CN101464458A - Micro-flow controlled chip used for detecting erythrocyte osmotic fragility - Google Patents

Micro-flow controlled chip used for detecting erythrocyte osmotic fragility Download PDF

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Publication number
CN101464458A
CN101464458A CN 200810218355 CN200810218355A CN101464458A CN 101464458 A CN101464458 A CN 101464458A CN 200810218355 CN200810218355 CN 200810218355 CN 200810218355 A CN200810218355 A CN 200810218355A CN 101464458 A CN101464458 A CN 101464458A
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hybrid
micro
channel
detection
chip
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CN101464458B (en
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叶大田
李雷
周亚浩
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Shenzhen Graduate School Tsinghua University
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Shenzhen Graduate School Tsinghua University
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Abstract

The invention relates to a micro-fluidic chip used for testing erythrocyte osmotic fragility, which comprises a chip main-body, wherein, two solution inlets and a blood sample inlet are formed on the chip main-body; and a plurality of detection pools arranged inside the chip main-body are communicated with the blood sample inlet through a blood sample microchannel and communicated with the two solution inlets through a microchannel network that generates concentration gradient automatically. Based on the micro-fluidic chip, a test method of erythrocyte osmotic fragility comprises the steps as follows: (1) blood samples are introduced into the different detection pools in the micro-fluidic chip through the blood sample inlet; (2) two NaCl solutions with different concentrations are introduced into the micro-fluidic chip through the two solution inlets at the same flow speed at the same time, and mixed with the blood samples in the detection pools; (3) erythrocytes in the detection pools are observed through a microscope and are shoot; and (4) the intact erythrocytes in each detection pool are distinguished in erythrocyte pictures and counted. The micro-fluidic chip reduces use of blood, reduces manual intervention, and has high detection speed and objective and accurate detection results.

Description

Be used to check the micro-fluidic chip of osmotic fragility
Technical field
The present invention relates to the osmotic fragility inspection technology, particularly a kind of micro-fluidic chip that can generate concentration gradient automatically and based on the osmotic fragility method of inspection of micro-fluidic chip.
Background technology
Haemolysis appears in red blood cell in hypotonic salt solusion characteristic is called osmotic fragility, is an important clinical blood test index.Endoerythrocytic osmotic pressure and plasma osmolarity place isotonic solution (0.9%NaCl) with red blood cell about equally under the normal condition, can keep normal size and form; If red blood cell is placed hypertonic solution (〉 0.9%), moisture can be overflowed outside the born of the same parents, and cell is because of the dehydration shrinkage; If red blood cell is placed hypotonic solution (<0.9%), moisture enters cell, and red blood cell expands and becomes sphere, and inflatable to breaking, haemoglobin is discharged in the solution, is called haemolysis.Normocyte generally begins to occur haemolysis in 0.42%NaCl solution, and complete hemolysis in 0.35%NaCl solution, so represent normocytic osmotic fragility scope with the NaCl solution of 0.42-0.35%.Osmotic fragility increases, common hereditary spherocytosis; Osmotic fragility reduces, common thalassemia, hypoferric anemia etc.Conventional checked operation method needs the NaCl solution of 12 variable concentrations of artificial preparation, does contrast test with normal person's blood sample and patient's blood sample, leaves standstill after 2 hours and obtains assay by naked-eye observation, and complicated operation, manual intervention are big.
Summary of the invention
The purpose of this invention is to provide a kind of osmotic fragility method of inspection that can realize quick test, reduce blood using amount and minimizing manual intervention, and the micro-fluidic chip that is used for this method of inspection.
Micro-fluidic chip is a Biochemical Lab that makes up on more than one square centimeters chip, it is based on MEMS (Micro Electro Mechanical systems) processing technology, at silicon chip, make the microchannel on the material such as glass or PDMS, and form network by the microchannel, run through total system with controlled fluid, realize related reaction in the biological and chemical field, separate, check, basic operations such as cellular incubation, in order to replace the breadboard various functions of conventional biological or chemical, therefore be also referred to as " laboratory on the chip (Lab-on-a-chip) ".Have characteristics such as tangible microminiaturization, integrated and portability.Compare with present breadboard common method, use the micro-fluidic chip pair cell to operate and analyze main advantage and be: 1) microchannel size and cell size are complementary, greatly about 10-100 μ m; 2) micro-fluidic chip provides the space of a relative closure, reduces the stimulation of external environment pair cell; 3) can directly examine under a microscope pair cell carries out on micro-fluidic chip the operation and the situation of cell; 4) the micro-fluidic chip volume is small, has saved the consumption of cell solution and other reagent, and the microchannel heat and mass is rapid, and the reaction time also significantly reduces; 5) operation of several steps such as cell sample, separation, analysis can be integrated in the chip piece last time finishes.
The concrete technical scheme of the present invention is as follows: a kind of osmotic fragility method of inspection based on micro-fluidic chip may further comprise the steps:
A, with micro-injection pump with in the different detection cells in the blood sample import input micro-fluidic chip of blood sample by micro-fluidic chip;
B, with micro-injection pump with the NaCl solution of two kinds of variable concentrations with identical flow velocity simultaneously in two solution inlet port input micro-fluidic chips of described micro-fluidic chip, make the NaCl solution of variable concentrations by the microchannel network of the automatic generation concentration gradient in the micro-fluidic chip, and send into respectively in the different detection cells, mix with blood sample in the detection cell;
C, with microscopic examination and take red blood cell photo in each detection cell;
D, from the red blood cell photo identification each detection cell in complete red blood cell and the counting.
In step b, in the NaCl solution of two kinds of variable concentrations that added, wherein a kind of concentration of NaCl solution is 0%~0.25%, and the concentration of another kind of NaCl solution is 0.5%~0.9%.
In steps d, preferred version is, discern complete red blood cell and counting in each detection cell by computing machine with image processing method, specifically may further comprise the steps: with threshold method the photo of microscope photographing is divided into red blood cell and non-red blood cell zone earlier, again with area-method statistics red blood cell number and calculating cell size.Under the situation that does not have the Computer Image Processing condition, also can go out macroscopic RBC number in the red blood cell photo by people's number.
Wherein, micro-fluidic chip comprises chip body, two solution inlet port and a blood sample import are set on the chip body, the plurality of detection pond is set in the chip body, described plurality of detection pond is in-line and arranges, they are communicated with the blood sample import by the blood sample microchannel, and are communicated with two solution inlet port by the microchannel network that generates concentration gradient automatically; The microchannel network of described automatic generation concentration gradient comprises some groups of hybrid channels that are arranged in parallel, from first group to last one group, the quantity of the hybrid channel that is comprised increases progressively successively, the inlet of all hybrid channels in every group, and the outlet of all hybrid channels all is connected on the overall channel in last one group, arrange in a triangle two hybrid channels adjacent with this hybrid channel in each hybrid channel and next group, and the overall channel equal in length in the outlet of each hybrid channel and next group between the inlet of two hybrid channels adjacent with this hybrid channel, the outlet correspondence of each hybrid channel is linked described plurality of detection pond in last group, the overall channel of first group of hybrid channel is communicated with described two solution inlet port in two diverse locations, and solution inlet port equates with passage length between the inlet of preceding two hybrid channels in first group, and another solution inlet port equates with passage length between the inlet of latter two hybrid channel in first group.
Described hybrid channel is a serpentine channel, and its shape can be S shape or zigzag or bow font etc., and its function is that two kinds of solution that flow in it are fully mixed.The hybrid channel can be designed to the passage of cross section for shapes such as circle, rectangle, square or triangles, and the passage internal diameter is 20~100 microns, and the degree of depth of detection cell is 20~100 microns.Preferred 30~60 microns of the detection cell degree of depth.
A kind of micro-fluidic chip that is used to check osmotic fragility, comprise chip body, it is characterized in that: two solution inlet port and a blood sample import are set on the described chip body, the plurality of detection pond is set in the chip body, described plurality of detection pond is in-line and arranges, the degree of depth of detection cell is 30~60 microns, and they are communicated with the blood sample import by the blood sample microchannel, and is communicated with two solution inlet port by the microchannel network that generates concentration gradient automatically; The microchannel network of described automatic generation concentration gradient comprises some groups of hybrid channels that are arranged in parallel, from first group to last one group, the quantity of the hybrid channel that is comprised increases progressively successively, the inlet of all hybrid channels in every group, and the outlet of all hybrid channels all is connected on the overall channel in last one group, arrange in a triangle two hybrid channels adjacent with this hybrid channel in each hybrid channel and next group, and the overall channel equal in length in the outlet of each hybrid channel and next group between the inlet of two hybrid channels adjacent with this hybrid channel, the outlet correspondence of each hybrid channel is linked described plurality of detection pond in last group, first group comprises three hybrid channels, the overall channel of first group of hybrid channel is communicated with described two solution inlet port in two diverse locations, and solution inlet port equates with passage length between the inlet of preceding two hybrid channels in first group, and another solution inlet port equates with passage length between the inlet of latter two hybrid channel in first group.
The present invention by the automatic compound concentration gradient of micro-fluidic chip NaCl solution and automatic subpackaging in different detection cells, mix with the blood sample in the detection cell, take pictures with microscope, realize the check of osmotic fragility then by computing machine with image processing method statistics intact cell number.Its whole solution process for preparation is all finished automatically in micro-fluidic chip inside, does not need artificial preparation, has reduced manual intervention, has shortened detection time widely, has reduced the stimulation of external environment pair cell simultaneously, has improved the accuracy that detects.And the blood sample of its use can reduce to micro updating, significantly reduced blood using amount.
Because it utilizes microscope to take pictures, add up the intact cell number by computing machine with image processing method then, further reduced manual intervention, make testing result more objective, accurate, and make detection speed faster.
Description of drawings
Fig. 1,2 is respectively the external structure synoptic diagram and the schematic internal view of micro-fluidic chip among the present invention;
Fig. 3 is the structural representation of the inner microchannel of its micro-fluidic chip network.
Embodiment
The present invention transfers to the related check of erythrocyte fragility on the micro-fluidic chip and carries out, can on chip, make concentration gradient automatically, red blood cell is sent in the detection cell of each variable concentrations, examined under a microscope change of red blood cell, and red blood cell is discerned with image processing method.Dividing method based on gray threshold is a kind of image partition method of classics, and it presses the some classes of gray scale fraction to pixel by threshold value is set, thereby realizes image segmentation.For our erythrogram, only need be divided into cell and the acellular district gets final product, add up the area of cell then, intact cell is counted.Be described further below in conjunction with accompanying drawing.
This may further comprise the steps based on the osmotic fragility method of inspection of micro-fluidic chip:
A, with micro-injection pump with in the different detection cells in the blood sample import input micro-fluidic chip of blood sample by micro-fluidic chip;
B, with micro-injection pump with the NaCl solution of two kinds of variable concentrations with identical flow velocity simultaneously in two solution inlet port input micro-fluidic chips of described micro-fluidic chip, make the NaCl solution of variable concentrations by the microchannel network of the automatic generation concentration gradient in the micro-fluidic chip, and send into respectively in the different detection cells, mix with blood sample in the detection cell;
C, with microscopic examination and take red blood cell photo in each detection cell;
D, handle described red blood cell photo with image processing method, discern complete red blood cell and counting in each detection cell by computing machine.Also can go out macroscopic RBC number in the red blood cell photo by people's number.
In step b, in the NaCl solution of two kinds of variable concentrations that added, a kind of concentration of NaCl solution is 0%~0.25%, and the concentration of another kind of NaCl solution is 0.5%~0.9%.There is a kind of concentration of solution to be 0 in two kinds of solution, that is to say, can add NaCl solution, add pure water from another solution inlet port from a solution inlet port.And the adding mode can be to use a double-channel trace syringe pump (as the KDS 200 of U.S. KDScience company), also can use two single channel micro-injection pumps (as the KDS 100 of U.S. KDScience company), can also use two passages of a hyperchannel micro-injection pump, but no matter which kind of mode of employing, must guarantee to add to two solution inlet port simultaneously, and the flow velocity of two solution inlet port is identical, and flow velocity generally is set in the order of magnitude of 0.1 μ l/min.
In steps d, the image processing method that is adopted comprises: with threshold method the photo of microscope photographing is divided into red blood cell and non-red blood cell zone earlier, adds up complete red blood cell number and calculate the cell size with area-method again.
With reference to Fig. 1-3, micro-fluidic chip comprises the chip body that is made of last substrate 1 and subtegulum 2 in the present embodiment, 3,4 and blood sample imports 5 of two solution inlet port are set on the chip body, eight detection cells 61~68 are set in the chip body, described eight detection cells 61~68 are in-line and arrange formation detection cell group 6, they are communicated with blood sample import 5 by blood sample microchannel 10, and are communicated with two solution inlet port 3,4 by the microchannel network 7 that generates concentration gradient automatically; The microchannel network 7 of described automatic generation concentration gradient comprises six groups of S shape serpentine channel 71~76 that are arranged in parallel, first group of S shape serpentine channel 71 contains three S shape serpentine channel 712,713,714, from first group 71 to last one group 76, the quantity of the S shape serpentine channel that is comprised increases progressively successively, the 6th group of S shape serpentine channel 76 contains eight S shape serpentine channel 762~769, the inlet of all S shape serpentine channel in every group, and the outlet of all S shape serpentine channel all is connected on the overall channel in last one group, as: in the 6th group in the inlet of all S shape serpentine channel 762~769 and the 5th group the outlet of all S shape serpentine channel all be connected on the overall channel 761, two the S shape serpentine channel adjacent with this S shape serpentine channel are arranged in a triangle in each S shape serpentine channel and next group, as wriggle S shape serpentine channel 722 in logical 712 and second groups of: the S shape in first group, 723 arrange in a triangle, S shape in the 5th group S shape serpentine channel 762 in logical 752 and the 6th groups of wriggling, 763 arrange in a triangle, and the passage length in the outlet of each S shape serpentine channel and next group between the inlet of two S shape serpentine channel adjacent with this S shape serpentine channel equates, as wriggle S shape serpentine channel 722 in logical 712 outlet and second group of: the S shape in first group, passage length between 723 the inlet equates, to guarantee flowing into S shape serpentine channel 722 from S shape sinuous logical 712,723 solution equates; The outlet correspondence of each S shape serpentine channel 762~769 is linked described eight detection cells 61~68 in last group, the overall channel 711 of first group of S shape serpentine channel 71 passes through two passages 8 respectively in two diverse locations, 9 are connected to described two solution inlet port 3,4, more particularly, solution inlet port 3 is connected in first group of S shape serpentine channel 71 on the overall channel 711 between preceding two S shape serpentine channel 712 and 713 by passage 8, and the passage length between the inlet of solution inlet port 3 and two S shape serpentine channel 712 and 713 equates, solution inlet port 4 is connected in first group of S shape serpentine channel 71 on the overall channel 711 between latter two S shape serpentine channel 713 and 714 by passage 9, and the passage length between the inlet of solution inlet port 4 and two S shape serpentine channel 713 and 714 equates.
Micro-fluidic chip can also be designed to have detection cell still less among the present invention, perhaps has more detection cell, and correspondingly, the group number that generates the S shape serpentine channel that the microchannel network 7 of concentration gradient comprised automatically also can change.
Described each passage (comprising S shape serpentine channel) can be designed to the passage of cross section for shapes such as circle, rectangle, square or triangles, and the passage internal diameter is 20~100 microns, and the degree of depth of detection cell 61~68 can be designed to 20~100 microns.The degree of depth of detection cell is preferably designed for 30~60 microns.
In the present embodiment, each detection cell also equates to equal in length, the internal diameter of the passage between the blood sample import 5.
Micro-fluidic chip is to be made by substrate 1,2 involutions of two printing opacities, form the micro-groove that constitutes described detection cell and each passage on one of them substrate 1, another substrate 2 is close to opening one side of described micro-groove, behind two substrate involutions the open side of described micro-groove is sealed described microchannel of formation and detection cell.Wherein, substrate 1,2 can adopt PDMS, glass or other macromolecular material to make.
Micro-fluidic chip also can be made by a PDMS substrate and a glass sheet bonding, and wherein the PDMS substrate is made by the microchannel mould, and the microchannel mould adopts the MEMS processing technology to make.A kind of concrete manufacturing process is as follows:
1. draw the design configuration of micro-fluidic chip with computerized mapping software, as shown in Figure 3;
2. making lay photoetching mask plate
Mask material can be selected 5 inches chromium plating glass plates for use, and electron beam exposure is transferred to design configuration on the mask.
3. exposure, development
Clean 4 cun silicon chips, be coated with last layer photoresist (positive glue) on its surface equably, the silicon chip that has photoresist exposes by the transparent part in the mask that is painted with predetermined pattern at a branch of light, and then pattern part has been protected by photoresist.
4. plasma etching (ICP)
High-energy plasma body with air inclusion molecule, free electron and gaseous ion clashes into silicon chip surface, and removes silicon from the surface, and so far, the microchannel mould is made and finished.
5. make the PDMS substrate
PDMS mixes according to mass ratio 10:1 with hardening agent, stir, remove bubble, be poured on the mould of microchannel, place baking oven to solidify about 1 hour back and take out, cool off, take the PDMS of shaping off with 70 degree, punch with boring cutter at passage blood sample injection port and solution inlet port, promptly make the PDMS substrate.
6. encapsulation
The PDMS substrate sticks together with glass sheet after the oxygen gas plasma surface treatment.
Utilize micro-fluidic chip among the present invention can generate the NaCl solution of concentration gradient automatically, and can determine the concrete concentration of NaCl solution in each detection cell, the following describes its principle: with reference to Fig. 3, being provided with concentration and being A and B volume is two kinds of NaCl solution of 1 and enters micro-fluidic chip from two solution inlet port 3,4 respectively, in the shunting of the point of crossing of microchannel, mix with diffusion way in S shape serpentine channel, the total flow of reagent is identical in each serpentine channel
First group of S shape serpentine channel 71, from left to right, the volume of three outlet outflow solution is 2/3, and concentration is respectively A, (A+B)/2, B;
Second group of S shape serpentine channel 72, from left to right, the volume of four outlet outflow solution is 1/2, and concentration is respectively A, (2A+B)/3, and (A+2B)/3, B;
The 3rd group of S shape serpentine channel 73, from left to right, the volume of five outlet outflow solution is 2/5, and concentration is respectively A, (3A+B)/4, (A+B)/2, (A+3B)/4, B;
The 4th group of S shape serpentine channel 74, from left to right, the volume of six outlet outflow solution is 1/3, and concentration is respectively A, (4A+B)/5, (3A+2B)/5, (2A+3B)/5, and (A+4B)/5, B;
The 5th group of S shape serpentine channel 75, from left to right, the volume of seven outlet outflow solution is 2/7, and concentration is respectively A, (5A+B)/6, (2A+B)/3, (A+B)/2, (A+2B)/3, (A+5B)/6, B;
The 6th group of S shape serpentine channel 76, from left to right, the volume of eight outlet outflow solution is 1/4, and concentration is respectively A, (6A+B)/7, (5A+2B)/7, (4A+3B)/7, (3A+4B)/7, (2A+5B)/7, and (A+6B)/7, B.
If from the NaCl solution of a solution inlet port 3 inputs 0.9%, another solution inlet port 4 input pure water, then the NaCl solution concentration of eight detection cells 61~68 is respectively: 0.90%, 0.77%, 0.64%, 0.51%, 0.39%, 0.26%, 0.13% and 0.
Method of inspection check speed of the present invention is fast, and manual intervention is few, has reduced blood using amount simultaneously, and the blood sample consumption can reduce to micro updating.
In the foregoing description, S shape serpentine channel is adopted in the hybrid channel in the micro-fluidic chip, in the reality, can also adopt the serpentine channel of other shapes such as zigzag or bow font, and its function is that two kinds of solution that flow in it are fully mixed.

Claims (10)

1, based on the osmotic fragility method of inspection of micro-fluidic chip, it is characterized in that may further comprise the steps:
A, with micro-injection pump with in the different detection cells in the blood sample import input micro-fluidic chip of blood sample by micro-fluidic chip;
B, with micro-injection pump with the NaCl solution of two kinds of variable concentrations with identical flow velocity simultaneously in two solution inlet port input micro-fluidic chips of described micro-fluidic chip, make the NaCl solution of variable concentrations by the microchannel network of the automatic generation concentration gradient in the micro-fluidic chip, and send into respectively in the different detection cells, mix with blood sample in the detection cell;
C, with microscopic examination and take red blood cell photo in each detection cell;
D, from the red blood cell photo identification each detection cell in complete red blood cell and the counting.
2, the method for inspection according to claim 1 is characterized in that: in step b, in the NaCl solution of two kinds of variable concentrations that added, wherein a kind of concentration of NaCl solution is 0%~0.25%, and the concentration of another kind of NaCl solution is 0.5%~0.9%.
3, the method for inspection according to claim 1 is characterized in that: in steps d, discern complete red blood cell and counting in each detection cell by computing machine with image processing method, perhaps people's number goes out macroscopic RBC number in the red blood cell photo; Wherein, may further comprise the steps: with threshold method the photo of microscope photographing is divided into red blood cell and non-red blood cell zone earlier, again with area-method statistics red blood cell number and calculating cell size with the method for computing machine with image processing method identification and counting.
4, according to each described method of inspection of claim 1-3, it is characterized in that: described micro-fluidic chip comprises chip body, two solution inlet port and a blood sample import are set on the chip body, the plurality of detection pond is set in the chip body, described plurality of detection pond is in-line and arranges, they are communicated with the blood sample import by the blood sample microchannel, and are communicated with two solution inlet port by the microchannel network that generates concentration gradient automatically; The microchannel network of described automatic generation concentration gradient comprises some groups of hybrid channels that are arranged in parallel, from first group to last one group, the quantity of the hybrid channel that is comprised increases progressively successively, the inlet of all hybrid channels in every group, and the outlet of all hybrid channels all is connected on the overall channel in last one group, arrange in a triangle two hybrid channels adjacent with this hybrid channel in each hybrid channel and next group, and the passage length in the outlet of each hybrid channel and next group between the inlet of two hybrid channels adjacent with this hybrid channel equates, the outlet correspondence of each hybrid channel is linked described plurality of detection pond in last group, the overall channel of first group of hybrid channel is communicated with described two solution inlet port in two diverse locations, and solution inlet port equates with passage length between the inlet of preceding two hybrid channels in first group, and another solution inlet port equates with passage length between the inlet of latter two hybrid channel in first group.
5, the method for inspection according to claim 4 is characterized in that: described hybrid channel is a serpentine channel, and it is shaped as S-shaped or zigzag or bow font.
6, the method for inspection according to claim 4 is characterized in that: the cross section of described hybrid channel is circle, rectangle, square or triangle, and the passage internal diameter is 20~100 microns, and the degree of depth of detection cell is 20~100 microns.
7, the method for inspection according to claim 4 is characterized in that: the cross section of described hybrid channel is circle, rectangle, square or triangle, and the passage internal diameter is 30~60 microns, and the degree of depth of described detection cell is 30~60 microns.
8, the method for inspection according to claim 4 is characterized in that: each detection cell also equates to equal in length, the internal diameter of the passage between the blood sample import.
9, the method for inspection according to claim 4, it is characterized in that: described micro-fluidic chip is to be made by PDMS substrate and glass sheet bonding, wherein the PDMS substrate is made by the microchannel mould, and the microchannel mould adopts the MEMS processing technology to make.
10, a kind of micro-fluidic chip that is used to check osmotic fragility, comprise chip body, it is characterized in that: two solution inlet port and a blood sample import are set on the described chip body, the plurality of detection pond is set in the chip body, described plurality of detection pond is in-line and arranges, the degree of depth of detection cell is 30~60 microns, and they are communicated with the blood sample import by the blood sample microchannel, and is communicated with two solution inlet port by the microchannel network that generates concentration gradient automatically; The microchannel network of described automatic generation concentration gradient comprises some groups of hybrid channels that are arranged in parallel, from first group to last one group, the quantity of the hybrid channel that is comprised increases progressively successively, the inlet of all hybrid channels in every group, and the outlet of all hybrid channels all is connected on the overall channel in last one group, arrange in a triangle two hybrid channels adjacent with this hybrid channel in each hybrid channel and next group, and the overall channel equal in length in the outlet of each hybrid channel and next group between the inlet of two hybrid channels adjacent with this hybrid channel, the outlet correspondence of each hybrid channel is linked described plurality of detection pond in last group, first group comprises three hybrid channels, the overall channel of first group of hybrid channel is communicated with described two solution inlet port in two diverse locations, and solution inlet port equates with passage length between the inlet of preceding two hybrid channels in first group, and another solution inlet port equates with passage length between the inlet of latter two hybrid channel in first group.
CN 200810218355 2008-12-12 2008-12-12 Micro-flow controlled chip used for detecting erythrocyte osmotic fragility Expired - Fee Related CN101464458B (en)

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