CN117718088A - Centrifugal microfluidic immunodetection chip - Google Patents
Centrifugal microfluidic immunodetection chip Download PDFInfo
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- CN117718088A CN117718088A CN202311736423.5A CN202311736423A CN117718088A CN 117718088 A CN117718088 A CN 117718088A CN 202311736423 A CN202311736423 A CN 202311736423A CN 117718088 A CN117718088 A CN 117718088A
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- 238000000746 purification Methods 0.000 claims abstract description 116
- 239000012188 paraffin wax Substances 0.000 claims abstract description 87
- 238000001514 detection method Methods 0.000 claims abstract description 56
- 239000007788 liquid Substances 0.000 claims abstract description 51
- 210000004369 blood Anatomy 0.000 claims abstract description 48
- 239000008280 blood Substances 0.000 claims abstract description 48
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 48
- 239000002699 waste material Substances 0.000 claims abstract description 31
- 238000002156 mixing Methods 0.000 claims description 77
- 239000011324 bead Substances 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 abstract description 10
- 238000004587 chromatography analysis Methods 0.000 abstract description 5
- 238000003018 immunoassay Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 19
- 239000003480 eluent Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 230000003287 optical effect Effects 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 238000003745 diagnosis Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 238000002032 lab-on-a-chip Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 238000007689 inspection Methods 0.000 description 1
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- 210000002966 serum Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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Abstract
The invention discloses a centrifugal microfluidic immunodetection chip, which comprises a spiral channel, wherein a blood chromatography cavity and a public reagent cavity are arranged on one side of the spiral channel close to a circle center, and a calibration unit and a plurality of magnetic purification units for detecting different indexes are arranged on one side of the spiral channel away from the circle center; the blood chromatographic cavity is connected with the spiral channel through a paraffin valve I, and the public reagent cavity is connected with the spiral channel through a paraffin valve II; the tail end of the spiral channel is connected with the waste liquid pool; the spiral channel is connected with the calibration unit through the first metering cavity, and the spiral channel is connected with the magnetic purification unit through the second metering cavity. The invention can realize the whole flow detection, integrates the whole flow of reagent addition, cleaning and result detection of blood centrifugal separation plasma and immunoassay on a chip, is beneficial to reducing detection steps, does not need sample pretreatment, result post-treatment and the like of blood, has simple patent detection flow, simple operation, uses less equipment, and reduces detection cost and the requirements on the speciality of detection personnel.
Description
Technical Field
The invention belongs to a detection chip, and particularly relates to a centrifugal microfluidic immunodetection chip.
Background
Microfluidic is a technology that uses micro-channels to control and handle very small amounts of fluids, with great scientific and commercial potential. The microfluidic device has small volume, low energy consumption and strong portability, and can complete biological or chemical experiments which can be realized by a conventional laboratory large instrument on a chip of a few square centimeters. By realizing the concept of Lab on a chip (LOC) or micro Total Analysis System (TAS), the microfluidic technology has great application prospect in the fields of molecular biology, analytical chemistry, on-site instant medical diagnosis and the like. The microfluidic chip adopting the immunoassay method has the advantages of high sensitivity, wide calibration range and the like.
The main techniques of immunodiagnosis include enzyme-linked immunosorbent assay, fluorescent immunosorbent assay, chemiluminescent immunosorbent assay, etc., and have the advantages of high sensitivity, strong specificity, etc. However. The traditional immunoassay device has the disadvantages of large reagent/sample consumption, long detection time and high cost, which limits the application of the products in the scenes of entry and exit, airports, railway stations, basic medical institutions and the like.
In clinical diagnosis, many indexes need to be detected by an immunoassay, and the confirmation diagnosis needs to be simultaneously used as a basis for multiple indexes. The current conventional method is to take blood for multiple times to detect multiple indexes, but when the method is aimed at certain indexes which need to be jointly described, diagnosis errors can be caused by extreme situations of high or low simultaneously, and the method needs to take blood for multiple times and wait for detection results for a long time, so that the method is inconvenient for patients. Therefore, there is a need to develop a detection device that can detect a plurality of indexes simultaneously using a small amount of blood.
Disclosure of Invention
The invention aims to: in order to overcome the defects in the prior art, the invention aims to provide a centrifugal microfluidic immunodetection chip which uses less blood volume, has full-flow multi-index joint inspection and has high accuracy.
The technical scheme is as follows: the centrifugal microfluidic immunodetection chip comprises a spiral channel, wherein a blood chromatography cavity and a public reagent cavity are arranged on one side of the spiral channel close to the center of a circle, and a plurality of magnetic purification units for detecting different indexes are arranged on one side of the spiral channel away from the center of the circle; the blood chromatographic cavity is connected with the spiral channel through a paraffin valve I, and the public reagent cavity is connected with the spiral channel through a paraffin valve II; the tail end of the spiral channel is connected with the waste liquid pool; the spiral channel is connected with the magnetic purification unit through the second metering cavity.
Further, the magnetic purification unit comprises a magnetic purification reagent cavity, a paraffin valve III, a magnetic purification mixing cavity, a paraffin valve IV, a primary magnetic purification capillary valve, a secondary magnetic purification capillary valve, a magnetic purification detection cavity and a magnetic purification waste liquid cavity; the magnetic purification reagent cavity is connected with the magnetic purification mixing cavity through the paraffin valve III, the metering cavity is connected with the magnetic purification mixing cavity through the first-stage magnetic purification capillary valve, the magnetic purification mixing cavity is connected with the magnetic purification detection cavity through the paraffin valve IV, and the metering cavity is connected with the magnetic purification waste liquid cavity through the second-stage magnetic purification capillary valve.
Further, the magnetic purification reagent cavity is positioned at one side of the center of the magnetic purification mixing cavity. The magnetic purification mixing cavity is internally provided with magnetic beads capable of realizing magnetic state and non-magnetic state conversion, and the bottom of the magnetic purification mixing cavity is provided with a magnet capable of realizing magnetic state and non-magnetic state conversion. The magnetic beads are coated with antibodies/antigens, when the magnet is in a magnetic state, the magnetic beads can be adsorbed and fixed at the bottom of the magnetic purification mixing cavity, and when the magnet is in a non-magnetic state, the magnetic beads are suspended in the liquid in the magnetic purification mixing cavity. The detection is assisted by a magnet.
Further, the difference value of the rotation speed breakthrough threshold values of the primary magnetic purification capillary valve and the secondary magnetic purification capillary valve is more than 50rpm, when the primary magnetic purification capillary valve is opened, the secondary magnetic purification capillary valve is kept closed, so that the mixing of liquid in the magnetic purification mixing cavity is realized, and the inlet and outlet sequences of liquid in the magnetic purification mixing cavity are realized.
In order to solve the problem of unified high index of the sample, one side of the spiral channel far away from the center of the circle is also provided with a calibration unit, the spiral channel is connected with the calibration unit through a first metering cavity, and the first metering cavity is positioned between a public reagent cavity and a blood chromatographic cavity on the spiral channel.
The calibration unit comprises a calibration reagent cavity, a paraffin valve five, a calibration mixing cavity, a paraffin valve six, a primary calibration capillary valve, a secondary calibration capillary valve, a calibration detection cavity, a calibration waste liquid cavity, a standard substance cavity and a paraffin valve seven, wherein the metering cavity I is connected with the calibration mixing cavity through the primary calibration capillary valve; the calibration mixing cavity is connected with the calibration reagent cavity through the paraffin valve five, is connected with the standard substance cavity through the paraffin valve six, is connected with the calibration detection cavity through the paraffin valve seven, and is connected with the calibration waste liquid cavity through the secondary calibration capillary valve.
Further, the calibration reagent cavity and the standard substance cavity are both positioned at one side of the calibration mixing cavity near the center of the circle. The magnet capable of realizing magnetic state and non-magnetic state conversion is arranged in the calibration mixing cavity, and the magnet capable of realizing magnetic state and non-magnetic state conversion is arranged at the bottom of the calibration mixing cavity. The magnetic beads are coated with antibodies/antigens, when the magnet is in a magnetic state, the magnetic beads can be adsorbed and fixed at the bottom of the calibration mixing cavity, and when the magnet is in a non-magnetic state, the magnetic beads are suspended in the liquid in the calibration mixing cavity.
The difference value of the rotation speed breakthrough threshold values of the primary calibration capillary valve and the secondary calibration capillary valve of the magnetic purification mixing cavity is more than 50rpm, and when the primary calibration capillary valve is opened, the secondary calibration capillary valve is kept closed, so that the mixing of liquid in the calibration mixing cavity is realized, and the inlet and outlet sequences of the liquid in the calibration mixing cavity are realized.
Further, all capillary valves and paraffin valves can be provided on the back of the chip, the paraffin valves being controllable. The common reagent chamber may be arranged in a plurality of volumes proportional to the sample for each assay, and disposed along the inside of the spiral channel.
Working principle: whole blood centrifugation is performed in a blood chromatography chamber. The blood chromatographic cavity is positioned at the near-center end of the chip and is provided with a paraffin valve for ensuring that blood cannot flow out when rotating at a high speed. The paraffin valve has the function of not opening under high-speed rotation and opening under low-speed rotation, and the valve opening of the paraffin valve I is arranged at any position on the side edge of the blood chromatographic cavity and is directly connected to the spiral channel. After long-time high-speed centrifugal rotation, the whole blood can be layered in a blood chromatographic cavity, the blood plasma separated from the whole blood is at the near-heart end, and blood cells are precipitated at the far-heart end. The rotation speed is reduced, the paraffin valve I is opened under the action of centrifugal force, and only the blood plasma at the near-center end is extracted and enters the spiral channel through the paraffin valve I.
Through the design of a reagent cavity, a blood chromatographic cavity, a spiral channel, a metering cavity, a capillary valve, a waste liquid pool and the like, the centrifugal force applied to the regulating device enables the blood plasma extracted by a public reagent or the blood chromatographic cavity to enter the spiral channel, and the flowing state of a liquid sample on the microfluidic chip is controlled through the regulation of the speed of the rotating platform, so that the sample enters and fills the metering cavity under the action of the centrifugal force, and redundant liquid flows into the waste liquid pool. And then the liquid in the metering cavity is released to enter the magnetic purification unit or the mixing cavity of the calibration unit by controlling the capillary micro valve (the primary calibration capillary valve, the secondary calibration capillary valve, the primary magnetic purification capillary valve and the secondary magnetic purification capillary valve). Through the dimension design of the first metering cavity and the second metering cavity, the volume entering the calibration mixing cavity and the magnetic purification mixing cavity is quantitatively controlled. Thus, plasma generated by centrifuging the same blood sample can enter a subsequent detection flow, or liquid used simultaneously in the subsequent detection flow can enter the flow, so that multi-index detection of full-flow integration is realized in the same chip. Simple structure and convenient operation.
The paraffin valve in the closed state can resist the rotating speed of 6000rpm/s, the paraffin valve in the open state has a lower opening threshold value, and the secondary capillary valve also has a larger opening threshold value which is higher than the paraffin valve threshold value in the open state and lower than the paraffin valve threshold value in the closed state. Therefore, under low-speed rotation, the two valves are closed, the liquid in the mixing cavity cannot flow out, and the mixing reaction can be carried out in the cavity; when the threshold value of the secondary capillary valve is broken through, the liquid flows to the waste liquid cavity; when the paraffin valve is opened, the liquid flows into the detection chamber at a low rotational speed.
And opening the paraffin valve I and the paraffin valve II to enable the blood plasma or other samples obtained after the common reagent or blood chromatography to enter the spiral channel, regulating the rotating speed to enable the liquid to enter and fill the metering cavity under the action of centrifugal force, and enabling the redundant liquid to flow into the waste liquid pool. And (3) releasing the liquid in the metering cavity to enter the mixing cavity of the magnetic purification unit or the calibration unit by controlling the primary magnetic purification capillary valve.
The using method of the magnetic purification mixing cavity comprises the following steps:
a. whole blood is injected into the blood chromatographic cavity;
b. rotating clockwise at 2000-5000rpm for 80-600 s, and layering after whole blood centrifugation.
c. Opening a paraffin valve I, reducing the rotating speed to 500rpm, enabling the blood plasma at the near-center end to enter a spiral channel, filling all metering cavities II, and enabling the redundant blood plasma to enter a waste liquid pool;
d. the rotating speed is increased to 600-700 rpm, the primary magnetic purification capillary valve is opened, and the plasma in the second metering cavity enters the magnetic purification mixing cavity;
e. the rotating speed is reduced to 200rpm, the clockwise and anticlockwise rotation is switched for 30s, and the plasma and the magnetic beads are fully mixed;
f. and switching the magnet to a magnetic state, and adsorbing the magnetic beads on the chip base plate. Rotating clockwise at 900-1000 rpm, opening a secondary magnetic purification capillary valve, and allowing unadsorbed magnetic beads and redundant blood plasma to enter a magnetic purification waste liquid cavity;
g. and opening a paraffin valve II of the public reagent cavity, enabling eluent to enter the spiral channel, filling all metering cavities II, opening a primary magnetic purification capillary valve, and entering the magnetic purification mixing cavity. Switching the magnet to a non-magnetic state, suspending the magnetic beads in a magnetic purification mixing cavity, switching the rotation clockwise and anticlockwise, fully cleaning, and opening a secondary magnetic purification capillary valve to enable redundant eluent to enter a magnetic purification waste liquid cavity;
h. and then, opening a paraffin valve III of the magnetic purification reagent cavity to enable the detected object to be fully combined with the antibody/antigen for optical detection, so as to form a sandwich structure.
i. Then cleaning for multiple times and adding reagent to make the sandwich structure be in proper substrate;
j. and opening a paraffin valve IV of the magnetic purification mixing cavity, rotating clockwise at 500rpm, enabling the sandwich structure with the detected object and the environmental substrates thereof to enter the magnetic purification detection cavity together, and then carrying out optical detection.
The using method of the calibration mixing cavity comprises the following steps:
a. opening a paraffin valve seven to enable the standard substance in the standard substance cavity to enter a calibration mixing cavity;
b. the rotation is switched between clockwise and anticlockwise for 30s, the rotation speed is up to 200rpm, and the standard substance and the magnetic beads are fully mixed;
c. and switching the magnet to a magnetic state, and adsorbing the magnetic beads on the chip base plate. Rotating clockwise at 900-1000 rpm, opening a secondary calibration capillary valve, and allowing unadsorbed magnetic beads and redundant standard substances to enter a calibration waste liquid cavity;
d. and opening a paraffin valve II of the public reagent cavity to enable eluent to enter the spiral channel, filling the metering cavity I, and entering the calibration mixing cavity after the primary calibration capillary valve is opened. Switching the magnet to a non-magnetic state, suspending the magnetic beads in the calibration mixing cavity, switching the magnet clockwise to rotate clockwise for full cleaning, and opening a secondary calibration capillary valve to enable redundant eluent to enter the calibration waste liquid cavity;
e. and then, opening a paraffin valve V of the calibration reagent cavity to enable the detected object to be fully combined with the antibody/antigen for optical detection, so as to form a sandwich structure.
f. Then cleaning for multiple times and adding reagent to make the sandwich structure be in proper substrate;
g. and opening a paraffin valve seven of the calibration mixing cavity, rotating clockwise at 500rpm, enabling the sandwich structure with the detection object and the environmental substrates thereof to enter the calibration detection cavity together, and then carrying out optical detection.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable characteristics:
1. the whole flow detection can be realized, the whole flow of reagent addition, cleaning and result detection of blood centrifugal separation plasma and immunoassay is integrated on a chip, the detection steps are reduced, sample pretreatment, result post-treatment and the like are not needed for blood, the patent detection flow is simple and convenient, the operation is simple, less equipment is used, and the detection cost and the requirements on the specificity of detection personnel are reduced;
2. the detection of multiple indexes can be realized simultaneously, the microfluidic chip is divided into a plurality of units, each unit can detect different indexes, blood is distributed by the spiral channel and the metering cavity, the units jointly use the same plasma sample for detection, the combined detection accuracy of each index can be improved, and the error of a certain index which is higher or lower is avoided;
3. in consideration of the condition of unified high index of the sample, the calibration unit is arranged, the detection result of the whole microfluidic chip can be determined in a certain range after calibration, and the diagnosis error can be reduced.
Drawings
FIG. 1 is an exploded view of the present invention;
FIG. 2 is a top view of the present invention;
FIG. 3 is a bottom view of the present invention;
FIG. 4 is a perspective view of the present invention;
FIG. 5 is a schematic diagram of a partition of the present invention;
FIG. 6 is a schematic diagram of the distribution of the calibration unit 4 and the magnetic purification unit 5 of the present invention;
FIG. 7 is a schematic diagram of the structure of the magnetic purification unit 5 of the present invention;
fig. 8 is a schematic structural view of the magnet 11 of the present invention;
fig. 9 is a schematic diagram of the structure of the calibration unit 4 of the present invention.
Detailed Description
For convenience of description, a position relatively close to the center of the detection chip is referred to as a proximal end, and a position relatively far from the center of the detection chip is referred to as a distal end, with the same radius line pointing from the center of the centrifugal microfluidic immunodetection chip to the edge as a reference.
As shown in fig. 1, the centrifugal microfluidic immunodetection chip is generally attached to a bottom plate 12 and a cover plate 13. The cover plate 13 is provided with vent holes, and the positions and the number of the vent holes can be adjusted according to specific application experimental scenes.
As shown in fig. 2-6, the spiral channel 1 of the three-index centrifugal microfluidic immunodetection chip starts from the near-center end, spirally extends to a position close to the edge of the chip and ends, and the ending end is provided with a channel connected to the waste liquid pool 8. Six public reagent chambers 3, a blood chromatographic chamber 2 and a calibration unit 4 and three magnetic purification units 5 for detecting different indexes are arranged along the near-center end of the spiral channel 1 on the inner side of the spiral channel 1. The blood chromatographic cavity 2 is connected with the spiral channel 1 through a paraffin valve I6, and the common reagent cavity 3 is connected with the spiral channel 1 through a paraffin valve II 7. The spiral channel 1 is connected with the calibration unit 4 through the first metering cavity 9, and the spiral channel 1 is connected with the magnetic purification unit 5 through the second metering cavity 10. The volumes of the first metering cavity 9 and the second metering cavity 10 are designed according to the requirement so as to realize that the public reagent or the sample quantitatively enters different magnetic purification units 5 or calibration units 4 according to the requirement. The blood chromatographic chamber 2 is arranged at the start of the spiral channel 1.
Whole blood centrifugation is performed in the blood chromatography chamber 2. The blood chromatographic cavity 2 is positioned at the near-center end of the chip, and a paraffin valve I6 is arranged to ensure that blood cannot flow out when rotating at a high speed. The paraffin valve I6 has the function of not opening under high-speed rotation and opening under low-speed rotation, and the valve opening of the paraffin valve I6 is arranged at the position, which is approximately 1/2 of the edge of the blood chromatographic cavity 2 and is close to the proximal end, and is directly connected to the spiral channel 1. After long-time high-speed centrifugal rotation, the whole blood can be layered in the blood chromatographic cavity 2, the blood plasma separated from the whole blood is arranged at the near-heart end, and the blood cell sediment is arranged at the far-heart end. The rotation speed is reduced, the paraffin valve I6 is opened under the action of centrifugal force, and only the blood plasma at the near-center end is extracted and enters the spiral channel 1 through the paraffin valve I6.
As shown in fig. 7 to 8, the magnetic purification unit 5 comprises a magnetic purification reagent chamber 501, a paraffin valve three 502, a magnetic purification mixing chamber 503, a paraffin valve four 504, a primary magnetic purification capillary valve 505, a secondary magnetic purification capillary valve 506, a magnetic purification detection chamber 507 and a magnetic purification waste liquid chamber 508; the magnetic purification reagent cavity 501 is connected with the magnetic purification mixing cavity 503 through the paraffin valve III 502, the metering cavity II 10 is connected with the magnetic purification mixing cavity 503 through the primary magnetic purification capillary valve 505, the magnetic purification mixing cavity 503 is connected with the magnetic purification detection cavity 507 through the paraffin valve IV 504, and is connected with the magnetic purification waste liquid cavity 508 through the secondary magnetic purification capillary valve 506. The magnetic purification reagent chamber 501 is located on the near-center side of the magnetic purification mixing chamber 503. The magnetic beads 11 are arranged in the magnetic purification mixing cavity 503, and a magnet capable of realizing magnetic state and non-magnetic state conversion is arranged at the bottom of the magnetic purification mixing cavity 503. The magnetic beads 11 are coated with antibodies/antigens, and when the magnetic beads 11 are in a magnetic state, the magnetic beads 11 can be adsorbed and fixed at the bottom of the magnetic purification mixing cavity 503, and when the magnetic beads are in a non-magnetic state, the magnetic beads 11 are suspended in the liquid in the calibration mixing cavity 403. The difference between the rotational speed breakthrough threshold values of the primary magnetic purification capillary valve 505 and the secondary magnetic purification capillary valve 506 is 50rpm or more.
As shown in fig. 9, the calibration unit 4 comprises a calibration reagent chamber 401, a paraffin valve five 402, a calibration mixing chamber 403, a paraffin valve six 404, a primary calibration capillary valve 405, a secondary calibration capillary valve 406, a calibration detection chamber 407, a calibration waste liquid chamber 408, a standard substance chamber 409 and a paraffin valve seven 410, and a metering chamber one 9 is connected with the calibration mixing chamber 403 through the primary calibration capillary valve 405; the calibration mixing chamber 403 is connected with the calibration reagent chamber 401 through a paraffin valve five 402, is connected with the standard substance chamber 409 through a paraffin valve six 404, is connected with the calibration detection chamber 407 through a paraffin valve seven 410, and is connected with the calibration waste liquid chamber 408 through a secondary calibration capillary valve 406. The calibration reagent chamber 401 and the standard substance chamber 409 are both positioned on the side near the center of the calibration mixing chamber 403. The magnetic beads 11 are arranged in the calibration mixing cavity 403, and a magnet capable of realizing magnetic state and non-magnetic state conversion is arranged at the bottom of the calibration mixing cavity 403. . The difference between the rotational speed breakthrough threshold values of the primary calibration capillary valve 405 and the secondary calibration capillary valve 406 is 50rpm or more.
The paraffin valves in the closed state (paraffin valve one 6, paraffin valve two 7, paraffin valve three 502, paraffin valve four 504, paraffin valve five 402, paraffin valve six 404, paraffin valve seven 410) can resist rotational speeds up to 6000rpm/s, the paraffin valve in the open state has a lower opening threshold, and the secondary capillary valve (secondary calibration capillary valve 406, secondary magnetic purification capillary valve 506) also has a larger opening threshold, which is higher than the paraffin valve threshold in the open state and lower than the paraffin valve threshold in the closed state. Therefore, under low-speed rotation, the two valves at the bottom are closed, and the liquid in the mixing cavity cannot flow out, so that the reaction can be mixed in the cavity. When the threshold value of the secondary capillary valve is broken through, the liquid flows to the waste liquid cavity; when the paraffin valve is opened, the liquid flows into the detection chamber at a low rotational speed.
For the early screening of gastric cancer, the three-index centrifugal microfluidic immunodetection chip is provided with 1 calibration unit 4 and 3 magnetic purification units 5. Wherein, the detection indexes of the 3 magnetic purification units 5 are respectively set as G-17, PGI and PGII, and the three indexes are important serum biomarkers in the gastric cancer early screening; 1 calibration unit 4 selects the G-17 index. The specific use method comprises the following steps:
a. whole blood is injected into the blood chromatographic cavity 2;
b. rotating clockwise at 2000-5000rpm for 80-600 s, and layering after centrifuging whole blood;
c. opening a paraffin valve I6, reducing the rotating speed to 500rpm, enabling the blood plasma at the near-center end to enter a spiral channel 1, filling all metering cavities II 10, and enabling the redundant blood plasma to enter a waste liquid pool 8;
d. the rotating speed is increased to 600-700 rpm, the primary magnetic purification capillary valve 505 is opened, and the blood plasma in the second metering cavity 10 enters the magnetic purification mixing cavity 503;
e. the rotation speed is reduced to 200rpm, the rotation is switched between clockwise and anticlockwise for 30s, and the plasma is fully mixed with the magnet 11;
f. the magnet is switched to a magnetic state, and the magnetic beads 11 are adsorbed on the bottom plate 12. Rotating clockwise at 900-1000 rpm, opening the secondary magnetic purification capillary valve 506, and allowing the non-adsorbed magnet 11 and redundant blood plasma to enter the magnetic purification waste liquid cavity 508;
g. the paraffin valve two 7 of the common reagent chamber 3 is opened, so that the eluent enters the spiral channel 1, fills all metering chambers two 10, and enters the magnetic purification mixing chamber 503 after the primary magnetic purification capillary valve 505 is opened. Switching the magnet to a non-magnetic state, suspending the magnetic beads 11 in the magnetic purification mixing cavity 503, and opening the secondary magnetic purification capillary valve 506 after full cleaning to enable the redundant eluent to enter the magnetic purification waste liquid cavity 508;
h. then, the paraffin valve III 502 of the magnetic purification reagent chamber 501 is opened to fully combine the detected object with the antibody/antigen for optical detection, so as to form a sandwich structure.
i. Then cleaning for multiple times and adding reagent to make the sandwich structure be in the substrate with proper chemiluminescence;
j. the paraffin valve IV 504 of the magnetic purification mixing cavity 503 is opened, the paraffin valve IV rotates clockwise at 500rpm, the sandwich structure with the detection object and the environmental substrates thereof enter the magnetic purification detection cavity 507 together, and then optical detection is carried out.
If the uniformity of the sample index is high, i.e. the data is abnormal or exceeds the normal order of magnitude, the calibration unit 4 needs to be used.
The using method of the calibration unit 4 specifically comprises the following steps:
a. opening a paraffin valve seven 410 to enable the standard in the standard cavity 409 to enter the calibration mixing cavity 403;
b. the rotation is switched between clockwise and anticlockwise for 30s, the rotation speed is up to 200rpm, and the standard substance and the magnetic beads 11 are fully mixed;
c. the magnet is switched to a magnetic state, and the magnetic beads 11 are adsorbed on the bottom plate 12 of the chip. Rotating clockwise at 900-1000 rpm, opening the second-stage calibration capillary valve 406, and allowing the non-adsorbed magnetic beads 11 and redundant standard substances to enter the calibration waste liquid cavity 408;
d. the paraffin valve two 7 of the common reagent chamber 3 is opened, so that the eluent enters the spiral channel 1 and fills the metering chamber one 9, and the primary calibration capillary valve 405 is opened and then enters the calibration mixing chamber 403. Switching the magnet to a non-magnetic state, suspending the magnetic beads 11 in the calibration mixing cavity 403, and opening the second-stage calibration capillary valve 406 after full cleaning to enable the redundant eluent to enter the calibration waste liquid cavity 408;
e. then, the paraffin valve five 402 of the calibration reagent chamber 401 is opened to fully combine the detected object with the antibody/antigen for optical detection, so as to form a sandwich structure.
f. Then cleaning for multiple times and adding reagent to make the sandwich structure be in proper substrate;
g. the paraffin valve seven 410 of the calibration mixing chamber 403 is opened, the paraffin valve seven is rotated clockwise at 500rpm, the sandwich structure with the detection object and its environmental substrates enter the calibration detection chamber 407 together, and then optical detection is performed.
The calibration unit 4 in this embodiment may be omitted or provided in plurality for collating calibration test data.
Claims (10)
1. A centrifugal microfluidic immunodetection chip is characterized in that: the device comprises a spiral channel (1), wherein a blood chromatographic cavity (2) and a public reagent cavity (3) are arranged on one side of the spiral channel (1) close to the center of a circle, and a plurality of magnetic purification units (5) for detecting different indexes are arranged on one side of the spiral channel away from the center of the circle; the blood chromatographic cavity (2) is connected with the spiral channel (1) through a paraffin valve I (6), and the public reagent cavity (3) is connected with the spiral channel (1) through a paraffin valve II (7); the tail end of the spiral channel (1) is connected with a waste liquid pool (8); the spiral channel (1) is connected with the magnetic purification unit (5) through a metering cavity II (10).
2. The centrifugal microfluidic immunodetection chip according to claim 1, wherein: the magnetic purification unit (5) comprises a magnetic purification reagent cavity (501), a paraffin valve III (502), a magnetic purification mixing cavity (503), a paraffin valve IV (504), a primary magnetic purification capillary valve (505), a secondary magnetic purification capillary valve (506), a magnetic purification detection cavity (507) and a magnetic purification waste liquid cavity (508); the magnetic purification reagent cavity (501) is connected with the magnetic purification mixing cavity (503) through the paraffin valve III (502), the metering cavity II (10) is connected with the magnetic purification mixing cavity (503) through the primary magnetic purification capillary valve (505), the magnetic purification mixing cavity (503) is connected with the magnetic purification detection cavity (507) through the paraffin valve IV (504), and is connected with the magnetic purification waste liquid cavity (508) through the secondary magnetic purification capillary valve (506).
3. The centrifugal microfluidic immunodetection chip according to claim 2, wherein: the magnetic purification reagent cavity (501) is positioned on the side near the center of the magnetic purification mixing cavity (503).
4. The centrifugal microfluidic immunodetection chip according to claim 2, wherein: magnetic beads (11) are arranged in the magnetic purification mixing cavity (503), and a magnet capable of realizing magnetic state and non-magnetic state conversion is arranged at the bottom of the magnetic purification mixing cavity (503).
5. The centrifugal microfluidic immunodetection chip according to claim 2, wherein: the difference value of the rotation speed breakthrough threshold values of the primary magnetic purification capillary valve (505) and the secondary magnetic purification capillary valve (506) is more than 50 rpm.
6. The centrifugal microfluidic immunodetection chip according to claim 1, wherein: the utility model discloses a blood chromatographic device, including spiral passageway (1), measuring chamber, blood chromatographic cavity (2), measuring chamber, calibration unit (4) are still set up to one side that the centre of a circle was kept away from to spiral passageway (1), spiral passageway (1) link to each other with calibration unit (4) through measuring chamber one (9), measuring chamber one (9) are located between public reagent chamber (3) and the blood chromatographic cavity (2) on spiral passageway (1).
7. The centrifugal microfluidic immunodetection chip according to claim 6, wherein: the calibration unit (4) comprises a calibration reagent cavity (401), a paraffin valve five (402), a calibration mixing cavity (403), a paraffin valve six (404), a primary calibration capillary valve (405), a secondary calibration capillary valve (406), a calibration detection cavity (407), a calibration waste liquid cavity (408), a standard substance cavity (409) and a paraffin valve seven (410), wherein the metering cavity I (9) is connected with the calibration mixing cavity (403) through the primary calibration capillary valve (404); the calibration mixing cavity (403) is connected with the calibration reagent cavity (401) through a paraffin valve five (402), is connected with the standard substance cavity (409) through a paraffin valve six (404), is connected with the calibration detection cavity (407) through a paraffin valve seven (410), and is connected with the calibration waste liquid cavity (408) through a secondary calibration capillary valve (406).
8. The centrifugal microfluidic immunodetection chip according to claim 7, wherein: the calibration reagent cavity (401) and the standard substance cavity (409) are both positioned at one side near the center of the calibration mixing cavity (403).
9. The centrifugal microfluidic immunodetection chip according to claim 7, wherein: the magnetic beads (11) are arranged in the calibration mixing cavity (403), and a magnet capable of realizing magnetic state and non-magnetic state conversion is arranged at the bottom of the calibration mixing cavity (403).
10. The centrifugal microfluidic immunodetection chip according to claim 7, wherein: the difference value of the rotation speed breakthrough threshold values of the primary calibration capillary valve (405) and the secondary calibration capillary valve (406) is more than 50 rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311736423.5A CN117718088A (en) | 2023-12-15 | 2023-12-15 | Centrifugal microfluidic immunodetection chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311736423.5A CN117718088A (en) | 2023-12-15 | 2023-12-15 | Centrifugal microfluidic immunodetection chip |
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| Publication Number | Publication Date |
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| CN117718088A true CN117718088A (en) | 2024-03-19 |
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|---|---|---|---|
| CN202311736423.5A Pending CN117718088A (en) | 2023-12-15 | 2023-12-15 | Centrifugal microfluidic immunodetection chip |
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| CN (1) | CN117718088A (en) |
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- 2023-12-15 CN CN202311736423.5A patent/CN117718088A/en active Pending
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