.

Further, the critical radius of the physically screening runner is 20~25 μm.

Further, the critical radius of the product runner is 20~30 μm.

Further, the critical radius of the product runner is 25 μm.

The advantages of the utility model and good effect:

The utility model combines traditional physics size screening technique and immunomagnetic beads negative itemsets method, passes through first Physically screening runner selects CTCs and biggish leucocyte, is then adsorbed by being coated with the immunomagnetic beads of CD45 and CD2 antibody Leucocyte, to isolate and purify to obtain CTCs.This method carries out preliminary screening, easy to operate, quick, cost using physical method It is cheap；The biggish leucocyte of volume in preliminary screening product is got rid of using the screening of immunomagnetic beads negative sense, on the one hand avoids producing The cell that the cell for not expressing EpCAM or EpCAM epitope are lost is lost in object, on the other hand avoid immunomagnetic beads for CTCs biological activity causes to damage.

Micro-fluidic chip provided by the utility model and its method of enrichment, purification cycle tumour cell, it is only necessary to one Experiment can be completed in centrifuge, and operation is quick, easy, without the use of expensive EpCAM antibody, therefore, relative inexpensiveness；Institute The circulating tumour cell obtained is not limited only to the type of expression EpCAM antibody, and it is more preferable to isolate and purify effect；Resulting circulation hemotoncus Without immunomagnetic beads, biological activity is more preferable on oncocyte surface.

Compared with prior art, the utility model has following features and progress:

Application No. is Chinese patent the methods of CN201711446591.5, and oscillator analog mainly to be utilized to be centrifuged situation, And utilize the location of different size cell when centrifugation difference, the isolated biggish circulating tumour cell of volume；No It is further screened, can not be separated compared with the biggish leucocyte of volume using magnetic bead.And shake is generated using piezoelectric ceramic piece The mode for swinging simulation centrifugation is higher for equipment requirement, operates also more cumbersome；And the utility model is separated by sieve pore and obtains body The biggish circulating tumour cell of product.

The utility model design principle is to pass through sieve pore according to cell partial size difference and separate the acquisition biggish circulating of volume Tumour cell has no intersection in principle and design channel design.The utility model by magnetic bead adsorb, negative sense remove volume compared with Big leucocyte, obtained circulating tumour cell purity are higher.It is special with the China application No. is CN201711446591.5 Benefit is compared, and equipment needed for the utility model is also simpler, and cost is lower, and it is more convenient to operate.

Application No. is Chinese patent the method for CN201610344918.7, the array screening method and this reality that use It is all different with novel structure and efficiency.The structure of microarray column used is more complicated in this method, for micro-fluidic chip Production requirement is finer, and cost is higher.And the chip of the utility model requires fineness lower, easier, the cost of production It is less expensive.And application No. is need to be using additional pump when Chinese patent the method feed liquor of CN201610344918.7 Sample apply fixed flow rate, it is higher to equipment requirement, whole flow process cannot a step complete, operate it is more complicated.And the utility model Only need centrifuge that experimentation can be completed, equipment requirement is simple, and it is more convenient that one step of whole flow process is completed, for operator It is required that it is lower, it more easily popularizes and is widely applied.

Application No. is Chinese patent the method for CN201610344920.4 and application No. is CN201610344918.7 Chinese patent the method it is similar, the structure and efficiency of the array screening method and the utility model that use are all different. The structure of microarray column used is more complicated in this method, the production of micro-fluidic chip is required finer, and cost is higher.And this The chip of utility model is lower for fineness requirement, and production is easier, and cost is less expensive.And application No. is Chinese patent the method for CN201610344920.4 lacks the process that magnetic bead is further screened, can not separated volume Biggish leucocyte.And the utility model is adsorbed by magnetic bead, negative sense removes the biggish leucocyte of volume, obtained circulating Tumour cell purity is higher.In addition to this, application No. is Chinese patent the method feed liquor of CN201610344920.4 is same It needs using additional pump, applies fixed flow rate for sample, it is higher to equipment requirement.And the utility model only needs centrifuge complete At experimentation, equipment requirement is simple, and it is more convenient that one step of whole flow process is completed, lower for operator's requirement, more easily In universal and be widely applied.

Detailed description of the invention

Fig. 1 is the positive overlooking structure diagram of micro-fluidic chip provided by the utility model；

Fig. 2 is that the physically screening runner is connected the partial cross-sectional cut-away view at place with the waste liquid runner.

Specific embodiment

It elaborates with reference to the accompanying drawing to specific embodiment of the utility model.

A kind of enrichment, purification cycle tumour cell micro-fluidic chip, it is described as shown in Fig. 1~2, including chip body Chip body includes location hole 1~4, well 5, example reaction chamber 6, the physically screening runner positioned at the chip body quadrangle 7, waste liquid runner 8, waste collection chamber 9, upper surface are coated with the magnetic-adsorption cavity 10, product runner 11, collection of products chamber of magnet 12 and product draw hole 13, the well 5 is located on the example reaction chamber 6, and product absorption hole 13 is located at the production On object collecting chamber 12, the physically screening runner 7 and the product runner 11 are pipeline configuration, the example reaction chamber 6 and institute It states and is connected between magnetic-adsorption cavity 10 by the physically screening runner 7, the magnetic-adsorption cavity 10 and the product It is connected between collecting chamber 12 by the product runner 11, the physically screening runner 7 is with the waste liquid runner 8 by being located at The notch of 7 lower wall of physically screening runner is connected, and the width of the notch 14 is 10 μm, the physically screening runner 7 Critical radius be 22 μm, the critical radius of the product runner 11 is 25 μm.

Above-mentioned micro-fluidic chip is applied to the method for enrichment, purification cycle tumour cell, includes the following steps:

Human whole blood sample is added into the example reaction chamber 6 from the well 5 by step S1, is then added and contains It has been coated with the reaction solution of the immunomagnetic beads of CD45, CD2 antibody, room temperature warm bath 20 minutes；

Step S2, by the location hole 1~4 of the chip body quadrangle, adapter corresponding to centrifuge is aligned insertion respectively, Fixed chip；

Step S3 is centrifuged 30min with the rate level of 300rpm；

Step S4 draws absorption in hole 13 from the product and isolates and purifies resulting circulating tumor cell.

The strength that the micro-fluidic chip provided in the utility model mainly utilizes is that horizontal centrifuge is generated when rotating Centrifugal force.Human whole blood sample is added into example reaction chamber 6 from well 5 first, containing being coated with CD45, CD2 antibody The reaction solution of immunomagnetic beads made leukocyte surface in blood sample combine coating CD45, CD2 antibody by room temperature warm bath 20 minutes Immunomagnetic beads.Then under the influence of centrifugal force, reaction solution flows into physically screening runner 7 from example reaction chamber 6.Equally from Under the action of mental and physical efforts, the haemocyte in physically screening runner 7 first passes through the notch of 7 lower wall of physically screening runner to waste liquid Flow, and finally flowed into waste collection chamber 9 in runner 8, but diameter be more than 10 μm circulating tumor cell and volume it is larger Leucocyte can then be intercepted by the side wall of physically screening runner 7, and oblique movement is into magnetic-adsorption cavity 10.Since magnetic force is inhaled The upper surface of attached cavity 10 is coated with magnet, and under the magnetic force, the biggish leucocyte of volume in conjunction with immunomagnetic beads is inhaled It is attached in magnetic-adsorption cavity 10, and remaining circulating tumor cell then continues under the influence of centrifugal force, by product runner 11 eventually arrive in collection of products chamber 12.The circulating tumor cell isolated and purified by this method can be drawn by product Hole 13 is taken out, the experiment for next step.

Above-described embodiment is only the more excellent embodiment of the utility model, all according to the technical essence of the utility model pair Any simple modification, modification and alternate variation made for any of the above embodiments, are all within the scope of the technical scheme of the utility model.

Claims (5)

1. the micro-fluidic chip of a kind of enrichment, purification cycle tumour cell, which is characterized in that including chip body, the chip Ontology includes the location hole (1,2,3,4) positioned at the chip body quadrangle, well (5), example reaction chamber (6), physics sieve Runner (7), waste liquid runner (8), waste collection chamber (9), upper surface is selected to be coated with magnetic-adsorption cavity (10), the product runner of magnet (11), collection of products chamber (12) and product draw hole (13), and the well (5) is located on the example reaction chamber (6), described Product is drawn hole (13) and is located on the collection of products chamber (12), the physically screening runner (7) and the product runner (11) For pipeline configuration, pass through the physically screening runner (7) between the example reaction chamber (6) and the magnetic-adsorption cavity (10) It is connected, is connected between the magnetic-adsorption cavity (10) and the collection of products chamber (12) by the product runner (11) Logical, the physically screening runner (7) and the waste liquid runner (8) are by being located at lacking for physically screening runner (7) lower wall Mouth is connected.

2. the micro-fluidic chip of a kind of enrichment according to claim 1, purification cycle tumour cell, which is characterized in that institute The width for stating notch is 8~12 μm.

3. the micro-fluidic chip of a kind of enrichment according to claim 1 or 2, purification cycle tumour cell, which is characterized in that The critical radius of the physically screening runner (7) is 20~25 μm.

4. the micro-fluidic chip of a kind of enrichment according to claim 3, purification cycle tumour cell, which is characterized in that institute The critical radius for stating product runner (11) is 20~30 μm.

5. the micro-fluidic chip of a kind of enrichment according to claim 4, purification cycle tumour cell, which is characterized in that institute The critical radius for stating product runner (11) is 25 μm.