WO2024234422A1 - Microfluidic chip for blood testing and testing method therefor - Google Patents

Abstract

Provided by the present invention is a microfluidic chip for blood testing. The chip comprises a chip body, the chip body comprising one or more separation and testing units, each of the separation and testing units comprising a micro-volume whole blood separation groove and one or more reaction test units, and each of the reaction test units comprising a first sample addition cavity, a first L-shaped microfluidic channel, a second sample addition cavity, a second L-shaped microfluidic channel, a Y-shaped microfluidic channel and a reaction detection cavity; the Y-shaped microfluidic channel comprises a first sample inlet, a second sample inlet and an outlet, and fluid flowing into the first sample inlet and the second sample inlet is mixed and then discharged from the outlet; the first L-shaped microfluidic channel is in communication with the first sample addition cavity and the first sample inlet of the Y-shaped microfluidic channel; the second L-shaped microfluidic channel is in communication with the second sample addition cavity and the second sample inlet of the Y-shaped microfluidic channel; the outlet of the Y-shaped microfluidic channel is in communication with the reaction detection cavity. The chip may be used for ABO blood group reverse typing, and can help improve immune agglutination reaction sensitivity; the chip may also be used for blood type IgG antibody titer detection in HDN pregnant women.

Description

A blood detection microfluidic chip and detection method thereof Technical Field

The present invention relates to blood testing technology, and in particular to a blood testing microfluidic chip and a testing method thereof.

Background Art

The ABO blood group system is the blood group system with the strongest antigen immunity in the human blood group system. The red blood cell ABO blood group identification test is divided into positive typing test and reverse typing test in serology. Among them, the reverse typing test uses A and B reagent red blood cells to check the anti-A antibodies and anti-B antibodies in the serum, which complements the positive typing test to improve the accuracy of ABO blood group identification. Commonly used methods include the slide method, test tube method, microplate method and microcolumn gel method. The slide method and the test tube method are manual operations with complicated operation processes. The accuracy of the results is greatly affected by human factors and has been gradually eliminated. The microplate method is suitable for large sample volume analysis, but it needs to be equipped with a large automatic sampler. The results are judged by using a microscope. The accuracy is greatly affected by human factors and is rarely used in clinical transfusion departments. The microcolumn gel method is currently a commonly used method, but it also has the following limitations: ① It is insensitive to the ABO weak antigen-antibody reaction. The inventor found that the reason for the insensitivity is that the gel card can only be centrifuged once to see the results and cannot be enhanced by repeated centrifugation. ② When the red blood cell antigen-antibody binding strength is weak, the shear force generated by the centrifugation of the gel card exceeds the affinity, and the antibody-dependent red blood cell agglutination is separated, resulting in a false negative result; ③ During transportation, the reagent gel is easy to deform and produce bubbles, and the temperature will also affect the size of the gel molecular sieve, thereby affecting the stability and reliability of the final test results. The above limitations will lead to the inconsistency of the positive and negative stereotypes of the microcolumn gel method. At this time, the sample needs to be specially treated to improve the sensitivity of the immune agglutination reaction, or the manual method is used to further clarify the identification.

Hemolytic disease of the newborn, referred to as HDN, refers to the incompatibility of the blood types of mother and baby. The fetus's red blood cells circulate in the mother's body, and the fetus's red blood cell circulation promotes the production of IgG antibodies in the mother's body. This IgG antibody can act on the fetus's red blood cells through the maternal placenta, causing different degrees of hemolysis. Clinically, the incidence of HDN caused by ABO blood type incompatibility and RhD blood type incompatibility is relatively high. Therefore, dynamic monitoring of maternal IgG antibody titers during pregnancy is of great significance for early intervention and treatment of the disease.

At present, clinical laboratories use the anti-human globulin test method for prenatal IgG antibody detection, which is applicable to the test tube method and the microcolumn gel method. The test tube method is a manual operation with a complicated operation process, and the accuracy of the results is greatly affected by human factors. The microcolumn gel method is currently a commonly used method in clinical laboratories. However, there are some limitations that are the same as the microcolumn gel method for ABO blood type reverse typing detection. These limitations include: ① When the red blood cell antigen-antibody binding strength is weak and the shear force generated by the centrifugation of the gel card exceeds the affinity, the antibody-dependent red blood cell agglutination is separated, resulting in a false negative result; ② During transportation, the reagent gel is easily deformed and bubbles are generated, and the temperature will also affect the size of the gel molecular sieve, thereby affecting the stability and reliability of the final test results.

Summary of the invention

Purpose of the invention: The technical problem to be solved by the present invention is to provide a blood detection microfluidic chip for the deficiencies of the prior art, which is suitable for ABO blood type reverse typing detection and HDN pregnant women blood type IgG antibody titer detection, and can improve the sensitivity of immune agglutination reaction detection; at the same time, it has a small sample usage, automated detection, micro-whole blood separation and hematocrit determination, and has a "micro-total analysis" function, which provides the conditions for the realization of a miniaturized, fully automated, high-throughput matching analyzer.

In order to solve the above technical problems, the first object of the present invention discloses a blood detection microfluidic chip. The chip includes a chip body, the chip body includes more than one separation and detection unit, each separation and detection unit includes a micro whole blood separation tank for receiving and separating a micro whole blood sample and more than one reaction test unit. When the chip body rotates, the red blood cells separated from the micro whole blood sample settle at one end of the micro whole blood separation tank away from the center of the chip body, and the plasma separated from the micro whole blood sample is located at the other end of the micro whole blood separation tank.

Each reaction test unit includes a first sample loading chamber for receiving plasma from a micro whole blood separation tank, a first L-shaped microfluidic channel, a second sample loading chamber for receiving a corresponding red blood cell reagent, a second L-shaped microfluidic channel, a Y-shaped microfluidic channel and a reaction detection chamber. The Y-shaped microfluidic channel includes a first injection port, a second injection port and an outflow port, and the fluids flowing into the first injection port and the second injection port, respectively, are mixed through the Y-shaped microfluidic channel and discharged from the outflow port. The first L-shaped microfluidic channel connects the first sample loading chamber with the first injection port of the Y-shaped microfluidic channel. The second L-shaped microfluidic channel connects the second sample loading chamber with the second injection port of the Y-shaped microfluidic channel. The outflow port of the Y-shaped microfluidic channel is connected to the reaction detection chamber.

Specifically, the first L-shaped microfluidic channel and the second L-shaped microfluidic channel are both composed of a lower microfluidic channel and a vertical microfluidic channel connected in sequence, and the lower microfluidic channel is arranged perpendicular to the vertical microfluidic channel. The inlet of the lower microfluidic channel in the first L-shaped microfluidic channel is connected to the bottom of the first sample loading chamber on the side away from the center of the chip body, and the inlet of the lower microfluidic channel in the second L-shaped microfluidic channel is connected to the bottom of the second sample loading chamber on the side away from the center of the chip body.

The first inlet, the second inlet and the outlet of the Y-shaped microfluidic channel are all located at the upper part of the chip body. The first inlet of the Y-shaped microfluidic channel is connected to the outlet of the vertical microfluidic channel in the first L-shaped microfluidic channel, and the second inlet of the Y-shaped microfluidic channel is connected to the outlet of the vertical microfluidic channel in the second L-shaped microfluidic channel.

The Y-shaped microfluidic channel includes two upper inlet microfluidic channels and an upper mixing microfluidic channel. The inlet of one of the upper inlet microfluidic channels is the first sample inlet of the Y-shaped microfluidic channel, the inlet of the other upper inlet microfluidic channel is the second sample inlet of the Y-shaped microfluidic channel, the outlets of the two upper inlet microfluidic channels meet at the inlet of the upper mixing microfluidic channel, and the outlet of the upper mixing microfluidic channel is the outlet of the Y-shaped microfluidic channel. Each upper inlet microfluidic channel is arranged vertically to the corresponding vertical microfluidic channel.

In some embodiments, the micro whole blood separation groove is arranged along the radial direction of the chip. The micro whole blood separation groove includes a plasma extraction groove and a straight tube groove connected to the plasma extraction groove. The straight tube groove is located at one end of the micro whole blood separation groove away from the center of the chip. The plasma extraction groove is located at the other end of the micro whole blood separation groove close to the center of the chip. Under the action of centrifugal force, all red blood cells obtained after the micro whole blood sample is separated are deposited at the end of the straight tube groove away from the center of the chip.

In some embodiments, the surface of the straight tube groove is provided with a scale mark for judging the hematocrit. The volume of the micro whole blood separation tank is 50-100ul.

In some embodiments, the number of the reaction test units is six. The chip also includes a plasma multiple dilution pretreatment tank corresponding to each reaction test unit.

In some embodiments, the reaction detection cavity includes a cylindrical cavity and a conical cavity with a gradually decreasing diameter, and the large diameter end of the conical cavity is connected to the bottom end of the cylindrical cavity.

The second object of the present invention is to provide a method for detecting a blood detection microfluidic chip. The detection method comprises the following steps:

The first microfluid in the first sample loading chamber flows into the first injection port of the Y-shaped microfluidic channel through the first L-shaped microfluidic channel. The second microfluid in the second sample loading chamber flows into the second injection port of the Y-shaped microfluidic channel through the second L-shaped microfluidic channel. After the first microfluid and the second microfluid are mixed in the Y-shaped microfluidic channel, a first mixture is formed, and the first mixture flows out to the reaction detection chamber through the outlet of the Y-shaped microfluidic channel.

The microchannels in the microfluidic chip are small in scale, and the fluid flow in the microchannels is laminar, and the corresponding Reynolds number is small, so the mixing between different microfluids mainly depends on diffusion. Therefore, in order to enhance mixing, it is necessary to increase the contact area between the solutes, and the way to increase the contact area can be achieved by stretching the fluid or shearing the fluid. The present application utilizes a pipeline geometric intersection design, by setting an L-type microchannel with a pipeline intersection characteristic and setting a vertical microchannel of the L-type microchannel and a Y-type microchannel to intersect in geometric space, when different microfluids flow through the corresponding L-type microchannel and the upper inlet microchannel of the Y-type microchannel respectively, the corresponding microfluid is first split into many microclusters, and then the upper mixing microchannel of the Y-type microchannel is used to promote the mixing between different microfluid microclusters.

The third object of the present invention is to provide a method for performing ABO blood type reverse typing detection using the above-mentioned blood detection microfluidic chip. The number of the reaction test units is three. The red blood cell reagents received by the second sample loading chamber of each reaction test unit are respectively A-type red blood cell reagent, B-type red blood cell reagent and O-type red blood cell reagent. The detection method comprises the following steps:

Step 1: The micro whole blood separation tank receives the micro whole blood sample to be tested.

Step 2: Under the action of centrifugal force, the red blood cells and plasma in the trace whole blood sample to be tested are separated, and the red blood cells obtained after separation are deposited to the end of the straight tube groove away from the center of the chip, the first part of the plasma obtained after separation is contained in the plasma extraction groove, and the second part of the plasma obtained after separation is contained in the side of the straight tube groove close to the center of the chip.

Step 3: After the centrifugation is completed, the plasma in the plasma extraction groove is sucked and transferred to the first sample loading chamber of each reaction test unit; the second sample loading chamber of each reaction test unit receives the corresponding red blood cell reagent.

Step 4: Under the action of centrifugal force, the plasma in the first sample loading chamber flows into the first injection port of the Y-shaped microfluidic channel through the first L-shaped microfluidic channel; the red blood cell reagent in the second sample loading chamber flows into the second injection port of the Y-shaped microfluidic channel through the second L-shaped microfluidic channel; the plasma and the red blood cell reagent are mixed in the Y-shaped microfluidic channel to form a first mixture, and the first mixture flows out to the reaction detection chamber through the outflow port of the Y-shaped microfluidic channel and fully reacts in the reaction detection chamber.

Step 5: Let it stand and read the test results.

If the blood type antibodies in the plasma to be tested undergo an immune agglutination reaction with the blood type antigens in the red blood cell reagent to form a red blood cell clot, the red blood cell clot will settle away from the center of the chip under the action of centrifugal force; when the chip is stationary, the red blood cell clot will remain adhered to the inner wall of the reaction detection cavity for a certain period of time.

If the blood type antibodies in the plasma to be tested do not undergo immune agglutination reaction with the blood type antigens in the red blood cell reagent, under the action of centrifugal force, the red blood cells that have not undergone immune agglutination reaction will settle in a direction away from the center of the chip; when the chip is stationary, the red blood cells that have not undergone immune agglutination reaction will naturally collapse and settle due to gravity.

The third object of the present invention is to provide a method for detecting the titer of blood type IgG antibodies of HDN pregnant women using the above-mentioned blood detection microfluidic chip. The number of the reaction test units is six. Each reaction test unit is also provided with an independent plasma multiple dilution pretreatment tank for forming a series of multiple dilutions. The second sample loading chamber of each reaction test unit receives the same red blood cell reagent, and the same red blood cell reagent is a type A red blood cell reagent, a type B red blood cell reagent or an O-type RhD positive red blood cell reagent. The detection method comprises the following steps:

Step 1: The micro whole blood separation tank receives the micro whole blood sample to be tested.

Step 2: Under the action of centrifugal force, the red blood cells and plasma in the trace whole blood sample to be tested are separated, and the red blood cells obtained after separation are deposited to the end of the straight tube groove away from the center of the chip, the first part of the plasma obtained after separation is contained in the plasma extraction groove, and the second part of the plasma obtained after separation is contained in the side of the straight tube groove close to the center of the chip.

Step 3: After centrifugation, the plasma in the plasma extraction groove is aspirated and transferred to each plasma multiple dilution pretreatment tank in sequence.

Step 4: pretreat the plasma to be tested in each of the plasma multiple dilution pretreatment tanks with a diluent containing dithiothreitol or dithiothioethanol, and let it stand for 15 to 30 minutes to destroy the activity of IgM antibodies; after the static reaction, add the sample diluent to complete the multiple dilution and obtain the multiple diluted plasma.

Step 5: aspirate the diluted plasma in each plasma dilution pretreatment tank and transfer it to the first sample loading chamber of the corresponding reaction test unit; the second sample loading chamber of each reaction test unit receives the red blood cell reagent.

Step 6: Under the action of centrifugal force, in each reaction test unit, the double-diluted plasma in the first sample loading chamber flows into the first sample inlet of the Y-shaped microfluidic channel through the first L-shaped microfluidic channel; the red blood cell reagent in the second sample loading chamber flows into the second sample inlet of the Y-shaped microfluidic channel through the second L-shaped microfluidic channel; the double-diluted plasma and the red blood cell reagent are mixed in the Y-shaped microfluidic channel to form the second sample loading chamber. A mixture, wherein the first mixture flows out of the outlet of the Y-shaped microchannel to the reaction detection chamber and fully reacts in the reaction detection chamber.

Step 7: Let it stand and read the test results.

If the blood type antibodies in the plasma to be tested undergo an immune agglutination reaction with the blood type antigens in the red blood cell reagent to form a red blood cell clot, the red blood cell clot will settle away from the center of the chip under the action of centrifugal force; when the chip is stationary, the red blood cell clot will remain adhered to the inner wall of the reaction detection cavity for a certain period of time.

If the blood type antibodies in the plasma to be tested do not undergo immune agglutination reaction with the blood type antigens in the red blood cell reagent, under the action of centrifugal force, the red blood cells that have not undergone immune agglutination reaction will settle in a direction away from the center of the chip; when the chip is stationary, the red blood cells that have not undergone immune agglutination reaction will naturally collapse and settle due to gravity.

Finally, the reciprocal of the dilution multiple at which no immune agglutination reaction occurred in the plasma sample with the highest dilution multiple was taken as the blood type IgG antibody titer against specific blood type antigens.

Preferably, the reaction detection chamber of each reaction test unit is pre-installed with anti-human globulin polyclonal anti-freeze-dried beads.

Beneficial effects:

(1) The present invention provides a blood testing microfluidic chip suitable for ABO blood type reverse typing detection and HDN pregnant women blood type IgG antibody titer detection. The blood testing microfluidic chip of the present application has a "micro-total analysis" function. Unlike the test tube method and micro-gel method, which require multiple steps and multiple vessels to complete, the present application completes the processes of mixing, entering the reaction chamber, centrifugal acceleration reaction, stopping centrifugation and standing, and displaying the reaction results in a separation detection unit of a blood testing microfluidic chip, which simplifies the operation process and provides the conditions for the realization of a miniaturized, fully automated, high-throughput matching analyzer.

(2) The present invention can complete the detection with a micro-amount of whole blood sample. The volume of the micro-amount whole blood separation tank is set to 50-100ul, and the sample injection volume is 40-80ul, which can well solve the applicability of those who have difficulty in blood collection, especially newborns, and is also conducive to saving reagent costs.

(3) The blood detection microfluidic chip of the present invention belongs to a centrifugal microfluidic chip, which is provided with an L-shaped microfluidic channel and a Y-shaped microfluidic channel. Under the action of centrifugal force, the first microfluid in the first sample loading chamber flows into the first inlet of the Y-shaped microfluidic channel through the first L-shaped microfluidic channel. The second microfluid in the second sample loading chamber flows into the second inlet of the Y-shaped microfluidic channel through the second L-shaped microfluidic channel. After the first microfluid and the second microfluid are mixed in the Y-shaped microfluidic channel, a first mixture is formed, and the first mixture flows out to the reaction detection chamber through the outlet of the Y-shaped microfluidic channel. The present application utilizes a pipeline geometric intersection design, by setting an L-shaped microfluidic channel with a pipeline intersection characteristic and setting a vertical microfluidic channel of the L-shaped microfluidic channel to intersect with the upper inlet microfluidic channel of the Y-shaped microfluidic channel in geometric space. When different microfluids flow through the corresponding L-shaped microfluidic channel and the upper inlet microfluidic channel of the Y-shaped microfluidic channel respectively, the corresponding microfluid is first split into many microclusters, and then the upper mixing microfluidic channel of the Y-shaped microfluidic channel is used to promote the mixing between different microfluidic microclusters. The present invention applies the above mixing process to the mixing of red blood cell suspension and other fluids such as plasma, and utilizes the fluid mechanics principle inside the microfluidic chip to The red blood cell suspension and the plasma are fully mixed and reacted to meet the conditions of the immune agglutination reaction. Compared with other mixing methods such as magnetic stirring, the mixing method of the present application has the advantages of no carryover pollution, no need to add additional materials, no need for structures such as magnetic stirring modules, and is conducive to simplifying the structure of the supporting analyzer.

(4) The blood detection microfluidic chip of the present invention allows the immune agglutination reaction of weak antigen-antibody to be enhanced by repeated centrifugation, thereby improving the sensitivity of weak antigen-antibody reaction detection, that is, improving the sensitivity of ABO blood type system reverse typing identification. In the traditional micro-column gel method, under the action of centrifugal force, the red blood cell agglutination inside the gel card will be subjected to shear stress. When the shear stress is greater than the affinity of the red blood cell antigen-antibody binding, the antibody-dependent red blood cell agglutination will be separated, resulting in a false negative test result. In order to avoid false negative test results, the micro-column gel method is limited to only interpreting the test results after a single centrifugation of the gel card. Although this method ensures the specificity of weak antigen-antibody reaction detection, it reduces the sensitivity of weak antigen-antibody reaction detection. Unlike the traditional micro-column gel method, if red blood cell antigen-antibody binding occurs in the blood detection microfluidic chip of the present application, during the centrifugation process, the red blood cell agglutination is not affected by the shear stress and is relatively stable, and there is no false negative risk in the micro-column gel method. Therefore, the blood detection microfluidic chip of the present application allows repeated centrifugation to enhance the immune agglutination reaction of weak antigen-antibody. If an immune agglutination reaction occurs and the reaction is sufficient, under the action of centrifugal force, the red blood cells will agglutinate and settle to the inner wall of the reaction chamber. The red blood cell agglutination will not collapse and settle naturally after standing for a certain period of time, and will remain vertically attached to the wall of the reaction chamber. There is no visible sedimented red blood cell button at the bottom of the reaction chamber, indicating that the plasma contains red blood cell blood type antibodies, that is, the result is positive. If no immune agglutination reaction occurs, the centrifugal force is removed and the reaction is left to stand for a period of time. The red blood cells that have not undergone immune agglutination reactions will naturally collapse and settle, and gather at the center of the bottom of the reaction chamber along the inverted cone slope at the bottom of the reaction chamber to form a red blood cell button, indicating that there are no red blood cell blood type antibodies in the plasma, that is, the result is negative.

(5) When the chip of the present invention is used to detect the titer of blood type IgG antibodies in HDN pregnant women, since there is no shear stress in the microchannel and reaction chamber of the chip during the centrifugation process as in the gel microcolumn method, the method of detecting the titer of blood type IgG antibodies in HDN pregnant women using the chip of the present invention avoids the risk of false negative results in the gel microcolumn method.

(6) When the chip of the present invention is used to detect the titer of IgG antibody in HDN pregnant women, anti-human globulin polyclonal freeze-dried balls can be pre-placed in the reaction detection chamber. The stability of freeze-dried ball reagents at room temperature is better than that of liquid reagents. Therefore, the transportation process and temperature have little effect on the stability of chip performance.

(7) The present invention can use microscopic image photography plus artificial intelligence to judge whether an immune agglutination reaction has occurred, thereby reducing the influence of human factors on the judgment and simplifying the operation steps; at the same time, the image can be permanently stored for easy review, thereby improving the accuracy of the test results.

(8) The structure of the micro whole blood separation tank of the present application includes a plasma extraction groove and a straight tube groove connected to the plasma extraction groove. By providing the straight tube groove, the micro whole blood separation function is provided while also having the function of measuring the hematocrit by the capillary method. By providing a scale mark for judging the hematocrit on the surface of the straight tube groove, it is convenient to manually read the value of the hematocrit. As a reference for blood transfusion.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and/or other advantages of the present invention will become more clear.

FIG1 is a schematic diagram of the three-dimensional structure of a chip body of a blood detection microfluidic chip for ABO blood type reverse typing detection according to the first embodiment of the present application;

FIG2 is a top view of the upper layer of a blood testing microfluidic chip for ABO blood type reverse typing detection according to the first embodiment of the present application;

FIG3 is a top view of the chip body shown in FIG1 ;

FIG4 is a partial enlarged view of a separation detection unit in the chip body shown in FIG3 ;

FIG5 is a perspective schematic cross-sectional view of FIG3 viewed along the line A;

FIG6 is a schematic diagram of the three-dimensional structure of a chip body of a blood testing microfluidic chip for detecting the titer of blood type IgG antibodies in HDN pregnant women according to the second embodiment of the present application;

FIG7 is a top view of the upper layer of a blood testing microfluidic chip for detecting the titer of blood type IgG antibodies in HDN pregnant women according to the second embodiment of the present application;

FIG8 is a top view of the chip body shown in FIG6 ;

FIG. 9 is a partial enlarged view of a separation detection unit in the chip body shown in FIG. 8 .

The reference numerals are as follows:
Chip body 1; first separation and detection unit 101; second separation and detection unit 102; micro whole blood separation tank 110; plasma extraction tank
111; straight tube groove 112; first sample adding chamber 120; first L-shaped microfluidic channel 130; second sample adding chamber 140; second L-shaped microfluidic channel 150; Y-shaped microfluidic channel 160; first injection port 161; second injection port 162; outflow port 163; reaction detection chamber 170; multiple dilution pretreatment tanks 180a, 180b, 180c, 180d, 180e, 180f; chip upper layer 2; micro whole blood separation tank injection hole 201; first sample adding chamber injection hole 202; second sample adding chamber injection hole 203; plasma multiple dilution pretreatment tank injection holes 204a, 204b, 204c, 204d, 204e, 204f.

DETAILED DESCRIPTION

The technical solution of the present application is described in detail below in conjunction with the accompanying drawings.

Example 1

This embodiment provides a blood testing microfluidic chip, which is used for reverse typing detection of ABO blood types. The chip includes a chip body 1 and a chip upper layer 2. FIG. 1 shows a three-dimensional structure diagram of the chip body 1 of this embodiment. intention.

2 shows a top view of the chip upper layer 2 of this embodiment. The chip upper layer 2 can be a transparent film, which covers the top surface of the chip body 1.

Fig. 3 shows a top view of the chip body 1 shown in Fig. 1. As shown in Fig. 3, the chip body 1 includes six separation detection units. The six separation detection units are evenly distributed along the circumferential direction of the rotation center axis of the chip body 1. The separation detection unit of this embodiment is equivalent to the first separation detection unit 101 in Fig. 3.

FIG4 shows a partial enlarged view of a separation detection unit in the chip body shown in FIG3 , wherein the dotted area represents a separation detection unit. As shown in FIG4 , each first separation detection unit 101 includes a micro whole blood separation tank 110 for receiving and separating a micro whole blood sample and three reaction test units. The volume of the micro whole blood separation tank 110 is fixed and can be set to 50 to 100 ul, and the sample injection volume is 40 to 80 ul, which can well solve the applicability problem of people who have difficulty in blood collection, especially newborns. When the chip body 1 rotates at a speed of 2000 to 5000 rpm, the red blood cells separated from the micro whole blood sample settle at one end of the micro whole blood separation tank 110 away from the center of the chip body 1, and the plasma separated from the micro whole blood sample is located at the other end of the micro whole blood separation tank 110.

As shown in Figure 4, each reaction test unit includes a first sample loading chamber 120 for receiving plasma from a micro-whole blood separation tank 110, a first L-shaped microfluidic channel 130, a second sample loading chamber 140 for receiving corresponding red blood cell reagents, a second L-shaped microfluidic channel 150, a Y-shaped microfluidic channel 160, and a reaction detection chamber 170. In this embodiment, the red blood cell reagents received by the second sample loading chamber 140 of the three reaction test units are respectively A-type red blood cell reagents, B-type red blood cell reagents, and O-type red blood cell reagents. In the same reaction test unit, the first sample loading chamber 120 and the second sample loading chamber 140 are adjacently arranged and are located on the same concentric circle of the rotation center axis of the chip body 1. The first sample loading chamber 120 and the second sample loading chamber 140 are closer to the side of the chip center position relative to the reaction detection chamber 170.

As shown in FIG4 , the Y-shaped microfluidic channel 160 includes a first injection port 161, a second injection port 162, and an outflow port 163. The fluids flowing into the first injection port 161 and the second injection port 162 respectively are mixed through the Y-shaped microfluidic channel 160 and then discharged from the outflow port 163. The first L-shaped microfluidic channel 130 connects the first sample adding chamber 120 with the first injection port 161 of the Y-shaped microfluidic channel 160. The second L-shaped microfluidic channel 150 connects the second sample adding chamber 140 with the second injection port 162 of the Y-shaped microfluidic channel 160. The outflow port 163 of the Y-shaped microfluidic channel 160 is connected to the reaction detection chamber 170.

During detection, under the action of centrifugal force, the first microfluid of the first sample loading chamber 120 flows into the first injection port 161 of the Y-type microfluidic channel 160 through the first L-type microfluidic channel 130. The second microfluid of the second sample loading chamber 140 flows into the second injection port 162 of the Y-type microfluidic channel 160 through the second L-type microfluidic channel 150. After the first microfluid and the second microfluid are mixed in the Y-type microfluidic channel 160, a first mixture is formed, and the first mixture flows out to the reaction detection chamber 170 through the outflow port 163 of the Y-type microfluidic channel 160. The first microfluid and the second microfluid can both be liquids, or at least one of the two can be a multiphase mixture. In a specific embodiment, the first microfluid is The plasma and the second microfluid are red blood cell suspensions. Under the centrifugal force of 300-2000rpm, the plasma and the red blood cell suspension are respectively introduced into the Y-shaped microfluidic channel 160 through the corresponding L-shaped microfluidic channels. The plasma and the red blood cell suspension are mixed and fully reacted by the mixing action of the Y-shaped microfluidic channel 160. The multiphase mixture formed after the mixing reaction enters and fills the reaction detection chamber 170.

Fig. 5 shows a three-dimensional schematic diagram of the cross section in the direction A shown in Fig. 3, in which the arrows indicate the flow direction of the fluid under the action of centrifugal force. As shown in Fig. 5, the first L-shaped microfluidic channel 130 and the second L-shaped microfluidic channel 150 are both composed of a lower layer microfluidic channel and a vertical microfluidic channel connected in sequence, and the lower layer microfluidic channel is arranged vertically with the vertical microfluidic channel. The lower layer microfluidic channel and the vertical microfluidic channel are vertically connected to form the pipeline intersection characteristic of the L-shaped microfluidic channel. The lower layer microfluidic channel is provided with a sealing film to prevent the sample from leaking out. The inlet of the lower layer microfluidic channel in the first L-shaped microfluidic channel 130 is connected to the bottom of the first sample loading chamber 120 away from the center of the chip body 1. The inlet of the lower layer microfluidic channel in the second L-shaped microfluidic channel 150 is connected to the bottom of the second sample loading chamber 140 away from the center of the chip body 1.

The first injection port 161, the second injection port 162 and the flow outlet 163 of the Y-shaped microfluidic channel 160 are all located at the upper part of the chip body 1. The first injection port 161 of the Y-shaped microfluidic channel 160 is connected to the outlet of the vertical microfluidic channel in the first L-shaped microfluidic channel 130, and the second injection port 162 of the Y-shaped microfluidic channel 160 is connected to the outlet of the vertical microfluidic channel in the second L-shaped microfluidic channel 150.

Specifically, as shown in Figure 5, the Y-type microfluidic channel 160 includes two upper inlet microfluidic channels and an upper mixing microfluidic channel. The inlet of one of the upper inlet microfluidic channels is the first sample inlet 161 of the Y-type microfluidic channel 160, and the inlet of the other upper inlet microfluidic channel is the second sample inlet 162 of the Y-type microfluidic channel 160. The outlets of the two upper inlet microfluidic channels meet at the inlet of the upper mixing microfluidic channel. The outlet of the upper mixing microfluidic channel is the outlet 163 of the Y-type microfluidic channel 160. The Y-type microfluidic channel 160 can be located in a plane perpendicular to the vertical microfluidic channel so that each upper inlet microfluidic channel is perpendicular to the corresponding vertical microfluidic channel.

The present application utilizes a pipeline geometric intersection design, by setting an L-shaped microfluidic channel that has its own pipeline intersection characteristics and setting a vertical microfluidic channel of the L-shaped microfluidic channel to intersect with the Y-shaped microfluidic channel in geometric space. When different microfluids flow through the corresponding L-shaped microfluidic channel and the upper inlet microfluidic channel of the Y-shaped microfluidic channel respectively, the corresponding microfluids are first split into many microclusters, and then the upper mixing microfluidic channel of the Y-shaped microfluidic channel is used to promote the mixing between different microfluidic microclusters.

As shown in FIG5 , the micro whole blood separation tank 110 is arranged along the radial direction of the chip. In order to have the function of detecting hematocrit, as shown in FIG5 , the micro whole blood separation tank 110 includes a plasma extraction groove 111 and a straight tube groove 112 connected to the plasma extraction groove 111. The straight tube groove 112 is located at one end of the micro whole blood separation tank 110 away from the center of the chip, and the plasma extraction groove 111 is located at the other end of the micro whole blood separation tank 110 close to the center of the chip. Specifically, the plasma extraction groove 111 in the micro whole blood separation tank 110 is used to receive the micro whole blood sample to be tested. Under the action of centrifugal force, the red blood cells obtained after the separation of the micro whole blood sample are all deposited at one end of the straight tube groove 112 away from the center of the chip, the first part of the plasma is contained in the plasma extraction groove 111, and the second part of the plasma is contained in the end of the straight tube groove 112 close to the center of the chip. In some examples, when the chip body 1 rotates at a speed of 2000-5000 rpm, the trace whole blood in the trace whole blood separation tank 110 is separated.

In order to facilitate manual reading, the surface of the straight tube groove 112 may be provided with a scale mark for reading the hematocrit. The scale mark is not shown in the figure.

In order to improve the sensitivity and accuracy of the reaction detection chamber, as shown in Figure 5, the reaction detection chamber 170 includes a cylindrical cavity and a tapered cavity with a gradually decreasing diameter. The large diameter end of the tapered cavity is connected to the bottom end of the cylindrical cavity.

As shown in FIG2 , a micro whole blood separation tank injection hole 201 for adding a micro whole blood sample is provided at one end of the top of the micro whole blood separation tank 110 near the center of the chip body 2. A first sample loading chamber injection hole 202 for adding plasma from the micro whole blood separation tank 110 is provided at one end of the top of the first sample loading chamber 120 near the center of the chip body 2. A second sample loading chamber injection hole 203 for adding a corresponding red blood cell reagent is provided at one end of the top of the second sample loading chamber 140 near the center of the chip body 2. The micro whole blood separation tank injection hole 201, the first sample loading chamber injection hole 202, and the second sample loading chamber injection hole 203 are all located on the upper layer 2 of the chip.

Preferably, as shown in FIG2 , the micro whole blood separation tank injection hole 201, the first sample loading chamber injection hole 202, and the second sample loading chamber injection hole 203 are all provided with a notch for ventilation. The effect is that when adding samples, the notch facilitates the exhaust of air, thereby preventing the sample from overflowing out of the hole. The shape of the notch can be U-shaped or V-shaped.

The detection method for reverse typing detection of ABO blood type using the blood detection microfluidic chip of this embodiment comprises the following steps:

Step 1: The micro whole blood separation tank 110 receives the micro whole blood sample to be tested.

Step 2: Under the action of centrifugal force, the red blood cells and plasma in the trace whole blood sample to be tested are separated, and the separated red blood cells are deposited on the end of the straight tube groove 112 away from the center of the chip, the first part of the plasma is contained in the plasma extraction groove 111, and the second part of the plasma is contained on the side of the straight tube groove 112 close to the center of the chip.

Step 3: After centrifugation, the plasma in the plasma extraction groove 111 is sucked and transferred to the first sample loading chamber 120 of each reaction test unit. The second sample loading chamber 140 of each reaction test unit receives the corresponding red blood cell reagent.

Step 4: Under the action of centrifugal force, the plasma in the first sample loading chamber 120 flows into the first sample inlet 161 of the Y-type microfluidic channel 160 through the first L-type microfluidic channel 130; the red blood cell reagent in the second sample loading chamber 140 flows into the second sample inlet 162 of the Y-type microfluidic channel 160 through the second L-type microfluidic channel 150. Under the action of centrifugal force, the plasma and the red blood cell reagent are mixed in the Y-type microfluidic channel 160 to form a first mixture, which flows out of the outlet 163 of the Y-type microfluidic channel 160 to the reaction detection chamber 170 and fully reacts in the corresponding reaction detection chamber 170 for 1 to 5 minutes. The action of centrifugal force can shorten the distance between red blood cells, promote the immune agglutination reaction of antibodies and red blood cell antigens, and enhance the strength of the immune agglutination reaction.

Step 5: Control the blood detection microfluidic chip to stop rotating, let it stand still, and read the detection result.

In this embodiment, the principle of judging whether an immune agglutination reaction occurs in each reaction detection chamber 170 is as follows: if the blood type antibody in the plasma to be tested reacts with the blood type antigen in the red blood cell reagent to form a red blood cell clot, the red blood cell clot will adhere vertically to the inner wall of the reaction detection chamber 170 due to centrifugal sedimentation under the action of centrifugal force. Afterwards, when the chip body is in a static state, the red blood cell clot will not collapse naturally within a certain period of time, that is, it will remain adhered to the inner wall of the reaction detection chamber 170 for a certain period of time. There are no unagglutinated red blood cells at the bottom of the reaction detection chamber 170, indicating that the result is positive.

If the blood type antibodies in the plasma to be tested do not undergo immune agglutination reaction with the blood type antigens in the red blood cell reagent, under the action of centrifugal force, the red blood cells that have not undergone immune agglutination reaction will also vertically adhere to the inner wall of the reaction detection chamber 170 due to centrifugal sedimentation. However, unlike red blood cell clots, the red blood cells that have not undergone immune agglutination reaction will naturally collapse and settle due to gravity after being left still for a period of time, that is, a large number of non-agglutinated red blood cells will form at the bottom of the reaction detection chamber 170, indicating that the result is negative.

For ABO blood type and Rh weak D antigen, during the static process after one centrifugation, the amount of red blood cell sedimentation or sedimentation speed at the bottom of the reaction detection chamber 170 is significantly less than that of the control reaction chamber. The control reaction chamber refers to the reaction detection chamber in the reaction test unit to which the O-type red blood cell reagent is added. At this time, the chip of the present application can directly enhance the immune agglutination reaction by repeating 2-3 centrifugation. Compared with the gel method, the chip of the present application can improve the sensitivity of immune agglutination reaction detection through repeated centrifugation to avoid the situation in which the positive and negative typing of the traditional microcolumn gel method is inconsistent due to weak antibodies.

In this embodiment, the test results can be obtained by naked eye interpretation or by microscopic photography combined with artificial intelligence analysis. Compared with naked eye judgment, the judgment method of microscopic photography combined with artificial intelligence analysis reduces the interference of human factors, and allows for review because the image can be permanently stored, which helps to improve the accuracy of the test results.

In this embodiment, since the bottom of the reaction detection chamber 170 is provided with a conical cavity, the unagglutinated red blood cells gather at the apex of the cone to form a sedimented red blood cell button. The sedimented red blood cell button provides a clearer, easier to read, more sensitive and accurate interpretation method.

The results of reverse typing blood typing are shown in Table 1.

Table 1. Judgment of blood typing results by reverse typing

Example 2

The present embodiment provides a blood detection microfluidic chip, which is used to detect the titer of IgG antibodies in HDN pregnant women. The chip includes a chip body 1 and a chip upper layer 2 covering the top surface of the chip body 1. FIG6 shows a schematic diagram of the three-dimensional structure of the chip body 1 in the blood detection microfluidic chip of the present embodiment. FIG7 shows a top view of the chip upper layer 2 of the present embodiment.

Fig. 8 shows a top view of the chip body 1 shown in Fig. 6. As shown in Fig. 8, the chip body 1 of this embodiment includes three separation detection units, and the three separation detection units are evenly distributed along the circumferential direction of the rotation center axis of the chip body 1. Each separation detection unit of this embodiment is equivalent to the second separation detection unit 102 in Fig. 8.

FIG9 shows a partial enlarged view of the second separation detection unit 102 in the chip body 1 shown in FIG8, wherein the dotted area represents a separation detection unit. As shown in FIG9, unlike Example 1, in each second separation detection unit 102 of this embodiment, the number of reaction test units is six. In addition, the chip body 1 of this embodiment also includes plasma multiple dilution pretreatment tanks 180a, 180b, 180c, 180d, 180e, 180f, which are arranged one by one with each reaction test unit, for preparing a series of plasmas with different dilution multiples, so that when the user uses the chip of this embodiment, there is no need to prepare plasma multiple dilution liquid preparation test tubes separately, and it helps to save consumables. Usually, plasmas with dilution multiples of 2, 4, 8, 16, 32, 64, etc. are used for anti-Rh blood type IgG antibody titer determination, and plasmas with dilution multiples of 64, 128, 256, 512, 1024, 2048, etc. are used for anti-A and anti-B blood type IgG antibody titer determination.

In this embodiment, the second sample loading chamber 140 of each reaction test unit receives the same red blood cell reagent, which is an A-type red blood cell reagent, a B-type red blood cell reagent, or an O-type RhD positive red blood cell reagent. The A-type red blood cell reagent and the B-type red blood cell reagent are suitable for detecting the titer of IgG antibodies for ABO blood types that do not conform to the HDN blood type. The O-type RhD positive red blood cell reagent is suitable for detecting the titer of IgG antibodies for Rh blood types that do not conform to the HDN blood type.

Specifically, the possible blood type of the fetus is first predicted based on the blood types of both parents, and then the corresponding known blood type red blood cell reagent is selected to determine the corresponding antibodies in the maternal blood. For example, if the pregnant woman is type A RhD negative and the father is type B RhD positive, the blood type of the fetus can be type A RhD positive, type A RhD negative, type B RhD positive, type B RhD negative, type AB RhD positive, type AB RhD negative, type O RhD positive or type O RhD negative. In this case, it is necessary to select type B red blood cell reagent and type O RhD positive red blood cell reagent to detect the anti-B and anti-RhD antibodies of the pregnant woman, that is, two separation detection units are required; if the father is type B RhD negative, the blood type of the fetus can be type A RhD negative, type B RhD negative, type AB RhD negative or type O RhD negative, then it is only necessary to select type B red blood cell reagent to detect the anti-B antibodies of the pregnant woman, that is, only one separation detection unit is required.

As shown in Fig. 7, in this embodiment, in addition to the micro-whole blood separation tank injection hole 201, the first sample loading chamber injection hole 202 and the second sample loading chamber injection hole 203, the chip upper layer 2 is also provided with six plasma multiple dilution pretreatment tank injection holes 204a, 204b, 204c, 204d, 204e, 204f. The plasma multiple dilution pretreatment tank injection holes 204a to 204f are sequentially arranged on the top of the plasma multiple dilution pretreatment tanks 180a to 180f, for example, the plasma multiple dilution pretreatment tank injection hole 204a is located at the top of the plasma multiple dilution pretreatment tank 180a, and the plasma multiple dilution pretreatment tank injection hole 204b is located at the top of the plasma multiple dilution pretreatment tank 180b, and the rest are similarly corresponding.

The detection method of using the blood detection microfluidic chip of this embodiment to detect the blood type IgG antibody titer of HDN pregnant women includes: Follow these steps:

Step 1: The micro whole blood separation tank 110 receives the micro whole blood sample to be tested.

Step 2: Under the action of centrifugal force, the red blood cells and plasma in the trace whole blood sample to be tested are separated, and the separated red blood cells are deposited on the end of the straight tube groove 112 away from the center of the chip, the first part of the plasma is contained in the plasma extraction groove 111, and the second part of the plasma is contained on the side of the straight tube groove 112 close to the center of the chip.

Step 3: After the centrifugation is completed, the plasma in the plasma extraction groove 111 is sucked and transferred to each plasma multiple dilution pretreatment tank in sequence.

Step 4: Pre-treat the plasma in the plasma dilution pre-treatment tank with a diluent containing dithiothreitol DTT or dithiothioethanol 2-ME, and let it stand for 15 to 30 minutes to destroy the activity of IgM antibodies. After the standing reaction is completed, add the sample diluent to complete the dilution to obtain the diluted plasma. The sample diluent can be PBS (phosphate buffered saline) or physiological saline.

Step 5: aspirate the diluted plasma in each plasma dilution pretreatment tank and transfer it to the first sample loading chamber 120 of the corresponding reaction test unit; the second sample loading chamber 140 of each reaction test unit receives the red blood cell reagent.

Step 6: Under the action of centrifugal force, in each reaction test unit, the doubly diluted plasma in the first sample loading chamber 120 flows into the first injection port 161 of the Y-type microfluidic channel 160 through the first L-type microfluidic channel 130; the red blood cell reagent in the second sample loading chamber 140 flows into the second injection port 162 of the Y-type microfluidic channel 160 through the second L-type microfluidic channel 150; under the action of centrifugal force, the doubly diluted plasma and the red blood cell reagent are mixed in the Y-type microfluidic channel 160 to form a first mixture, and the first mixture flows out to the reaction detection chamber 170 through the outflow port 163 of the Y-type microfluidic channel 160 and fully reacts in the reaction detection chamber 170 for 1 to 5 minutes.

Step 7: Control the blood detection microfluidic chip to stop rotating, let it stand still, and read the detection result.

In this embodiment, the judgment principle of whether an immune agglutination reaction occurs in each reaction detection chamber 170 is the same as the judgment principle in the first embodiment of the present application. If the blood type antibodies in the diluted plasma react with the blood type antigens in the red blood cell reagent to form red blood cell clots, the red blood cell clots will adhere vertically to the inner wall of the reaction detection chamber 170 due to centrifugal sedimentation under the action of centrifugal force. The red blood cell clots will not collapse naturally after being left standing for a certain period of time, that is, they will remain adhered to the inner wall of the reaction detection chamber 170. There are no unagglutinated red blood cells at the bottom of the reaction detection chamber 170, indicating that the result is positive.

If the blood type antibodies in the diluted plasma do not undergo an immune agglutination reaction with the blood type antigens in the red blood cell reagent, under the action of centrifugal force, the red blood cells that do not undergo an immune agglutination reaction will also vertically adhere to the inner wall of the reaction detection chamber 170 due to centrifugal sedimentation. However, unlike red blood cell clots, the red blood cells that do not undergo an immune agglutination reaction will naturally collapse and settle due to gravity after standing for a period of time, that is, non-agglutinated red blood cells are formed at the bottom of the reaction detection chamber 170, indicating a negative result. The reciprocal of the dilution multiple at which the plasma sample with the highest multiple dilution does not undergo an immune agglutination reaction is used as the blood type IgG antibody titer of the specific blood type antigen.

In this embodiment, the reaction detection chamber 170 of each reaction test unit may be pre-installed with anti-human globulin polyclonal antibody freeze-dried beads. The stability of freeze-dried ball reagents at room temperature is better than that of liquid reagents. Anti-human globulin antibodies are used as the second antibody to achieve the purpose of bridge It acts by linking specific antibodies that bind to red blood cell antigens, causing red blood cells to agglutinate.

In this embodiment, a conical cavity may also be provided at the bottom of the reaction detection chamber 170. Unagglutinated red blood cells gather at the apex of the cone to form a sedimented red blood cell button. The sedimented red blood cell button provides a clearer, easier to read, more sensitive and accurate interpretation method.

The present invention provides a blood detection microfluidic chip and its detection method. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims (10)

A blood detection microfluidic chip, characterized in that the chip comprises a chip body (1), the chip body (1) comprises more than one separation detection unit, each of the separation detection units comprises a micro whole blood separation tank (110) for receiving and separating a micro whole blood sample and more than one reaction test unit; when the chip body (1) rotates, the red blood cells separated from the micro whole blood sample settle at one end of the micro whole blood separation tank (110) away from the center of the chip body (1), and the plasma separated from the micro whole blood sample is located at the other end of the micro whole blood separation tank (110); each of the reaction test units comprises a first sample loading chamber (120) for receiving the plasma from the micro whole blood separation tank (110), a first L-shaped microfluidic channel (130), and a second sample loading chamber (120) for receiving a corresponding red blood cell reagent. 40), a second L-shaped microfluidic channel (150), a Y-shaped microfluidic channel (160) and a reaction detection chamber (170); the Y-shaped microfluidic channel (160) comprises a first injection port (161), a second injection port (162) and an outflow port (163); fluids respectively flowing into the first injection port (161) and the second injection port (162) are mixed through the Y-shaped microfluidic channel (160) and then discharged from the outflow port (163); the first L-shaped microfluidic channel (130) connects the first sample addition chamber (120) and the first injection port (161) of the Y-shaped microfluidic channel (160); the second L-shaped microfluidic channel (150) connects the second sample addition chamber (140) and the second injection port (162) of the Y-shaped microfluidic channel (160); the outflow port (163) of the Y-shaped microfluidic channel (160) is connected to the reaction detection chamber (170). A blood detection microfluidic chip according to claim 1, characterized in that the first L-shaped microfluidic channel (130) and the second L-shaped microfluidic channel (150) are both composed of a lower microfluidic channel and a vertical microfluidic channel connected in sequence, and the lower microfluidic channel is arranged vertically to the vertical microfluidic channel; the inlet of the lower microfluidic channel in the first L-shaped microfluidic channel (130) is connected to the bottom of the first sample loading chamber (120) on the side away from the center of the chip body (1); the inlet of the lower microfluidic channel in the second L-shaped microfluidic channel (150) is connected to the bottom of the second sample loading chamber (140) on the side away from the center of the chip body (1); The first sample inlet (161), the second sample inlet (162) and the outlet (163) of the Y-shaped microfluidic channel (160) are all located at the upper part of the chip body (1); the first sample inlet (161) of the Y-shaped microfluidic channel (160) is connected to the outlet of the vertical microfluidic channel in the first L-shaped microfluidic channel (130), and the second sample inlet (162) of the Y-shaped microfluidic channel (160) is connected to the outlet of the vertical microfluidic channel in the second L-shaped microfluidic channel (150); The Y-shaped microfluidic channel (160) comprises two upper inlet microfluidic channels and an upper mixing microfluidic channel; the inlet of one of the upper inlet microfluidic channels is the first sample inlet (161) of the Y-shaped microfluidic channel (160), the inlet of the other upper inlet microfluidic channel is the second sample inlet (162) of the Y-shaped microfluidic channel (160), the outlets of the two upper inlet microfluidic channels meet at the inlet of the upper mixing microfluidic channel, and the outlet of the upper mixing microfluidic channel is the outlet (163) of the Y-shaped microfluidic channel (160); each upper inlet microfluidic channel is arranged perpendicular to the corresponding vertical microfluidic channel. The blood detection microfluidic chip according to claim 2 is characterized in that the micro whole blood separation groove (110) is arranged along the radial direction of the chip; the micro whole blood separation groove (110) includes a plasma extraction groove (111) and a plasma extraction groove (112) connected to the plasma extraction groove (113). The groove (111) is connected to a straight tube groove (112); the straight tube groove (112) is located at one end of the micro whole blood separation groove (110) away from the center of the chip, and the plasma extraction groove (111) is located at the other end of the micro whole blood separation groove (110) close to the center of the chip; under the action of centrifugal force, the red blood cells obtained after the separation of the micro whole blood sample are all deposited at the end of the straight tube groove (112) away from the center of the chip. A blood detection microfluidic chip according to claim 3, characterized in that the surface of the straight tube groove (112) is provided with a scale mark for judging the hematocrit; and the volume of the micro whole blood separation groove (110) is 50-100ul. The blood testing microfluidic chip according to claim 1 is characterized in that the number of the reaction test units is six; and it also includes a plasma multiple dilution pretreatment tank arranged in a one-to-one correspondence with each of the reaction test units. A blood detection microfluidic chip according to any one of claims 1 to 5, characterized in that the reaction detection cavity (170) comprises a cylindrical cavity and a conical cavity with a gradually decreasing diameter, and the large diameter end of the conical cavity is connected to the bottom end of the cylindrical cavity. A detection method based on a blood detection microfluidic chip according to claim 2, characterized in that it comprises the following steps: the first microfluid in the first sample loading chamber (120) flows into the first injection port (161) of the Y-shaped microfluidic channel (160) through the first L-shaped microfluidic channel (130); the second microfluid in the second sample loading chamber (140) flows into the second injection port (162) of the Y-shaped microfluidic channel (160) through the second L-shaped microfluidic channel (150); After the first microfluid and the second microfluid are mixed in the Y-shaped microfluidic channel (160), a first mixture is formed, and the first mixture flows out to the reaction detection chamber (170) through the outlet (163) of the Y-shaped microfluidic channel (160). A method for performing ABO blood type reverse typing detection using a blood detection microfluidic chip according to claim 3, characterized in that the number of the reaction test units is three; the red blood cell reagents received by the second sample loading chamber (140) of each of the reaction test units are respectively A-type red blood cell reagent, B-type red blood cell reagent and O-type red blood cell reagent; the detection method comprises the following steps: Step 1: The micro whole blood separation tank (110) receives a micro whole blood sample to be tested; Step 2: under the action of centrifugal force, the red blood cells and plasma in the trace whole blood sample to be tested are separated, and the red blood cells obtained after separation are deposited on the end of the straight tube groove (112) away from the center of the chip, the first part of the plasma obtained after separation is contained in the plasma extraction groove (111), and the second part of the plasma obtained after separation is contained on the side of the straight tube groove (112) close to the center of the chip; Step 3: After the centrifugation is completed, the plasma in the plasma extraction groove (111) is sucked and transferred to the first sample loading chamber (120) of each reaction test unit; the second sample loading chamber (140) of each reaction test unit receives the corresponding red blood cell reagent; Step 4: Under the action of centrifugal force, the plasma in the first sample loading chamber (120) flows into the first sample inlet (161) of the Y-shaped microfluidic channel (160) through the first L-shaped microfluidic channel (130); the red blood cell reagent in the second sample loading chamber (140) flows into the second sample inlet (162) of the Y-shaped microfluidic channel (160) through the second L-shaped microfluidic channel (150); the plasma and the red blood cell reagent are mixed. After the reagents are mixed in the Y-shaped microfluidic channel (160), a first mixture is formed, and the first mixture flows out to the reaction detection chamber (170) through the outflow port (163) of the Y-shaped microfluidic channel (160) and fully reacts in the reaction detection chamber (170); Step 5: Let it stand and read the test results; If the blood type antibodies in the plasma to be tested undergo an immune agglutination reaction with the blood type antigens in the red blood cell reagent, a red blood cell clot is formed; under the action of centrifugal force, the red blood cell clot settles in a direction away from the center of the chip; when the chip is stationary, the red blood cell clot remains adhered to the inner wall of the reaction detection cavity (170) for a certain period of time; if the blood type antibodies in the plasma to be tested do not undergo an immune agglutination reaction with the blood type antigens in the red blood cell reagent, under the action of centrifugal force, the red blood cells that have not undergone an immune agglutination reaction settle in a direction away from the center of the chip; when the chip is stationary, the red blood cells that have not undergone an immune agglutination reaction collapse and settle naturally due to the action of gravity. A method for detecting the titer of blood type IgG antibodies of HDN pregnant women using a blood detection microfluidic chip as described in claim 3, characterized in that the number of the reaction test units is six; each reaction test unit is also provided with an independent plasma multiple dilution pretreatment tank for forming a series of multiple dilutions; the second sample loading chamber (140) of each reaction test unit receives the same red blood cell reagent, and the same red blood cell reagent is an A-type red blood cell reagent, a B-type red blood cell reagent or an O-type RhD positive red blood cell reagent; the detection method comprises the following steps: Step 1: the micro whole blood separation tank (110) receives the micro whole blood sample to be tested; Step 2: under the action of centrifugal force, the red blood cells and plasma in the trace whole blood sample to be tested are separated, and the red blood cells obtained after separation are deposited on the end of the straight tube groove (112) away from the center of the chip, the first part of the plasma obtained after separation is contained in the plasma extraction groove (111), and the second part of the plasma obtained after separation is contained on the side of the straight tube groove (112) close to the center of the chip; Step 3: After the centrifugation is completed, the plasma in the plasma extraction groove (111) is sucked and transferred to each of the plasma multiple dilution pretreatment grooves in sequence; Step 4: pretreating the plasma in each of the plasma multiple dilution pretreatment tanks with a diluent containing dithiothreitol (DTT) or dimercaptoethanol (2-ME), and allowing the reaction to stand for 15 to 30 minutes to destroy the activity of IgM antibodies; after the reaction is completed, adding the sample diluent to complete multiple dilution to obtain multiple diluted plasma; Step 5: aspirating the diluted plasma in each of the plasma dilution pretreatment tanks and transferring it to the first sample loading chamber (120) of the corresponding reaction test unit; the second sample loading chamber (140) of each of the reaction test units receives the red blood cell reagent; Step 6: Under the action of centrifugal force, in each of the reaction test units, the double-diluted plasma in the first sample loading chamber (120) flows into the first sample inlet (161) of the Y-shaped microfluidic channel (160) through the first L-shaped microfluidic channel (130); the red blood cell reagent in the second sample loading chamber (140) flows into the second sample inlet (162) of the Y-shaped microfluidic channel (160) through the second L-shaped microfluidic channel (150); the double-diluted plasma and the red blood cell reagent are mixed in the Y-shaped microfluidic channel (160) to form a first A mixture, wherein the first mixture flows out through the outlet (163) of the Y-shaped microfluidic channel (160) to the reaction detection chamber (170) and fully reacts in the reaction detection chamber (170); Step 7: Let it stand and read the test results; If the blood type antibodies in the plasma to be tested react with the blood type antigens in the red blood cell reagent to form a red blood cell clot, the red blood cell clot will settle in a direction away from the center of the chip under the action of centrifugal force; when the chip is stationary, the red blood cell clot will remain adhered to the inner wall of the reaction detection cavity (170) for a certain period of time; If the blood type antibodies in the plasma to be tested do not undergo immune agglutination reaction with the blood type antigens in the red blood cell reagent, under the action of centrifugal force, the red blood cells that have not undergone immune agglutination reaction will settle in the direction away from the center of the chip; when the chip is stationary, the red blood cells that have not undergone immune agglutination reaction will naturally collapse and settle due to gravity; the reciprocal of the dilution multiple at which the plasma sample with the highest dilution multiple does not undergo immune agglutination reaction is taken as the blood type IgG antibody titer against the specific blood type antigen. The method according to claim 9 is characterized in that anti-human globulin polyclonal anti-freeze-dried beads are pre-installed in the reaction detection chamber (170) of each reaction test unit.