CN111644216B - Microfluidic structures for plasma separation and detection - Google Patents
Microfluidic structures for plasma separation and detection Download PDFInfo
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- CN111644216B CN111644216B CN202010780990.0A CN202010780990A CN111644216B CN 111644216 B CN111644216 B CN 111644216B CN 202010780990 A CN202010780990 A CN 202010780990A CN 111644216 B CN111644216 B CN 111644216B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/12—Specific details about materials
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
Abstract
Microfluidic structures for plasma separation and detection are provided herein that employ U-shaped channels having partially hydrophobic inner surfaces and partially hydrophilic inner surfaces for the purpose of separating plasma from a blood sample and using the separated plasma directly for detection by centrifugation.
Description
Technical Field
The present invention relates to microfluidic structures, and in particular to microfluidic structures useful for performing plasma separation and detection.
Background
Methods for detecting the concentration or presence of certain biological components (e.g., antibodies) in blood are often used clinically for blood typing, disease diagnosis, drug efficacy determination, and the like. Most of these methods require the use of plasma or serum depleted of blood cells to eliminate the effect of blood cells on the detection process. For example, for some methods that require a color change to determine the concentration of a test substance in a sample, the presence of red blood cells in whole blood often renders detection impossible. For another example, it is also apparent that red blood cells in blood to be tested need to be removed for blood group countertyping by cell agglutination. Therefore, in clinical examination, it is necessary to first separate plasma from whole blood and then examine the separated plasma.
There are various methods for separating plasma from whole blood, including centrifugation, natural sedimentation, membrane filtration, and the like. These methods either require large blood collection volumes (e.g., above a few milliliters), or the separation process is complicated and time consuming, or costly (e.g., using membrane filtration). In addition, how to integrate the plasma separation process and the detection process to simplify the detection operation and reduce the detection cost is also a problem to be solved.
Disclosure of Invention
In one aspect, provided herein is a microfluidic structure for plasma separation and detection, comprising
1) A microfluidic structure body;
2) the U-shaped channel is arranged in the microfluidic body and comprises a left branch and a right branch;
3) a first hole as a sample adding hole of the blood sample is communicated with the end part of the left branch of the U-shaped channel from the surface of the microfluidic structure body;
4) the second hole is used as an air hole and communicated with the end part of the right branch of the U-shaped channel from the surface of the microfluidic structure body; and
5) a reaction area channel and at least one reaction area which are arranged in the microfluidic body, wherein one end of the reaction area channel is communicated with the U-shaped channel near the end part of the right branch of the U-shaped channel, and the other end of the reaction area channel is communicated with the reaction area,
wherein the U-shaped channel is used for separating plasma from the blood sample under centrifugation conditions, at least a portion of the left branch of the U-shaped channel having a hydrophobic inner surface that prevents the blood sample from flowing into the right branch when the blood sample is added from the addition well; at least a portion of the right branch of the U-shaped channel has a hydrophilic inner surface for facilitating flow of the plasma separated from the blood sample to the reaction zone channel.
In some embodiments, a sample application slot is further disposed in the microfluidic structure body, and is communicated with the sample application hole and the end of the left branch of the U-shaped channel, and is used for accommodating the blood sample applied from the sample application hole, and the blood sample can enter the left branch of the U-shaped channel from the sample application slot under the centrifugal condition.
In some embodiments, the inner surface of the left branch of the U-shaped channel is a hydrophobic surface and the inner surface of the right branch of the U-shaped channel is a hydrophilic surface.
In some embodiments, the inner diameter of the U-shaped channel is not less than 0.8 mm.
In some embodiments, the reaction zone channel has an inner diameter of no greater than 0.5 mm.
In some embodiments, the body is made of polycarbonate.
In some embodiments, the hydrophilic inner surface of the right branch of the U-shaped channel is obtained by Hydro 300 treatment.
In some embodiments, the reaction zones are at least two, one of which is pre-loaded with retrocommitted a cells and the other of which is pre-loaded with retrocommitted B cells.
The microfluidic structure provided by the invention can directly complete two operations of plasma separation and sample addition, so that the detection efficiency is improved, and the experimental operation is simplified.
Drawings
Fig. 1 is a schematic top view of a microfluidic structure provided herein.
Fig. 2 is a schematic perspective view of a microfluidic structure provided herein.
Fig. 3 is a schematic top view of the lower layer of the microfluidic structure provided by the present invention.
Fig. 4 is a schematic diagram of the flow of a blood sample in a microfluidic structure according to the present invention, wherein the arrows in the U-shaped channel indicate the flow direction of the blood sample, and the black arrows F indicate the centrifugal force direction during centrifugation.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
As used herein, a "microfluidic structure," when it is a stand-alone entity, may be referred to as a "microfluidic chip" or a "microfluidic test card"; alternatively, it may be part of a microfluidic chip.
As used herein, a "U-shaped channel," which may also be referred to as a "U-shaped channel," means that the channel is shaped like a "U". Meanwhile, for convenience of description, a left portion of the U-shaped channel is referred to as a left branch, and a right portion is referred to as a right branch. The left branch and the right branch are divided by taking the middle of the lower part of the U-shaped channel as a boundary. It will be appreciated that the left and right branches need not be completely symmetrical in construction, as long as the entire channel is guaranteed to be substantially U-shaped.
In this context, "inner diameter" is used to describe the cross-sectional inner circle diameter of the various channels. It will be appreciated by those skilled in the art that the cross-section of the channels may also be other shapes, such as rectangular or square, in which case their average length and width may simply be taken as the internal diameter.
The present invention will be described in detail below with reference to the accompanying drawings.
Referring to fig. 1-4, a microfluidic structure 1 provided herein can include an upper layer 10a and a lower layer 10 b. The surface of the upper layer 10a is provided with two holes communicating with the lower layer 10 b: a loading hole 11 and an air hole 12. A section of U-shaped channel 13 is provided on the lower layer 10b between the loading hole 11 and the air hole 12. The U-shaped channel 13 is divided into a left branch 13a and a right branch 13b (their junction can be considered to be in the middle of the bottom of the U-shaped channel 13). The left branch 13a communicates with the loading hole 11 through its end 13a 1; the right branch 13b communicates with the air hole 12 through its end 13b 1. A reaction zone channel 14 is provided at the right side branch 13b of the U-shaped channel 13 near the gas hole 12. The reaction zone channel 14 communicates at one end with the right side branch 13b of the U-shaped channel 13 and at the other end with one or more reaction zones 15. The inner surface of the left branch 13a of the U-shaped channel 13 is a hydrophobic surface, and the right branch 13b comprises at least one section of hydrophilic region 131 (shown in fig. 3, partially in the dashed box), and the inner surface is a hydrophilic surface.
The sample adding hole 11 is an inlet of the microfluidic structure 1 for receiving a blood sample to be tested. The gas holes 12 are used to release gas pressure and facilitate the flow of liquid (e.g. separated plasma) in the U-shaped channel 13, especially in the right branch 13 b. The inner surface of the left branch 13a of the U-shaped channel 13 is hydrophobic, which is not favorable for the sample to be tested (such as whole blood or processed (such as diluted) blood) fed from the feeding hole 11 to flow therein. Thus, when the sample is added, the sample is distributed only in the sample addition hole 11 and the left branch 13a of the U-shaped channel 13, and does not enter the right branch 13b, as long as the amount of the sample added is not excessive. In order to further prevent the sample added during the sample addition from entering the left branch 13a, a sample addition well 16 may be provided at a position (e.g., below) of the sample addition well 11, and the volume of the sample addition well 16 is suitable for containing most or all of the blood sample.
Various methods known in the art may be used to make a portion of the inner surface of the U-shaped channel 13 hydrophilic and another portion hydrophobic. At present, in the field of microfluidic chip manufacturing, high molecular polymer materials are widely used, such as Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), Polycarbonate (PC), and the like. These materials are hydrophobic in nature, and thus the channels of microfluidic chips made with these materials have hydrophobic inner surfaces. Methods for hydrophilizing partially hydrophobic interior surfaces are also known in the art and include plasma modification, ultraviolet light irradiation modification, nanocoating modification, and the like. In addition, in order to promote the flow of the separated plasma in the reaction-area channels 14 and the reaction areas 15, the U-shaped channel 13 may be subjected to hydrophilic treatment, and at the same time, the reaction-area channels 14 and/or the reaction areas 15 may be subjected to hydrophilic treatment.
Plasma separation was performed by centrifugation. Referring to FIG. 4, upon centrifugation (the direction of centrifugal force is shown by the thick arrow F in FIG. 4), the added blood sample S flows into the lower part of the U-shaped channel 13 from the well 11, the sample addition slot 16 (if any), and the left branch 13a by the centrifugal force, and forms two equal liquid levels 17. Under the centrifugal force, blood cells are deposited below the U-shaped channel 13, and separated plasma is deposited above the U-shaped channel. Meanwhile, the separated plasma does not enter the upper portion of the left branch 13a and does not reach the reaction region passage 14 under the centrifugal force. In order to ensure that the liquid surface 17 does not reach the reaction region channel 14 at the time of centrifugation, the approximate position of the liquid surface 17 may be calculated based on the inner diameter of the U-shaped channel and the amount of the sample to be added, and the reaction region channel 14 may be disposed at least 1cm, for example, 2cm or more apart from the liquid surface 17. The reaction zone channel 14 may be generally disposed near the end 13b1 of the U-shaped channel 13 (e.g., at a distance of 0.2cm or more, e.g., 0.5cm, 1cm, 1.5cm, etc., from the end 13b 1). After the centrifugation is stopped, the separated plasma tends to be distributed uniformly in the U-shaped channel 13 under the action of gravity, i.e. the plasma on the right side of the U-shaped channel 13 tends to flow towards the air holes 12, and the presence of the hydrophilic region 131 promotes this flow. The length of the hydrophilic region 131 may be set from below the liquid level 17 to the reaction-zone channel 14. Of course, the entire inner surface of the right branch 13b may be subjected to hydrophilic treatment. The separated plasma flows from the right branch 13b of the U-shaped channel 13 into the reaction-zone channel 14 and then into the respective reaction zones 15 by capillary action. Various reagents may be pre-loaded into reaction zone 15. For example, for blood typing, counterlabeling reagent A cells and counterlabeling reagent B cells may be added, respectively, and the blood type of blood to be tested is determined by agglutination. The reaction zone 15 may also be provided with various test strips, such as a tumor marker test strip, for determining whether each tumor marker in the sample exceeds the standard. By providing a plurality of reaction regions 15, detection of a plurality of items can be performed simultaneously.
The centrifugal force used to separate the plasma can be set as desired. In some cases, only erythrocytes need to be pelleted from whole blood by centrifugation. In other cases, it may be desirable to precipitate out fine cellular components, including platelets. It is known to those skilled in the art that different centrifugal forces are required for the precipitation of different cells and will not be described in detail herein.
The upper and lower layers of the microfluidic structure 1 may be bonded to each other in various ways, for example, by bonding, using an adhesive, or the like.
As can be seen from the above description, the microfluidic structure 1 of the present invention skillfully utilizes gravity, centrifugal force and capillary action to achieve plasma separation and directional flow, and can complete the detection of multiple blood items in a simple microfluidic structure 1.
In some specific embodiments, the inner diameter of the U-shaped channel 13 is 0.8mm or more, such as 1, 1.2, 1.5, 1.8mm, or more. The two sides of the U-shaped channel 13 are not necessarily symmetrically arranged, for example, the inner diameters of the two sides may be different. The inner diameter of the right branch 13b of the U-shaped channel 13 should be not less than 0.8mm to prevent plasma from entering the reaction zone channel 14 by capillary action during centrifugation. Understandably, by properly setting the inner diameter and length of the U-shaped channel 13, the purpose of separating plasma and preventing the sample from directly entering the reaction area channel 14 during sample loading can be realized. To utilize capillary action, the inner diameter of the reaction zone channel 14 can generally be selected to be between 0.1mm and 0.5mm, such as 0.3mm, 0.4mm, or 0.5 mm. The volume of the sample addition well 16 can be set according to the amount of sample addition, and for example, the volume can be 20. mu.L to 200. mu.L, or larger.
Examples
The upper layer and the lower layer of the microfluidic structure are prepared by adopting Polycarbonate (PC) materials through injection molding, and the upper layer and the lower layer are bonded for final molding. Before bonding, isopropanol is used for diluting Hydro 300 (Shanghai Sangjing chemical Co., Ltd.) to 30% concentration, the isopropanol is uniformly coated on the right side of the U-shaped channel (forming a hydrophilic region), the reaction region channel and the reaction region are dried in the ventilation place for 2 hours, and then the upper layer and the lower layer are bonded to form a complete microfluidic structure.
The inner diameter of the U-shaped channel is 1.5mm, the total length is 30mm, the lengths of two sides are respectively 15mm, and the inner diameter of the channel in the reaction zone is 0.5 mm. The sample addition well volume was 30. mu.L. When the device is used, 30 mu L of whole blood sample is added from the sample adding port, and after the centrifugation of 700gX2min, plasma is separated from the whole blood at the lower part of the U-shaped channel, wherein the liquid level does not reach the reaction area channel. After the centrifugation is stopped, since the right branch of the U-shaped channel is subjected to hydrophilic treatment, the plasma reaches the reaction-zone channel under the action of gravity and hydrophilicity. The reaction area channel divides the plasma into the reaction areas, wherein the two reaction areas are added with the antideterminal reagent A cells and the antideterminal reagent B cells, so that the blood type of the sample can be determined according to the generated agglutination reaction.
Advantages of the microfluidic structures of the present invention include, but are not limited to:
1. at present, most of experiments for detecting blood plasma all need to centrifuge a whole blood sample, and then sample adding experiments are carried out on upper layer blood plasma. The microfluidic structure provided by the invention can directly complete two operations of plasma separation and sample addition, thereby improving the detection efficiency and simplifying the experimental operation;
2. the hydrophobic and hydrophilic areas of the U-shaped channel of the microfluidic structure provided by the invention ensure that a whole blood sample does not contact the reaction area channel during sample addition, and separated plasma after centrifugation is easy to enter the reaction area channel, so that the use of a filtering membrane is avoided;
3. the microfluidic structure provided by the invention has the advantages of small sample demand, simple structure and low processing cost.
Claims (8)
1. Microfluidic structures for plasma separation and detection comprising
1) A microfluidic structure body;
2) the U-shaped channel is arranged in the microfluidic body and comprises a left branch and a right branch;
3) a first hole as a sample adding hole of the blood sample is communicated with the end part of the left branch of the U-shaped channel from the surface of the microfluidic structure body;
4) the second hole is used as an air hole and communicated with the end part of the right branch of the U-shaped channel from the surface of the microfluidic structure body; and
5) a reaction area channel and at least one reaction area which are arranged in the microfluidic body, wherein one end of the reaction area channel is communicated with the U-shaped channel near the end part of the right branch of the U-shaped channel, and the other end of the reaction area channel is communicated with the reaction area,
wherein the U-shaped channel is used for separating plasma from the blood sample under centrifugation conditions, at least a portion of the left branch of the U-shaped channel having a hydrophobic inner surface that prevents the blood sample from flowing into the right branch when the blood sample is added from the addition well; at least a portion of the right branch of the U-shaped channel has a hydrophilic inner surface for facilitating flow of the plasma separated from the blood sample to the reaction zone channel.
2. The microfluidic structure according to claim 1, wherein a sample loading slot is further disposed in the microfluidic structure body, and is communicated with the sample loading hole and an end of the left branch of the U-shaped channel, for accommodating the blood sample loaded from the sample loading hole, and the blood sample can enter the left branch of the U-shaped channel from the sample loading slot under centrifugation.
3. The microfluidic structure of claim 1 or 2, wherein the inner surface of the left branch of the U-shaped channel is a hydrophobic surface and the inner surface of the right branch of the U-shaped channel is a hydrophilic surface.
4. The microfluidic structure of claim 1 or 2, wherein the inner diameter of the U-shaped channel is not less than 0.8 mm.
5. The microfluidic structure of claim 1 or 2, wherein the reaction zone channel has an inner diameter of no greater than 0.5 mm.
6. The microfluidic structure of claim 1 or 2, wherein the body is made of polycarbonate.
7. The microfluidic structure according to claim 1 or 2, wherein the hydrophilic inner surface of the right branch of the U-shaped channel is obtained by Hydro 300 treatment.
8. The microfluidic structure of claim 1 or 2, wherein the reaction zones are at least two, one of which is pre-loaded with anti-committed A cells and the other of which is pre-loaded with anti-committed B cells.
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| CN116298347B (en) * | 2023-05-12 | 2023-07-25 | 北京未神生物科技有限公司 | Centrifugal microfluidic full-automatic blood type detection card and detection method thereof |
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| EP1900432A2 (en) * | 2006-08-30 | 2008-03-19 | Samsung Electronics Co., Ltd. | Magnetic bead-packing unit using centrifugal force, microfluidic device including the same, and immunoassay method using the microfluidic device |
| CN103097029A (en) * | 2010-09-07 | 2013-05-08 | 多检测技术有限公司 | Assay device and reader |
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| CN105562130A (en) * | 2015-12-11 | 2016-05-11 | 中国科学院苏州生物医学工程技术研究所 | Micro-fluidic chip and method for detecting endotoxins by using micro-fluidic chip |
| WO2018220135A1 (en) * | 2017-05-31 | 2018-12-06 | Biosurfit, S.A. | Liquid handling, in particular metering |
| CN107796933A (en) * | 2017-10-10 | 2018-03-13 | 绍兴普施康生物科技有限公司 | Micro-fluidic centrifugation disc and Hemostasis examination method |
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| EP1900432A2 (en) * | 2006-08-30 | 2008-03-19 | Samsung Electronics Co., Ltd. | Magnetic bead-packing unit using centrifugal force, microfluidic device including the same, and immunoassay method using the microfluidic device |
| CN103097029A (en) * | 2010-09-07 | 2013-05-08 | 多检测技术有限公司 | Assay device and reader |
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