CN103834558A - Blood cell rapid sorting device and manufacturing method thereof - Google Patents
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Abstract
Relating to the technical field of blood cell detection, the invention provides a blood cell rapid sorting device, which includes a microfluidic chip. The microfluidic chip comprises a pipeline for blood flow, and the center line of the pipeline is parallel to the fluid direction. Micro-column arrays are arranged in the microfluidic chip, and are in symmetrical arrangement at certain angle with the center line of the pipeline as the central axis. The invention also provides a manufacturing method of the blood cell rapid sorting device. According to the invention, by designing symmetrical micro-column arrays in the microfluidic chip, the micro-column arrays can separate particles of different sizes on the basis of the deterministic lateral displacement principle, and large cells can be screened out, for instance, circulating tumor cells or circulating endothelial cells larger than ordinary blood cells can be enriched and separated. The blood cell rapid sorting device has the advantages of rapid detection speed, simple operation, and low cost.
Description
Technical field
The present invention relates to blood cell detection technique field, particularly relate to the quick sorting unit of a kind of blood cell and manufacture method thereof.
Background technology
Circulating tumor cell is the tumour cell that enters peripheral blood from tumour primary lesion or metastatic lesion, detects circulating tumor cell significant to prevention and control of cancer, can be effectively applied to rapid evaluation, the individualized treatment etc. of external early diagnosis, chemotherapeutics.Traditional diagnosing tumor method comprises blood serum tumor markers, iconography and histopathology, the advantage of blood serum tumor markers detection method is to can be imaging diagnosis to provide reference data, expense cheap and be applicable to popular examination, shortcoming is the mark that there is no high degree of specificity, often occurs false positive and false negative result; The advantage of imaging examination is to make tumour visual, shortcoming be to patient have radiotherapy damage, the minimum diameter that can detect tumour is 2～3mm, the grade malignancy of tumor size and tumour is inconsistent; The advantage of histopathology is to be conducive to make a definite diagnosis, shortcoming be have the inspection of wound property, can not for detection of with INFORMATION OF INCOMPLETE for shifting.Compare above traditional method, the advantage of circulating tumor cell detection method is that transfer index, the rapid evaluation of chemotherapy and radiation effect, prognosis evaluation, detection recurrence, expense that non-dipped type, susceptibility are high, good are low and be applicable to popular examination, and therefore circulating tumor cell detection method has broad application prospects in clinical tumor prevention and control field.
Circulating tumor cell proportion in peripheral blood is very little, contains 1～10 circulating tumor cell in every milliliter of blood, accounts for 1/109 of total hemocytes count, and it is very difficult therefore detecting circulating tumor cell.Unique commercial product is the detection of CellSearch circulating tumor cell and the analytical system of American I mmunicon company exploitation at present, this system can use immune nano magnetic granule technology to catch analysis to cell, but the shortcoming of the method is complicated operation, need multistep to complete, often occur false positive and false-negative result.
Micro-fluidic chip technology is to grow up the nineties in 20th century, the advantages such as sample reagent consumption is few, analyzing and testing is highly sensitive owing to having, high-throughput and high-level efficiency, current had a research and utilization micro-fluidic chip technology for detection circulating tumor cell, the principle detecting is mainly divided into two classes: the one, utilize the feature that circulating tumor cell is larger than hemocyte, and it is screened from blood; The 2nd, utilize the specific effect of antigen-antibody, by chip surface grafting antibody capture circulating tumor cell.These are studied at present also in the laboratory study stage, its advantage is that reagent consumption is few, cost is low, high-throughput high-level efficiency, although it can be used for screening detection circulating tumor cell, but detection speed is slow, blood testing amount hourly is in milliliter rank, do not reach clinical demand, hindered its application in clinical detection field.
Summary of the invention
The present invention is directed to the above-mentioned defect of prior art, provide the fast blood cell of a kind of detection speed quick sorting unit.The present invention adopts following technical scheme:
The quick sorting unit of a kind of blood cell, comprises micro-fluidic chip, and described micro-fluidic chip comprises a pipeline for blood circulation, and the center line of described pipeline is parallel with flow direction; In described micro-fluidic chip, be provided with micro-pillar array, described micro-pillar array axle centered by the center line of described pipeline is angle symmetric offset spread.
Preferably, the spacing between described microtrabeculae is 10～500 microns.
Preferably, the spacing between described microtrabeculae is 20～30 microns.
Preferably, the spacing between described microtrabeculae is 25 microns.
Preferably, the angle ranging from 0～90 degree.
Preferably, the angle ranging from 2.5～3.2 degree.
Preferably, the angle ranging from 3 degree.
Preferably, the cross-sectional shape of described microtrabeculae is circle or Polygons, and the external diameter of described circle or polygonal circumcircle diameter are 10～500 microns.
Preferably, the external diameter of described circle or polygonal circumcircle diameter are 40 microns.
Preferably, the height of described pipeline is 10～500 microns.
Preferably, the height of described pipeline is 40 microns.
Preferably, be provided with at least one entrance and at least two outlets on described micro-fluidic chip, described entrance connects one end of described pipeline, and described outlet connects the other end of described pipeline.
Preferably, described cell is circulating tumor cell or circulating endothelial cells.
The present invention also provides the manufacture method of the quick sorting unit of a kind of blood cell, and described method comprises the steps:
Utilize photolithography to prepare the photoetching agent pattern that contains the micro-pillar array in the quick sorting unit of blood cell as above at silicon chip surface;
Build polydimethylsiloxane at described patterned surfaces, under 60～100 DEG C of conditions, toast 15 minutes～2 hours;
Described polydimethylsiloxane is taken off from silicon chip surface, and the Chemical bond after Cement Composite Treated by Plasma by the polydimethylsiloxane of taking off and a sheet glass, forms the quick sorting unit of described blood cell.
The present invention by designing symmetrical micro-pillar array in micro-fluidic chip, this micro-pillar array can separate based on determinacy sidesway principle (Deterministic lateral displacement) particle of different sizes, filter out larger-size cell, for example can be by than the large circulating tumor cell of common hemocyte size or circulating endothelial cells concentration and separation, detection speed is fast, simple to operate, cost is low.
Brief description of the drawings
Fig. 1 is the structural representation of 1 one kinds of quick sorting units of blood cell of the embodiment of the present invention;
Fig. 2 is I place partial enlarged drawing in Fig. 1;
Fig. 3 is II place partial enlarged drawing in Fig. 1;
Fig. 4 is the local structure schematic diagram of 2 one kinds of quick sorting units of blood cell of the embodiment of the present invention;
Fig. 5 is circulating tumor cell separating effect and tensionvelocity curve figure;
Fig. 6 is solution enlarged view before separating;
Fig. 7 is solution enlarged view after separating.
Reference numeral:
1-micro-fluidic chip; 11-pipeline;
12-microtrabeculae; 13-entrance;
14-outlet; 14a-first exports;
14b-second exports; 14c-the 3rd outlet;
2-circulating tumor cell; 3-white corpuscle;
4-red corpuscle; A-flow direction;
B-circulating tumor cell sidesway direction.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
embodiment 1:
As shown in Figure 1, be the structural representation of 1 one kinds of quick sorting units of blood cell of the embodiment of the present invention, Fig. 2 is I place partial enlarged drawing in Fig. 1; Fig. 3 is II place partial enlarged drawing in Fig. 1.In Fig. 2, Fig. 3,2 is circulating tumor cell, and 3 is white corpuscle, and 4 is red corpuscle.The present embodiment provides a kind of blood cell quick sorting unit, for detection of the circulating tumor cell 2 in blood, be one embodiment of the present invention, this device comprises micro-fluidic chip 1, micro-fluidic chip 1 comprises a pipeline 11 for blood circulation, and the center line of pipeline 11 is parallel with flow direction A; In micro-fluidic chip 1, be provided with microtrabeculae 12 arrays, microtrabeculae 12 arrays axle centered by the center line of pipeline 11 is angle symmetric offset spread.Microtrabeculae 12 arrays of the present embodiment have the effect of determinacy sidesway to circulating tumor cell 2, and (determinacy sidesway principle refers to: due to hydromechanical principle, the direction that the micro-pillar array of arranging with certain orientation makes the ducted particle that exceedes critical size trend towards micro-pillar array flows, finally converged to one side of array), circulating tumor cell 2 in can separation and concentration blood, the direction of motion (be circulating tumor cell sidesway direction B) of circulating tumor cell 2 in microtrabeculae 12 arrays is consistent with the orientation of microtrabeculae 12.On micro-fluidic chip 1, be provided with at least one entrance 13 and at least two outlets 14, one end of entrance 13 connecting tubes 11, export the other end of 14 connecting tubes 11, in the present embodiment, be provided with 3 outlets 14, be respectively the first outlet 14a, the second outlet 14b and the 3rd outlet 14c, wherein the second outlet 14b is positioned on the center line of pipeline 11, and the blood after the separation that contains circulating tumor cell 2 i.e. from then on outlet flows out.
In the present embodiment, the cross-sectional shape of microtrabeculae 12 is circular, and the external diameter of circle is 40 microns, and the spacing between microtrabeculae 12 is 25 microns, and the arrangement of microtrabeculae 12 arrays and the angle that center line is of pipeline 11 are 3 degree, and the height of pipeline 11 is 40 microns.
The present embodiment utilizes determinacy sidesway principle, in micro-fluidic chip 1, separate and compare the larger-size circulating tumor cell 2 of hemocyte (comprising white corpuscle 3 and red corpuscle 4) by microtrabeculae 12 arrays, can reach the blood testing amount of per minute milliliter rank, significantly improve the feasibility of circulating tumor cell 2 detection techniques in clinical medicine application aspect.The present embodiment does not need multistep operation, energy continuous sample introduction, single stepping, and simple to operate, detection speed is fast, and because chip size is little and do not use antibody, testing cost is low, can provide effective technique means for peripheral blood detects circulating tumor cell 2.
embodiment 2:
As shown in Figure 4, be the local structure schematic diagram of 2 one kinds of quick sorting units of blood cell of the embodiment of the present invention.The present embodiment is one embodiment of the present invention, and wherein, the cross-sectional shape of microtrabeculae 12 is trilateral, its circumcircle diameter is 40 microns, spacing between microtrabeculae 12 is 20 microns, and the arrangement of microtrabeculae 12 arrays and the angle that center line is of pipeline 11 are 3.2 degree, and the height of pipeline 11 is 40 microns.
Other structures, with embodiment 1, are not described in detail in this.
embodiment 3:
The present embodiment also provides a kind of blood cell quick sorting unit, for one embodiment of the present invention, wherein, the cross-sectional shape of microtrabeculae 12 is rectangle, its circumcircle diameter is 40 microns, spacing between microtrabeculae 12 is 30 microns, and the arrangement of microtrabeculae 12 arrays and the angle that center line is of pipeline 11 are 2.5 degree, and the height of pipeline 11 is 40 microns.
Other structures, with embodiment 1, are not described in detail in this.
embodiment 4:
The present embodiment also provides a kind of blood cell quick sorting unit, wherein, the cross-sectional shape of microtrabeculae 12 is square, its circumcircle diameter is 80 microns, spacing between microtrabeculae 12 is 10 microns, the arrangement of microtrabeculae 12 arrays and the angle that center line is of pipeline 11 are 0 degree, and the height of pipeline 11 is 100 microns.
Other structures, with embodiment 1, are not described in detail in this.
embodiment 5:
The present embodiment also provides a kind of blood cell quick sorting unit, wherein, the cross-sectional shape of microtrabeculae 12 is trapezoidal, its circumcircle diameter is 500 microns, spacing between microtrabeculae 12 is 500 microns, the arrangement of microtrabeculae 12 arrays and the angle that center line is of pipeline 11 are 15 degree, and the height of pipeline 11 is 10 microns.
Other structures, with embodiment 1, are not described in detail in this.
embodiment 6:
The present embodiment also provides a kind of blood cell quick sorting unit, wherein, the cross-sectional shape of microtrabeculae 12 is rhombus, its circumcircle diameter is 10 microns, spacing between microtrabeculae 12 is 60 microns, the arrangement of microtrabeculae 12 arrays and the angle that center line is of pipeline 11 are 20 degree, and the height of pipeline 11 is 500 microns.
The above is 6 embodiment of the quick sorting unit of blood cell of the present invention.Certainly, protection scope of the present invention is not limited to above-described embodiment, and the spacing between microtrabeculae can be 10～500 microns, or 20～30 microns; The arrangement of microtrabeculae 12 arrays and the angle that center line is of pipeline 11 can be 0～90 degree, or 2.5～3.2 degree; The cross-sectional shape of microtrabeculae can be circle or Polygons, and external diameter or the polygonal circumcircle diameter of circle can be 10～500 microns; The height of pipeline can be 10～500 microns; Cell can be circulating tumor cell or circulating endothelial cells, or other larger-size cells that need to screen.
embodiment 7:
The present embodiment provides the manufacture method of the quick sorting unit of a kind of blood cell, is one embodiment of the present invention, and the method comprises the steps:
Step S1: silicon chip surface utilize photolithography preparation contain embodiment 1～6 in the photoetching agent pattern of micro-pillar array in the quick sorting unit of blood cell described in any one.
Wherein, photoresist material can be eurymeric photoresist material or negative photoresist, is preferably negative photoresist, for example Su-8 series.Micro-pillar array has the effect of determinacy sidesway to circulating tumor cell.
Step S2: build polydimethylsiloxane (polydimethylsiloxane, PDMS) at patterned surfaces, toast 1 hour under 80 DEG C of conditions.
Step S3: described polydimethylsiloxane is taken off from silicon chip surface, and the Chemical bond after Cement Composite Treated by Plasma by the polydimethylsiloxane of taking off and a sheet glass, forms the quick sorting unit of described blood cell.
Wherein, the quick sorting unit of the blood cell of formation has small pipeline, for blood flow mistake to be measured.
embodiment 8:
The present embodiment also provides the manufacture method of the quick sorting unit of a kind of blood cell, wherein, builds after polydimethylsiloxane (polydimethylsiloxane, PDMS) at patterned surfaces, under 60 DEG C of conditions, toasts 2 hours.
Other steps, with embodiment 7, are not described in detail in this.
embodiment 9:
The present embodiment also provides the manufacture method of the quick sorting unit of a kind of blood cell, wherein, builds after polydimethylsiloxane (polydimethylsiloxane, PDMS) at patterned surfaces, under 100 DEG C of conditions, toasts 15 minutes.
Other steps, with embodiment 7, are not described in detail in this.
The above is 3 embodiment of the manufacture method of the quick sorting unit of blood cell of the present invention.Certainly; protection scope of the present invention is not limited to above-described embodiment; build after polydimethylsiloxane at patterned surfaces; under 60～100 DEG C of conditions, toast 15 minutes～2 hours all can, wherein in the time of 60 DEG C, baking is greater than 1 hour, 80 DEG C of time bakings are greater than half hour, baking is greater than 15 minutes 100 DEG C time.The method that embodiment 7～9 provides can be used for manufacturing the quick sorting unit of blood cell of embodiment 1～6.
experiment:
As shown in Fig. 5～Fig. 7, in figure, white represents the breast cancer tumour cell after fluorescent dye, by experiment, breast cancer tumour cell is joined to the blood of simulating cancer patient in phosphate buffer soln or normal human blood, detected result shows: exceed 95% tumour cell all by concentration and separation, before tumour cell concentration after separation separates, improved 50 times, detection speed reaches 2 milliliters of per minutes.Although along with flow velocity increases, separation efficiency has trace to decline, and all approaches 100%.
The quick sorting unit of blood cell that the embodiment of the present invention provides not only can be used for partitioning cycle tumour cell, can also be used to the circulating endothelial cells in separating blood, to assess the risk of cardiovascular disorder.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (14)
1. the quick sorting unit of blood cell, comprises micro-fluidic chip, it is characterized in that, described micro-fluidic chip comprises a pipeline for blood circulation, and the center line of described pipeline is parallel with flow direction; In described micro-fluidic chip, be provided with micro-pillar array, described micro-pillar array axle centered by the center line of described pipeline is angle symmetric offset spread.
2. device according to claim 1, is characterized in that, the spacing between described microtrabeculae is 10～500 microns.
3. device according to claim 2, is characterized in that, the spacing between described microtrabeculae is 20～30 microns.
4. device according to claim 3, is characterized in that, the spacing between described microtrabeculae is 25 microns.
5. device according to claim 1, is characterized in that, the angle ranging from 0～90 degree.
6. device according to claim 5, is characterized in that, the angle ranging from 2.5～3.2 degree.
7. device according to claim 6, is characterized in that, the angle ranging from 3 degree.
8. device according to claim 1, is characterized in that, the cross-sectional shape of described microtrabeculae is circle or Polygons, and the external diameter of described circle or polygonal circumcircle diameter are 10～500 microns.
9. device according to claim 8, is characterized in that, the external diameter of described circle or polygonal circumcircle diameter are 40 microns.
10. device according to claim 1, is characterized in that, the height of described pipeline is 10～500 microns.
11. devices according to claim 10, is characterized in that, the height of described pipeline is 40 microns.
12. devices according to claim 1, is characterized in that, are provided with at least one entrance and at least two outlets on described micro-fluidic chip, and described entrance connects one end of described pipeline, and described outlet connects the other end of described pipeline.
13. devices according to claim 1, is characterized in that, described cell is circulating tumor cell or circulating endothelial cells.
The manufacture method of 14. 1 kinds of quick sorting units of blood cell, is characterized in that, described method comprises the steps:
Utilize photolithography preparation containing the photoetching agent pattern just like the micro-pillar array in the quick sorting unit of blood cell described in claim 1～9 any one at silicon chip surface;
Build polydimethylsiloxane at described patterned surfaces, under 60～100 DEG C of conditions, toast 15 minutes～2 hours;
Described polydimethylsiloxane is taken off from silicon chip surface, and the Chemical bond after Cement Composite Treated by Plasma by the polydimethylsiloxane of taking off and a sheet glass, forms the quick sorting unit of described blood cell.
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