CN103464230B - Centrifugal whole blood analysis micro-fluidic chip, preparation method as well as application method thereof - Google Patents
Centrifugal whole blood analysis micro-fluidic chip, preparation method as well as application method thereof Download PDFInfo
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Abstract
The invention relates to a centrifugal micro-fluidic chip based on the Euler force assisting effect, a preparation method as well as an application method thereof. The invention aims to provide a polymer centrifugal micro-fluidic chip based on Euler force suffered by liquid in accelerated rotation of a chip, a preparation method and an application method thereof. The integration of multiple functions of blood separation, serum extraction, serum quantification, reagent prepackaging, reagent extraction, reagent quantification, serum and reagent mixing, and mixed liquid distribution is realized; the dependence on surface tension in polymer centrifugal micro-fluidic chip surface treatment and flow control is avoided; the functional reliability and long-time stability of the chip are ensured. The centrifugal whole blood analysis micro-fluidic chip, the preparation method as well as the application method thereof, which are provided by the invention, have the advantages of high integration level, simple technology, high reliability and easiness in implementation.
Description
Technical field
The present invention relates to based on the centrifugal type microfludic chip of Euler force booster action, preparation method and application process thereof.
Background technology
Micro-fluidic chip is the hot fields of current micro-total analysis system development, is the main platform that microflow control technique realizes.Resulting structure (microchannel, micro-valve, reaction chamber and other functional part) size of its device characteristic mainly its containing fluid is in micron dimension.Due to micron-sized structure, fluid shows wherein and creates the property different from macro-scale, therefore develops unique analytical performance.Its application purpose produced is the ultimate aim realizing micro-total analysis system---chip lab.At present, the emphasis application of micro-fluidic chip development is life science.Microfluidic chip technology has the advantages such as high flux, microminiaturization, automation, cost be low, anti-pollution compared with traditional instrument detection method.Micro-fluidic chip is divided into active and passive type two kinds.Passive type chip is mainly made up of High Density Molecular microarray, comprises DNA chip, peptide chips etc., is applied in gene structure and functional study, is also biochip the most general at present.But there is the shortcomings such as complicated operation, probe synthetic work amount is large, with high costs, monolithic chip function is more single in this kind of chip.Active chip is then based on various structure microarray, introduces the effect of external force field, thus the examination and analysb of the process such as cell separation, chemical reaction and biological information efficiently, is rapidly carried out automatically on sheet in the structure and application of chip.Compared with the former, active chip is unique on 26S Proteasome Structure and Function, can realize the function of chip lab, process integration, and it develops and studies noticeable.It is adopt Micropump to provide drive source for liquid flow that chip introduces one of method of the effect of external force field, but Micropump exists the shortcoming of integrated difficulty on chip.And on centrifugal type microfludic chip, centrifugal force can realize the driving of microfluidic, thus efficiently avoid the integrated difficulty brought of Micropump.Micro-fluidic chip requires to realize each microfluidic cell (microchannel, micro-valve and liquid storage cavity) and corresponding function is integrated.The essential characteristic of micro-fluidic chip and sharpest edges are by integrated to various monotechnics flexible combination, scale on the small platform that entirety is controlled.
At present, polymer is the micro-fluidic chip rapidoprint (as: PC, PMMA) generally adopted.On the micro-fluidic chip of polymeric material, the processing method of micro-fluidic structure comprises hot pressing, Laser Processing etc.Wherein, laser processing has high, little to the constraint of the chip structure design advantage of efficiency.On polymer centrifugal type microfludic chip, blood separation, serum are extracted and occurs in quantitative vital siphon valve to need spontaneous capillary flow occurs before siphon is flowed, is full of siphonal to realize liquid; And except centrifugal force, in chip, the control of microfluidic adopts the surface tension strong to material surface character dependence more, and corresponding micro-fluidic device comprises capillary Passive microvalve.But surface of polymer material character mostly is hydrophobic and unstable.Therefore, polymer centrifugal type microfludic chip often needs surface treatment.The problem of current existing process for treating surface ubiquity long-time stability aspect, and material surface process can cause the difficulty of chip manufacture, assembling aspect.
Reagent is pre-packaged, be separated, extract, quantitatively, mixing and Liquid distribution be the household function that micro-fluidic chip is applied at portable analysis detection field.The above-mentioned each function of development need of current microfluidic chip technology integrated, thus provide a kind of technology platform for the various functions that replace conventional chemical or biology laboratory.
Summary of the invention
The object of the invention is the polymer centrifugal type microfludic chip of a kind of applicable whole blood test of Euler force proposition be subject to based on liquid during chip acceleration rotation, preparation method and application process thereof, achieve that blood is separated, serum extracts, serum is quantitative, reagent is pre-packaged, reagent extractions, reagent quantitative, serum mix with actual, mixing material distributes the integrated of several functions, and avoid the dependence of effects on surface tension force in the surface treatment of polymer centrifugal type microfludic chip and flowing control, ensure that functional reliability and the long-time stability of chip.
Centrifugal whole blood micro-fluidic chip, comprise two functional units, each functional unit comprises reagent inlet opening, the pre-packaged chamber of reagent, reagent fluid hole, reagent sample introduction microchannel, reagent quantitative chamber, reagent waste liquid chamber, reagent siphon pipe, blood sample holes, blood sample cavity, blood sample introduction microchannel, blood disengagement chamber, blood waste liquid chamber, serum siphon pipe, hybrid chamber, mixing material siphon pipe, mixing material distribution cavity, mixing material waste liquid chamber and passage; Also comprise package strip, by the two ends inflection of package strip, and be pasted onto on reagent fluid hole by the two ends of inflection, pre-packaged reagent is injected in the pre-packaged chamber of reagent through reagent inlet opening;
Described blood sample cavity is connected with blood sample holes and passage A; Blood sample cavity is connected with blood disengagement chamber by blood sample introduction microchannel, and the junction of described blood sample introduction microchannel and blood disengagement chamber arranges expansive valve A; The both sides of blood disengagement chamber are connected with blood waste liquid chamber and serum siphon pipe respectively; The siphonal end of described serum is connected with hybrid chamber; On described hybrid chamber, one end siphonal with reagent, the siphonal one end of mixing material and passage B are connected respectively; The siphonal other end of reagent is connected with reagent quantitative chamber; Described reagent quantitative chamber is connected with reagent waste liquid chamber and reagent sample introduction microchannel, arranges expansive valve B in the junction in reagent sample introduction microchannel and reagent quantitative chamber; The siphonal sidewall of described mixing material connects multiple mixing material distribution cavity; The siphonal other end of mixing material connects mixing material waste liquid chamber.
The preparation method of centrifugal whole blood micro-fluidic chip, adopts six PMMA disks to paste successively and makes; Concrete steps are:
Step one, employing laser engraving machined PMMA sheet material, obtain a PMMA disk, a described PMMA disk be processed with passage and the blood sample holes of microcontroller chip; Adopt laser engraving machined one side with the PMMA sheet material of pressure sensitive adhesive, obtain the 2nd PMMA disk, the 3rd PMMA disk, the 4th PMMA disk, the 5th PMMA disk and the 6th PMMA disk; Described 2nd PMMA disk is processed to form micro-fluidic structure and the passage corresponding with a PMMA disk and blood sample holes; 3rd PMMA disk is formed the reagent fluid hole in the pre-packaged chamber of reagent; 4th PMMA disk is formed the pre-packaged chamber of reagent; 5th PMMA disk is formed passage and the reagent inlet opening in the pre-packaged chamber of reagent;
Step 2, by the two ends inflection of package strip, then two inflection ends to be pasted on the 3rd PMMA disk with the side of pressure sensitive adhesive, the reagent fluid hole in pre-packaged for reagent chamber to be closed simultaneously;
Step 3, a PMMA disk and the 2nd PMMA disk one side with pressure sensitive adhesive to be pasted; The another side of the 2nd PMMA disk and the 3rd PMMA disk are pasted with the one side of pressure sensitive adhesive; The another side of the 3rd PMMA disk and the 4th PMMA disk are pasted with the one side of pressure sensitive adhesive; The another side of the 4th PMMA disk and the 5th PMMA disk are pasted with the one side of pressure sensitive adhesive;
Step 4, pre-packaged reagent to be injected by the reagent inlet opening on the 5th PMMA disk, then the another side of the 5th PMMA disk and the 6th PMMA disk are pasted with the side of pressure sensitive adhesive, the reagent realized on micro-fluidic chip is pre-packaged, completes the making of centrifugal whole blood micro-fluidic chip.
The application process of centrifugal whole blood micro-fluidic chip, the method is realized by following steps:
Steps A, micro-fluidic chip to be arranged on centrifugal arbor, to be specially: by centrifugal arbor through the installing hole on chip; Through in process, centrifugal arbor exerts a force to package strip, opens reagent fluid hole after making the two ends tension of package strip inflection;
Step B, by blood sample holes, blood is injected in blood sample cavity, start centrifuge, stop after micro-fluidic chip being rotated 120 seconds with the rotating speed of 3000 revs/min, reagent reagent being sealed in chamber in advance by centrifugal force flows into reagent quantitative chamber by reagent fluid hole, blood simultaneously in blood sample cavity flows into blood disengagement chamber, completes the separation quantitatively and blood disengagement chamber in blood of reagent quantitative chamber to reagent;
Step C, with the angular acceleration of 2000 revs/min of seconds start centrifuge, continue rotation after making rotating speed reach 2000 revs/min to stop after 30 seconds, respectively by serum siphon pipe and reagent siphon pipe inflow hybrid chamber under the Euler force effect making the serum in the blood disengagement chamber in step 2 and the reagent in reagent quantitative chamber produce at centrifuge acceleration;
Step D, startup centrifuge accelerate to after centrifuge reaches the rotating speed of 3000 revs/min with the angular acceleration of 3000 revs/min of seconds and stop, and return multiple circulation N time, the serum in completing steps C in hybrid chamber mixes with the concussion of reagent, obtains mixing material;
Step e, to start centrifuge to centrifuge with the angular acceleration of 6000 revs/min of seconds and reach after the rotating speed of 6000 revs/min and continue rotation 30 seconds, multiple mixing material distribution cavity is flowed into successively under the Euler force effect that mixing material in hybrid chamber is produced when centrifuge accelerates, unnecessary mixing material flows into mixing material waste liquid chamber, finally completes the whole blood function to micro-fluidic chip.
Beneficial effect of the present invention: the present invention, in order to ensure functional reliability for the polymer centrifugal type microfludic chip of whole blood test and long-time stability, proposes the centrifugal whole blood micro-fluidic chip and preparation method thereof based on Euler force booster action.In the present invention, in chip, microfluidic will be broken away from material surface character and the capillary dependence of liquid level, the substitute is the Euler force perpendicular to chip radial direction that chip accelerates to be subject to when rotating, thus effectively avoid the dependence of effects on surface tension force in the surface treatment of polymer centrifugal type microfludic chip and flowing control, and ensure functional reliability and the long-time stability of chip; And the blood separation of institute's invention integrated chip, serum extracts, serum is quantitative, reagent is pre-packaged, reagent extractions, reagent quantitative, serum mix with actual, mixing material distributes several functions.The present invention has that integrated level is high, technique is simple, and reliability is high, is easy to the advantage realized.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of a PMMA disk in centrifugal whole blood micro-fluidic chip of the present invention, preparation method and application process;
Fig. 2 is the schematic diagram of the 2nd PMMA disk in centrifugal whole blood micro-fluidic chip of the present invention, preparation method and application process;
Fig. 3 is the schematic diagram of the 3rd PMMA disk in centrifugal whole blood micro-fluidic chip of the present invention, preparation method and application process;
Fig. 4 is the schematic diagram of the 4th PMMA disk in centrifugal whole blood micro-fluidic chip of the present invention, preparation method and application process;
Fig. 5 is the schematic diagram of the 5th PMMA disk in centrifugal whole blood micro-fluidic chip of the present invention, preparation method and application process;
Fig. 6 is the schematic diagram of the 6th PMMA disk in centrifugal whole blood micro-fluidic chip of the present invention, preparation method and application process;
Fig. 7 is the schematic diagram of the package strip of two ends inflection in centrifugal whole blood micro-fluidic chip of the present invention, preparation method and application process;
Fig. 8 is the centrifugal whole blood micro-fluidic chip schematic diagram that a PMMA disk to the 6th PMMA disk pastes formation successively;
Fig. 9 is the pre-packaged and schematic diagram of chip of the reagent opened in centrifugal whole blood micro-fluidic chip by the effect of centrifugal arbor to package strip;
In Figure 10, a ~ g is respectively process schematic fluid operated in centrifugal type microfludic chip whole blood process of the present invention.
Detailed description of the invention
Detailed description of the invention one, composition graphs 1 to Fig. 8 illustrate present embodiment, centrifugal whole blood micro-fluidic chip, by a PMMA disk, the 2nd PMMA disk B, package strip, the 3rd PMMA disk D, the 4th PMMA disk E, the 5th PMMA disk F and the 6th PMMA disk G, pasted by pressure sensitive adhesive successively and form, pre-packaged reagent is before stickup the 6th PMMA disk G, package strip C is injected by reagent inlet opening 19, 3rd PMMA disk D, 4th PMMA disk E and the 5th PMMA disk F pastes the pre-packaged chamber 18 of reagent formed, the stickup of last 6th PMMA disk G completes closing reagent inlet opening 19, comprise two functional units, each functional unit comprises reagent inlet opening 19, the pre-packaged chamber 18 of reagent, reagent fluid hole 17, reagent sample introduction microchannel 9, reagent quantitative chamber 12, reagent waste liquid chamber 10, reagent siphon pipe 16, blood sample holes 3, blood sample cavity 4, blood sample introduction microchannel 6, expansive valve A, blood disengagement chamber 7, blood waste liquid chamber 5, serum siphon pipe 8, hybrid chamber 14, mixing material siphon pipe 15, mixing material distribution cavity 13, mixing material waste liquid chamber 11, and passage several.
Described blood sample cavity 4 is connected with blood sample holes 3 and passage A; Blood sample cavity 4 is connected with blood disengagement chamber 7 by blood sample introduction microchannel 6, and described blood sample introduction microchannel 6 arranges expansive valve A with the junction of blood disengagement chamber 7; Be connected with serum siphon pipe 8 in blood waste liquid chamber 5 respectively in blood disengagement chamber 7 both sides; The end of described serum siphon pipe 8 is connected with hybrid chamber 14; Described hybrid chamber 14 is connected with one end of reagent siphon pipe 16, one end of mixing material siphon pipe 15 and passage B respectively; One end of reagent siphon pipe 16 is connected with reagent quantitative chamber 12; Described reagent quantitative chamber 12 is connected with reagent waste liquid chamber 10 and reagent sample introduction microchannel 9, arranges expansive valve B in reagent sample introduction microchannel 9 and the junction in reagent quantitative chamber 12; The sidewall of described mixing material siphon pipe 15 connects multiple mixing material distribution cavity 13; The other end of mixing material siphon pipe 15 connects mixing material waste liquid chamber 11.
Detailed description of the invention two, composition graphs 1 to Fig. 8 illustrate present embodiment, and present embodiment is the preparation method of the centrifugal whole blood micro-fluidic chip described in detailed description of the invention one: its detailed process is:
One, by laser engraving machined PMMA sheet material, obtain a PMMA wafer A, composition graphs 1, with the through hole that several obtain by cutting threading material on it, gained through hole is for the formation of the passage of micro-fluidic chip, locating hole 1 and installing hole 2;
Two, by the PMMA sheet material of laser engraving machined one side with pressure sensitive adhesive, obtain the 2nd PMMA disk B, composition graphs 2, with by cutting the micro-fluidic structure that threading material obtains on it, comprise blood sample cavity 4, blood waste liquid chamber 5,6, two, blood sample introduction microchannel expansive valve, two passages, blood disengagement chamber 7, serum siphon pipe 8, reagent sample introduction microchannel 9, reagent waste liquid chamber 10, mixing material waste liquid chamber 11, reagent siphon pipe 16, reagent quantitative chamber 12, mixing material siphon pipe 15, hybrid chamber 14, mixing material distribution cavity 13;
Three, by the PMMA sheet material of laser engraving machined one side with pressure sensitive adhesive, obtain the 3rd PMMA disk D, composition graphs 3, with by cutting the through hole that threading material obtains on it, gained through hole is for the formation of the installing hole 2 of the reagent fluid hole 17 in the pre-packaged chamber of reagent, locating hole 1 and micro-fluidic chip;
Four, by the PMMA sheet material of laser engraving machined one side with pressure sensitive adhesive, obtain the 4th PMMA disk E, composition graphs 4, with by cutting the through hole that threading material obtains on it, gained through hole is for the formation of the installing hole 2 of the pre-packaged chamber 18 of reagent, micro-fluidic chip and locating hole 1;
Five, by the PMMA sheet material of laser engraving machined one side with pressure sensitive adhesive, obtain the 5th PMMA disk F, composition graphs 5, with by cutting the through hole that threading material obtains on it, gained through hole is for the formation of the installing hole 2 of the passage in the pre-packaged chamber of reagent, reagent inlet opening 19 and micro-fluidic chip and locating hole 1;
Six, by laser engraving machined one side with the PMMA sheet material (thickness 0.8mm) of pressure sensitive adhesive, obtain the 6th PMMA disk G, composition graphs 6, on it, through hole is for the formation of the installing hole 2 of locating hole 1 and micro-fluidic chip;
Seven, by package strip C, (kraft paper strip with PET film) two ends inflection, composition graphs 7, is then pasted on the side with pressure sensitive adhesive on the 3rd PMMA disk D, the reagent fluid hole 17 on the 3rd PMMA disk is closed by the two inflection end M of package strip C by the two ends M of inflection;
Eight, located by locating hole, a PMMA wafer A and the 2nd PMMA disk B are pasted with the side of pressure sensitive adhesive;
Nine, located by locating hole, the 2nd PMMA disk B and the 3rd PMMA disk D is pasted with the side of pressure sensitive adhesive;
Ten, by the pre-packaged chamber of reagent on the 4th PMMA disk E and installing hole location, the 3rd PMMA disk D and the 4th PMMA disk E is pasted with the side of pressure sensitive adhesive;
11, located by locating hole, the 4th PMMA disk E and the 5th PMMA disk F is pasted with the side of pressure sensitive adhesive;
12, inject appropriate reagent by the inlet opening on the 5th PMMA disk F, then by locating hole location 1, the 5th PMMA disk F and the 6th PMMA disk G is pasted with the side of pressure sensitive adhesive, thus the reagent completed on micro-fluidic chip is pre-packaged.
A PMMA wafer A described in present embodiment, 3rd PMMA disk D, the thickness of the 5th PMMA disk F and the 6th PMMA disk G is 0.8mm, 2nd PMMA disk B, the thickness of the 4th PMMA disk E is 1mm, one PMMA wafer A, 2nd PMMA disk B, the diameter of the 3rd PMMA disk D is 11cm, 4th PMMA disk E, 5th PMMA disk F, the diameter of the 6th PMMA disk G is 4cm, on each disk, the size of through hole is respectively: passage diameter is 1mm, the diameter 2mm of reagent inlet opening 19, reagent fluid hole 17 diameter 2mm, the diameter 1cm in the pre-packaged chamber 18 of reagent, the diameter 2mm of locating hole 1, installing hole 2 diameter 8mm in one PMMA wafer A, installing hole diameter 6mm on all the other disks, the width of serum siphon pipe 8 and mixing material siphon pipe 15 is 600 μm.Adopt pressure sensitive adhesive thickness to be 250 μm.
Detailed description of the invention three, composition graphs 9 and Figure 10 illustrate present embodiment, and present embodiment is the application process of the centrifugal whole blood micro-fluidic chip described in detailed description of the invention one, and the method is realized by following steps:
Steps A, centrifugal whole blood micro-fluidic chip to be installed on as chip provides on the centrifugal arbor H of centrifugal force.Be fitted through axle to realize through the installing hole on chip; Through in process, centrifugal arbor H, to package strip C force, makes the inflection end M tension of package strip C, and then opens the reagent fluid hole 17 on the 3rd PMMA disk D;
Step B, by blood sample holes 3, blood is injected in blood sample cavity 4, start centrifuge I, stop after the micro-fluidic chip be installed on centrifugal arbor H being rotated 120 seconds with the rotating speed of 3000 revs/min, thus make the reagent in the pre-packaged chamber 18 of reagent flow into reagent quantitative chamber 12 by reagent fluid hole 17 by centrifugal force, and make blood in blood sample cavity 4 flow into blood disengagement chamber 7, and complete the 12 pairs of reagent in reagent quantitative chamber quantitatively with being separated of blood disengagement chamber 7 inner blood;
Step C, to start with the 2000 turns/angular acceleration of (every minute and second) and continue rotation after centrifuge I reaches 2000 revs/min to rotating speed and stop after 30 seconds, and then make the reagent in the serum in blood disengagement chamber 7 and reagent quantitative chamber 12 flow into hybrid chamber 14 respectively by serum siphon pipe 8 and reagent siphon pipe 16 under the Euler force effect produced with centrifuge I acceleration;
Step D, start after centrifuge I accelerates to the rotating speed of 3000 revs/min with the 3000 turns/angular acceleration of (every minute and second) and be decelerated to stopping, so circulation 5 times, mixes with the concussion of reagent to complete serum in hybrid chamber 14;
Step e, to start centrifuge I with the 6000 turns/angular acceleration of (every minute and second) continue rotation 30 seconds to the rotating speed reaching 6000 revs/min, thus flow into five reagent distribution cavity 13 successively under the Euler force effect that the mixing material in hybrid chamber 14 is produced when accelerating, unnecessary mixing material finally flows into mixing material waste liquid chamber 11, and then completes the whole blood function of chip.
In conjunction with Figure 10, present embodiment is described further, in blood sample cavity 4, appropriate blood is injected by blood sample holes 3, there is size and expand and form expansive valve A in blood sample introduction microchannel 6 and blood disengagement chamber 7 junction, blood ends in blood sample cavity 4 by this expansive valve A, with prevent chip rotate before blood enter blood disengagement chamber 7, composition graphs 10a, wherein, line X blood, line Y is pre-packaged reagent; Then, open centrifuge I, for chip provides rotating speed, the reagent in the pre-packaged chamber 18 of reagent and the blood in blood sample cavity 4 is made to enter reagent quantitative chamber 12 and blood disengagement chamber 7 under the influence of centrifugal force respectively, and after the quantitative chamber 12 of candidate agent and blood disengagement chamber 7 be filled respectively, unnecessary reagent and blood spill in reagent waste liquid chamber 10 and blood waste liquid chamber 5 respectively, composition graphs 10b; Continue rotary chip, the blood in blood disengagement chamber 7 is separated under the influence of centrifugal force, composition graphs 10c; After the blood in blood disengagement chamber 7 is separated completely, accelerate rotary chip, for chip provides angular acceleration, thus make liquid in chip by the Euler force perpendicular to chip radial direction, and then make the portion of reagent in reagent quantitative chamber 12 continue to flow into reagent siphon pipe 16, and be full of reagent siphon pipe 16, the serum simultaneously in blood disengagement chamber 7 also continues to flow into serum siphon pipe 8 under Euler force effect, and be full of serum siphon pipe 8, composition graphs 10d; There is siphon flowing in the chip being full of for lasting rotation of reagent siphon pipe 16 and serum siphon pipe 8 and provide condition, thus under the influence of centrifugal force, reagent in reagent quantitative chamber 12 and blood disengagement chamber 7 and serum, respectively by the siphon flowing in reagent siphon pipe 16 and serum siphon pipe 8, continue to flow in hybrid chamber 14, until siphon flowing stops when the liquid level in reagent quantitative chamber 12 and blood disengagement chamber 7 arrives the porch of reagent siphon pipe 16 and serum siphon pipe 8, composition graphs 10e; Then, concussion rotary chip, makes liquid in hybrid chamber 14 under the effect of coriolis force and Euler force, concussion flowing, thus completes the mixing of reagent and serum in hybrid chamber 14, composition graphs 10f; At acceleration rotary chip, the mixing material in hybrid chamber 14 is made to be subject to Euler force effect, and be full of mixing material siphon pipe, and continue rotary chip, make siphon flowing occurs in mixing material siphon pipe, thus make the mixing material in hybrid chamber 14 flow in mixing material distribution microchannel, then each mixing material distribution cavity 13 is flowed into successively, unnecessary mixing material overflows last distribution cavity and flows into mixing material waste liquid chamber 11, and then complete the whole flowing controlling functions of invention chip, composition graphs 10g.
Claims (7)
1. centrifugal whole blood micro-fluidic chip, comprise two functional units, each functional unit comprises reagent inlet opening (19), the pre-packaged chamber of reagent (18), reagent fluid hole (17), reagent sample introduction microchannel (9), reagent quantitative chamber (12), reagent waste liquid chamber (10), reagent siphon pipe (16), blood sample holes (3), blood sample cavity (4), blood sample introduction microchannel (6), blood disengagement chamber (7), blood waste liquid chamber (5), serum siphon pipe (8), hybrid chamber (14), mixing material siphon pipe (15), mixing material distribution cavity (13), mixing material waste liquid chamber (11) and passage, it is characterized in that, also comprise package strip (C), by the two ends inflection of package strip (C), and two inflection ends (M) are pasted onto on reagent fluid hole (17), pre-packaged reagent is injected in the pre-packaged chamber of reagent (18) through reagent inlet opening (19),
Described blood sample cavity (4) is connected with blood sample holes (3) and passage A (c); Blood sample cavity (4) is connected with blood disengagement chamber (7) by blood sample introduction microchannel (6), and described blood sample introduction microchannel (6) arranges expansive valve A (a) with the junction of blood disengagement chamber (7); The both sides of blood disengagement chamber (7) are connected with blood waste liquid chamber (5) and serum siphon pipe (8) respectively; The end of described serum siphon pipe (8) is connected with hybrid chamber (14); Described hybrid chamber (14) is connected with one end of reagent siphon pipe (16), one end of mixing material siphon pipe (15) and passage B (d) respectively; The other end of reagent siphon pipe (16) is connected with reagent quantitative chamber (12); Described reagent quantitative chamber (12) is connected with reagent waste liquid chamber (10) and reagent sample introduction microchannel (9), arranges expansive valve B (b) in reagent sample introduction microchannel (9) and the junction in reagent quantitative chamber (12); The sidewall of described mixing material siphon pipe (15) connects multiple mixing material distribution cavity (13); The other end of mixing material siphon pipe (15) connects mixing material waste liquid chamber (11).
2. make the method for centrifugal whole blood micro-fluidic chip according to claim 1, it is characterized in that, adopt six PMMA disks to paste successively and make; Concrete steps are:
Step one, employing laser engraving machined PMMA sheet material, obtain a PMMA disk (A), a described PMMA disk (A) be processed with passage and the blood sample holes (3) of micro-fluidic chip; Adopt laser engraving machined one side with the PMMA sheet material of pressure sensitive adhesive, obtain the 2nd PMMA disk (B), the 3rd PMMA disk (D), the 4th PMMA disk (E), the 5th PMMA disk (F) and the 6th PMMA disk (G); Described 2nd PMMA disk (B) is processed to form micro-fluidic structure and the passage corresponding with a PMMA disk (A) and blood sample holes (3); At the upper reagent fluid hole (17) forming the pre-packaged chamber of reagent (18) of the 3rd PMMA disk (D); At the 4th PMMA disk (E) the upper formation pre-packaged chamber of reagent (18); At upper passage and the reagent inlet opening (19) forming the reagent pre-packaged chamber (18) corresponding with a PMMA disk (A) of the 5th PMMA disk (F);
Step 2, by the two ends inflection of package strip (C), then the two ends of inflection are pasted on the upper side with pressure sensitive adhesive of the 3rd PMMA disk (D), the reagent fluid hole (17) of pre-packaged for reagent chamber (18) are closed simultaneously;
Step 3, a PMMA disk (A) and the 2nd PMMA disk (B) to be pasted with the one side of pressure sensitive adhesive; The another side of the 2nd PMMA disk (B) and the 3rd PMMA disk (D) are pasted with the one side of pressure sensitive adhesive; The another side of the 3rd PMMA disk (D) and the 4th PMMA disk (E) are pasted with the one side of pressure sensitive adhesive; The another side of the 4th PMMA disk (E) and the 5th PMMA disk (F) are pasted with the one side of pressure sensitive adhesive;
Step 4, pre-packaged reagent to be injected by the reagent inlet opening (19) on the 5th PMMA disk (F), then the another side of the 5th PMMA disk (F) and the 6th PMMA disk (G) are pasted with the side of pressure sensitive adhesive, the reagent realized on micro-fluidic chip is pre-packaged, completes the making of centrifugal whole blood micro-fluidic chip.
3. the preparation method of centrifugal whole blood micro-fluidic chip according to claim 2, it is characterized in that, a described PMMA disk (A), 2nd PMMA disk (B), 3rd PMMA disk (D), 4th PMMA disk (E), 5th PMMA disk (F) and the 6th PMMA disk (G) are processed with installing hole (2) and locating hole (1) respectively, and installing hole (2) on each disk is corresponding, a described PMMA disk (A), locating hole (1) on 2nd PMMA disk (B) and the 3rd PMMA disk (D) is corresponding respectively, 4th PMMA disk (E), locating hole (1) on 5th PMMA disk (F) and the 6th PMMA disk (G) is corresponding respectively.
4. the preparation method of centrifugal whole blood micro-fluidic chip according to claim 2, it is characterized in that, a described PMMA disk (A), 3rd PMMA disk (D), 5th PMMA disk (F) is identical with the thickness of the 6th PMMA disk (G), 2nd PMMA disk (B) is identical with the thickness of the 4th PMMA disk (E), a described PMMA disk (A), 2nd PMMA disk (B) is identical with the diameter of the 3rd PMMA disk (D), 4th PMMA disk (E), 5th PMMA disk (F) is identical with the diameter of the 6th PMMA disk (G).
5. the preparation method of centrifugal whole blood micro-fluidic chip according to claim 2, it is characterized in that, describedly on the 2nd PMMA disk (B), be processed to form micro-fluidic structure specifically comprise: the blood sample cavity (4) be connected with blood disengagement chamber (7) by blood sample introduction microchannel (6), the blood sample holes (3) be connected with blood sample cavity (4) and passage A (c); Be arranged on expansive valve A (a) of blood sample introduction microchannel (6) and blood disengagement chamber (7) junction; The blood waste liquid chamber (5) be connected respectively with blood disengagement chamber (7) both sides and serum siphon pipe (8); Be connected to the hybrid chamber (14) of serum siphon pipe (8) end; The reagent siphon pipe (16) be connected with hybrid chamber (14), mixing material siphon pipe (15) and passage B (d); Reagent quantitative chamber (12) is connected with reagent siphon pipe (16) end; The reagent waste liquid chamber (10) be connected with reagent quantitative chamber (12) and reagent sample introduction microchannel (9), be arranged on expansive valve B (b) of reagent sample introduction microchannel (9) and reagent quantitative chamber (12) junction; The multiple mixing material distribution cavity (13) connected at mixing material siphon pipe (15) sidewall and be connected to the mixing material waste liquid chamber (11) of mixing material siphon pipe (15) end.
6. application rights requires the method for the centrifugal whole blood micro-fluidic chip described in 1, and it is characterized in that, the method is realized by following steps:
Steps A, micro-fluidic chip to be arranged on centrifugal arbor, to be specially: by centrifugal arbor through the installing hole (2) on chip; Through in process, centrifugal arbor, to package strip (C) force, opens reagent fluid hole (17) after making two inflection end (M) tensions of package strip (C);
Step B, by blood sample holes (3), blood is injected in blood sample cavity (4), start centrifuge, stop after micro-fluidic chip being rotated 120 seconds with the rotating speed of 3000 revs/min, reagent reagent being sealed in chamber in advance by centrifugal force flows into reagent quantitative chamber (12) by reagent fluid hole (17), blood simultaneously in blood sample cavity (4) flows into blood disengagement chamber (7), completes the separation quantitatively and blood disengagement chamber (7) in blood of reagent quantitative chamber (12) to reagent;
Step C, with the angular acceleration of 2000 revs/min of seconds start centrifuge, continue rotation after making rotating speed reach 2000 revs/min to stop after 30 seconds, respectively by serum siphon pipe (8) and reagent siphon pipe (16) inflow hybrid chamber (14) under the Euler force effect making the serum in the blood disengagement chamber (7) in step 2 and the reagent in reagent quantitative chamber (12) produce at centrifuge acceleration;
Step D, startup centrifuge accelerate to after centrifuge reaches the rotating speed of 3000 revs/min with the angular acceleration of 3000 revs/min of seconds and stop, and return multiple circulation N time, the serum in completing steps C in hybrid chamber (14) mixes with the concussion of reagent, obtains mixing material;
Step e, to start centrifuge to centrifuge with the angular acceleration of 6000 revs/min of seconds and reach after the rotating speed of 6000 revs/min and continue rotation 30 seconds, multiple mixing material distribution cavity (13) is flowed into successively under the Euler force effect that mixing material in hybrid chamber (14) is produced when centrifuge accelerates, unnecessary mixing material flows into mixing material waste liquid chamber (11), finally completes the whole blood function to micro-fluidic chip.
7. the application process of centrifugal whole blood micro-fluidic chip according to claim 6, is characterized in that, the value of the N in step D is 5.
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