An automated washing microfluidic chip for irregular antibody detection

Abstract

This invention relates to the field of irregular antibody detection technology, specifically to an automated washing microfluidic chip for irregular antibody detection. The chip includes a chip body, a membrane, and a detection component comprising a sample application chamber and a detection chamber arranged sequentially away from the center of rotation. The sample application chamber and the detection chamber are connected by a washing channel. The detection component is pre-filled with a washing solution that can fill the detection chamber and the washing channel under centrifugation. The washing solution contains anti-human immunoglobulin that reacts with erythrocyte antigen-antibody complexes. The specific gravity of the washing solution is configured to be greater than the specific gravity of the sample to be tested but less than the specific gravity of the erythrocyte antigen. This configuration solves the problems of cumbersome procedures, excessive time consumption, high operator dependence, and difficulty in standardization caused by multiple manual washing steps required in traditional test tube methods for detecting irregular antibodies. It effectively ensures detection sensitivity and accuracy and reduces the false negative rate.

Description

Technical Field

[0001] This invention relates to an automated washing microfluidic chip for irregular antibody detection, belonging to the field of irregular antibody detection technology. Background Technology

[0002] Irregular antibodies (accidental antibodies) refer to blood group antibodies other than anti-A and anti-B (such as Rh, Kidd, MNSs, etc.), mostly produced by immune stimulation such as blood transfusion, pregnancy, or transplantation. If these antibodies are not detected, they will bind to the recipient's red blood cells after transfusion of blood containing the corresponding antigens, triggering a hemolytic reaction. For example, the "Clinical Transfusion Technical Specifications" clearly stipulates that patients with a history of blood transfusion, pregnancy, or requiring multiple blood transfusions in a short period must undergo irregular antibody screening. Currently, the most commonly used detection method is to specifically bind red blood cells with known blood group antigens to irregular antibodies in the test sample (serum), forming red blood cell immune complexes. After washing to remove non-specific antibodies and other components from the sample, anti-human immunoglobulin (also known as secondary antibody) is added to bind with the immune complexes, ultimately forming a red blood cell-antibody-anti-human globulin antibody "sandwich" complex, which causes red blood cell agglutination (hemagglutination). The presence of irregular antibodies in the sample is determined by whether red blood cells agglutinate. In this reaction system, anti-human immunoglobulin antibodies can react not only with specific antibodies (irregular antibodies) bound to red blood cells, but also with non-specific immunoglobulins in the sample. This means that the abundant non-specific immunoglobulins in the sample also react with anti-human immunoglobulin antibodies, significantly consuming them and greatly reducing the number of anti-human immunoglobulins bound to red blood cells. This results in a substantial decrease in detection sensitivity, thus affecting detection accuracy. Therefore, in actual testing, red blood cell antigens specifically bind to irregular antibodies in the sample to form an immune response. After the antibody complex is detected, multiple washing steps are required to remove interfering components such as unbound non-specific immunoglobulins before adding anti-human immunoglobulins. This avoids interference, but the operation is time-consuming, has low detection efficiency, and requires manual intervention, increasing the complexity of the process and making it highly dependent on the operator. It is difficult to standardize and is prone to human error or contamination from contact with the external environment, which can affect the accuracy of the detection. Therefore, it is of great practical significance to study a novel automated washing microfluidic chip for the detection of irregular antibodies. Summary of the Invention

[0003] This invention addresses the shortcomings of existing technologies by providing an automated washing microfluidic chip for the detection of irregular antibodies.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: An automated washing microfluidic chip for irregular antibody detection includes a chip body with a mounting hole at its center. A film is attached and fixed to the surface of the chip body. The chip body also includes a detection component, which has several groups arranged in a ring array around the rotation center of the chip body. The detection component includes sample dispensing chambers and detection chambers arranged sequentially away from the rotation center. The sample dispensing chambers and the detection chambers are connected by a washing channel. The film has a sample dispensing hole corresponding to the position of the sample dispensing chamber. The detection component is pre-filled with a washing solution that can fill the detection chambers and washing channels under centrifugation. The washing solution contains anti-human immunoglobulin that reacts with the red blood cell antigen-antibody complex. The specific gravity of the washing solution is configured to be greater than the specific gravity of the sample to be tested and less than the specific gravity of the red blood cell antigen.

[0005] Furthermore, the centerline of the sample loading chamber, the centerline of the washing channel, and the centerline of the detection chamber are collinear with the same radial line passing through the center of rotation;

[0006] The sample loading chamber comprises a first zone, a second zone, and a third zone from near to far from the rotation center. The sidewalls of the first zone and the third zone are both arc-shaped, and the radius of curvature of the sidewall of the first zone is smaller than that of the sidewall of the third zone. The sidewall of the second zone is straight.

[0007] Furthermore, the second region sidewall gradually moves away from the radial line collinear with the center of rotation from near to far, and the angle α1 between the second region sidewall and the radial line collinear with the center of the sample dispensing cavity ranges from 6° to 11°.

[0008] Furthermore, the relationship between the horizontal projection length L1 from the first region to the center line of the sample dispensing cavity, the horizontal projection length L2 from the second region to the center line of the sample dispensing cavity, and the horizontal projection length L3 from the bottom edge contour of the third region to the center line of the sample dispensing cavity satisfies L1:L2:L3=1:(1.9-2.2):(1.55-1.75), and the sample dispensing hole is connected to the first region.

[0009] Furthermore, both the washing channel and the sample dispensing chamber are formed by a downward recess of a predetermined depth on the surface of the chip body, and the recess depth of the washing channel is less than the recess depth of the sample dispensing chamber.

[0010] The inlet end of the washing channel is connected to the third region, and the sidewall of the third region is inclined at a predetermined angle relative to the central axis of the chip body towards the washing channel.

[0011] Furthermore, a drainage section is provided at the proximal end of the washing channel, the drainage section extends obliquely downward and connects to the third zone, and the drainage section is composed of an inclined plane and arc-shaped sidewalls located on both sides of the inclined plane;

[0012] The inclined plane gradually tapers inward from the third zone toward the washing channel. The inclined plane is connected to both the third zone and the washing channel in an arc transition, and its bottom edge connected to the third zone is set to be an arc facing the washing channel.

[0013] Furthermore, the arc-shaped sidewall is an inward-facing arc, which connects with the bottom edge of the inclined plane to form a pointed auxiliary drainage channel.

[0014] Furthermore, the angle β between the inclined plane and the central axis of the chip body is 1.4-1.8 times the inclination angle α2 of the third region sidewall relative to the central axis of the chip body, and the inclination angle α2 satisfies: 15°≤α2≤20°.

[0015] Furthermore, the relationship between the horizontal projection length H1 of the inclined plane on the central axis of the chip body, the washing channel depth H2, and the sample dispensing chamber depth H3 satisfies H1:H2:H3=(0.35-0.45):(0.1-0.2):1.

[0016] Furthermore, the relationship between the washing channel length S, the washing channel width K, and the angle β between the inclined plane and the central axis of the chip body satisfies... In the formula, This is a correction factor, with a value of 1.0; The standard included angle is 90°.

[0017] The beneficial effects of this invention are as follows: This application integrates sample addition, reaction, washing, and detection functions into one unit through the above-described configuration. Automatic washing is achieved through centrifugation. Within a closed microfluidic channel, unbound non-specific immunoglobulins and other interfering substances can be efficiently and automatically isolated and removed. This solves the problems of cumbersome procedures, excessive time consumption, strong operator dependence, and difficulty in standardization caused by the multiple manual washing steps required in traditional test tube methods for detecting irregular antibodies. Without sacrificing the high throughput and simplicity advantages of microfluidic technology itself, it effectively ensures detection sensitivity and accuracy while reducing the false negative rate. Secondly, by simplifying multiple manual washing operations into a single centrifugation process, a one-stop detection workflow can be achieved without manual intervention, eliminating the tedium of manual operation, simplifying the process, reducing operational difficulty, minimizing the impact of human error on test results, significantly shortening detection time, effectively improving detection efficiency, and reducing the risk of contamination caused by contact between the sample and the external environment. Finally, the unified centrifugation operating parameters among the various detection components of the microfluidic chip ensure consistent washing conditions for each component, achieving detection standardization and improving the consistency and repeatability of test results. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the three-dimensional structure of the microfluidic chip provided in Embodiment 1 of the present invention;

[0020] Figure 2 This is a schematic diagram of the three-dimensional structure of the chip body provided in Embodiment 1 of the present invention;

[0021] Figure 3 This is a cross-sectional view of the chip body provided in Embodiment 1 of the present invention;

[0022] Figure 4 This is a schematic diagram of the detection component structure provided in Embodiment 1 of the present invention;

[0023] Figure 5 for Figure 2 Enlarged view of the structure at point A in the middle;

[0024] Figure 6 This is a partial cross-sectional view of the chip body provided in Embodiment 1 of the present invention.

[0025] Reference numerals: 0, Rotation center; 1, Film application; 2, Chip body; 3, Mounting hole; 4, Detection component; 41, Sample application chamber; 411, First zone; 412, Second zone; 413, Third zone; 42, Detection chamber; 43, Washing channel; 44, Drainage section; 441, Inclined plane; 4411, Bottom edge; 442, Arc-shaped sidewall; 443, Auxiliary drainage channel. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0028] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances. Furthermore, the terms "first," "second," and "third," etc., are used only for distinguishing descriptions and should not be construed as indicating or implying relative importance.

[0029] In this invention, unless otherwise expressly specified and limited, "above or below" a first feature may include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on" the first feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the first feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0030] Example 1

[0031] like Figure 1-5As shown, the present invention provides an automated washing microfluidic chip for irregular antibody detection, comprising a chip body 2 with a mounting hole 3 at its center, a film 1 fixedly attached to the surface of the chip body 2, and a detection component 4 having several groups arranged in a ring around the rotation center O of the chip body 2, including a sample application chamber 41 and a detection chamber 42 arranged sequentially away from the rotation center O, the sample application chamber 41 and the detection chamber 42 being connected by a washing channel 43, the film 1 having a sample application hole corresponding to the position of the sample application chamber 41, and the detection component 4 pre-filled with a washing solution that can fill the detection chamber 42 and the washing channel 43 under centrifugation, the washing solution containing anti-human immunoglobulin that reacts with the red blood cell antigen-antibody complex, the specific gravity of the washing solution being configured to be greater than the specific gravity of the sample to be tested and less than the specific gravity of the red blood cell antigen.

[0032] It should be noted that the mounting hole 3 is used to connect a centrifuge device, which is used to provide centrifugal power for the automatic washing microfluidic chip. The centrifuge device is a technology known to those skilled in the art and will not be described in detail here. In this invention, the "distal end" and "proximal end" are determined by their distance from the rotation center O, which will not be described in detail hereafter. The specific gravity of the serum sample to be tested is 1.025-1.030, the specific gravity of the red blood cells is 1.090-1.092, and the specific gravity of the washing solution is configured to be 1.035-1.040.

[0033] During testing, erythrocyte antigen is first added to the serum sample to be tested. This application uses an irregular antibody screening reagent for human erythrocytes. The samples are then mixed. If irregular antibodies are present in the sample, they react with the erythrocyte antigen to form an erythrocyte antigen-irregular antibody complex. Then, under centrifugal force, the specific gravity of non-specific antibodies and other interfering substances in the sample is less than that of the washing solution. Due to the liquid-liquid interface, these substances cannot enter the washing channel 43, effectively isolating non-specific antibodies and other interfering substances. The erythrocyte antigen-antibody complex, being greater than the washing solution, can pass through the washing channel 43 filled with washing solution and react with anti-human immunoglobulin during its passage through the washing channel 43, thus forming an erythrocyte antigen-antibody-anti-human immunoglobulin complex. The sandwich-like complex of immunoglobulin undergoes hemagglutination (hemagglutination) at this point. Under centrifugal force, the complex passes through the washing channel 43 and then adheres to the detection chamber 42 after centrifugation. After standing for a period of time, the red blood cells remain agglutinated on the inner wall of the detection chamber 42 without collapsing and settling, and the result is judged as positive. If there are no irregular antibodies in the sample to be tested, no immune reaction will occur. The red blood cell antigen passes through the washing channel 43 alone and adheres to the detection chamber 42 after centrifugation. After standing for a period of time, the red blood cells that have not undergone an immune reaction will not agglutinate on the inner wall of the detection chamber 42, but will naturally collapse and settle at the bottom of the detection chamber 42, and the result is judged as negative. It should be noted that anti-human immunoglobulin can also be pre-placed in the detection chamber 42, and its specific gravity can be adjusted to be no less than that of the washing solution.

[0034] This application integrates sample addition, reaction, washing, and detection functions into one unit through the above settings. Automatic washing is achieved through centrifugation, and within a closed microfluidic channel, unbound non-specific immunoglobulins and other interfering substances are efficiently and automatically isolated and removed. This solves the problems of cumbersome procedures, excessive time consumption, strong operator dependence, and difficulty in standardization caused by the multiple manual washing steps required in traditional test tube methods for detecting irregular antibodies. Without sacrificing the high throughput and simplicity advantages of microfluidic technology, it firstly effectively ensures detection sensitivity and accuracy, reducing the false negative rate; secondly, because... The process simplifies multiple manual washing operations into a single centrifugation operation, enabling a one-stop detection process from sample input to result output without human intervention. This avoids the tedium of manual operation, simplifies the process, reduces operational difficulty, minimizes the impact of human error on test results, significantly shortens detection time, effectively improves detection efficiency, and reduces the risk of contamination caused by contact between the sample and the external environment. Finally, the unified centrifugation operating parameters among the various detection components 4 of the microfluidic chip ensure consistent washing conditions for each component 4, achieving detection standardization and improving the consistency and repeatability of test results.

[0035] Specifically, such as Figure 4As shown, the centerline of the sample loading chamber 41, the centerline of the washing channel 43, and the centerline of the detection chamber 42 are collinear with the same radial line passing through the rotation center O. The dashed line X in the attached figure represents the radial line passing through the rotation center O. The sample loading chamber 41, relative to the rotation center O, includes, from near to far, a first region 411, a second region 412, and a third region 413. The sidewalls of the first region 411 and the third region 413 are both arc-shaped, and the radius of curvature of the sidewall of the first region 411 is smaller than that of the sidewall of the third region 413. The sidewall of the second region 412 is straight. It should be noted that the dashed lines in the figure are only used to distinguish between the first region 411 and the second region 412. Zones 412 and 413 are not part of the structure; by setting the centerlines collinear with the same radial line, they can match the radial outward action direction of centrifugal force, effectively reducing the resistance of the sample flow between the chamber and the channel, making the flow velocity more stable. Furthermore, by setting the first zone 411 with an arc-shaped sidewall 442 with a small radius of curvature, it can guide the sample to gather in a low centrifugal force region during the initial stage of centrifugation and generate a relatively stable vortex field, which can gently disturb the sample, ensuring that the erythrocyte antigen remains suspended, thus enabling sufficient binding between the erythrocyte antigen and irregular antibodies within a limited time. The first step involves setting the sidewall of the second zone 412 to be linear, which can buffer the flow state of the sample to be tested. Within the second zone 412, the sample to be tested tends to transition from a vortex state to a laminar flow state, guiding the erythrocyte antigen and its complexes towards the washing channel 43. During this process, it continues to bind with irregular antibodies, preventing the erythrocyte antigen and its complexes from being thrown towards and retained on the sidewall of the first zone 411 due to excessive vortex. Furthermore, by setting the third zone 413 to have an arc-shaped sidewall 442 with a large radius of curvature, the sample to be tested is guided into the washing channel 43 by the rotational centrifugation action of the microfluidic chip. In the third zone 413, the sample under test, whose flow state gradually stabilizes, generates a low-energy vortex field. This field is used to agitate and suspend the erythrocyte antigen-antibody complex, ensuring that the erythrocyte antigen-antibody complex can smoothly enter the washing channel 43. This prevents the erythrocyte antigen-antibody complex from settling in the third zone 413 due to its excessive density. Furthermore, because the sidewall of the third zone 413 is designed with a large radius of curvature, the dynamic stability of the washing solution interface is not affected when the complex is agitated. This design allows the erythrocyte antigen to fully combine with the irregular antibody to form a complex before entering the washing channel 43, achieving an orderly process of first combining and then washing.

[0036] Specifically, such as Figure 4As shown, the sidewall of the second region 412 gradually moves away from the radial line collinear with the center of rotation 0 from near to far, and the angle α1 between the sidewall of the second region 412 and the radial line collinear with the center of the sample filling cavity 41 ranges from 6° to 11°. Driven by centrifugal force, the sample flowing out of the first zone 411 already possesses an initial radial velocity. However, at this point, the flow velocity and the distribution of erythrocyte antigens and complexes are uneven. If the second zone 412 has a radial straight wall (α1 = 0°), the sample will maintain its original state and pass through at high speed, preserving or even amplifying its internal inhomogeneity. If the second zone 412 is designed to gradually converge, although it can accelerate the sample, it will exacerbate shear stress and cause excessive aggregation of erythrocyte antigens and their mixtures at the center line, failing to provide a buffering effect. This application, through the above settings, gives the second zone 412 a small divergence angle, enabling it to better serve as a flow stabilizer and buffer, forming an acceleration-buffering-re-acceleration flow pattern in conjunction with the first zone 411 and the third zone 413. When the sample enters the second zone 412, the flow velocity will decrease slightly, and the dynamic pressure will be converted into static pressure, allowing the flow field to expand and stabilize. This setting both homogenizes the flow velocity distribution and weakens the velocity gradient between the center and edge of the sample fluid, resulting in a more uniform distribution of erythrocyte antigens and their mixtures. Within the flow channel cross-section, this further promotes the binding between erythrocyte antigens and irregular antibodies, ensuring they enter the subsequent washing and reaction stages in a uniform and dispersed form. It also releases shear stress, reducing the shear rate within the sample fluid and minimizing the risk of mechanical dissociation of the fragile erythrocyte antigen-irregular antibody complex due to high-speed shearing, thus further ensuring detection accuracy. Furthermore, it optimizes the flow direction, providing a more ideal incident flow state for the sample fluid entering the subsequent third zone 413, preventing the sample from directly impacting the arc wall of the third zone 413, thereby further avoiding excessive external force damage to the complex. If α1 is less than 6°, the dispersion effect is too weak, almost like a straight wall, and the flow rate homogenization and decompression effects are not significant, failing to effectively solve the problem of uneven distribution of erythrocyte antigens and their mixtures within the sample. If α1 is greater than 11°, the diffusion is too fast, the flow rate drops too much, resulting in excessive flow energy loss, and even generating flow separation and backflow zones at the sidewalls, disrupting laminar flow and introducing new unstable factors and complex retention zones.

[0037] Specifically, such as Figure 4As shown, the horizontal projection length L1 from the first region 411 to the center line of the sample dispensing cavity 41, the horizontal projection length L2 from the second region 412 to the center line of the sample dispensing cavity 41, and the horizontal projection length L3 from the bottom edge 4411 of the third region 413 to the center line of the sample dispensing cavity 41 satisfy L1:L2:L3=1:(1.9-2.2):(1.55-1.75), and the sample dispensing hole is connected to the first region 411.Through the above settings, the reaction pathway of rapid mixing-mild extended reaction-continuous suspension for binding is further optimized. The first zone 411, serving as the inlet and initial mixing zone, ensures that the test sample and erythrocyte antigen rapidly enter the main chamber under centrifugal force from the start, avoiding prolonged static diffusion at the inlet and achieving initial rapid mixing. If the first zone 411 is too long, it will cause ineffective residence of the test sample in the low centrifugal force zone, hindering rapid entry into the main processing flow. Then, by setting the second zone 412 to have sufficient length, it works in conjunction with the 6°-11° divergence angle set above to form a relatively low-shear, long-residence-time reaction extension, allowing the test sample... This fluid completes the transition from turbulent to laminar flow, fully releasing shear stress. Erythrocyte antigens, irregular antibodies, and their complexes are situated in a mild environment of low shear, long path, and parallel flow. The lower shear force protects the already formed fragile antigen-antibody complexes, especially ensuring that weakly bound complexes are not destroyed. The longer side-by-side flow path provides more opportunities for low-concentration or low-affinity irregular antibodies in the sample to collide and bind with erythrocyte surface antigens, directly increasing the yield of effective complexes and improving detection sensitivity, particularly crucial for detecting weak reactions, thus improving detection accuracy. If the length of the second zone 412 is too small, for example less than 1.9L... 1. If the sample fluid is rushed into the third zone 413 at high speed before it has had sufficient time to stabilize, the third zone 413 will not only fail to effectively generate beneficial micro-vortices for suspension, but will also cause destructive impacts and secondary separation due to the unstable incoming flow. This will cause the red blood cell antigen-antibody complex to settle in the third zone 413 or even be destroyed by violent collisions. By setting the third zone 413 to have a central length, the micro-vortices generated when the sample enters the third zone 413 are not violent stirring, but a gentle disturbance. This can prevent the heavier red blood cell antigens and complexes from settling due to gravity before reaching the inlet of the washing channel 43, thus ensuring that most of the red blood cell antigens and complexes settle. All materials can be transported to subsequent stages, further ensuring detection accuracy. Gentle agitation can also agitate erythrocyte antigens, exposing unbound antigen sites on their surface and allowing them to come into contact with residual irregular antibodies, further consolidating the binding. If the third region 413 is too short, for example, less than 1.55L3, its guiding and suspending effects are insufficient, easily leading to sedimentation problems. If L3 is too long, for example, greater than 1.75L1, energy loss may occur due to flow path redundancy, and an excessively long arc-shaped path can easily cause excessively strong micro-vortices or evolve into unfavorable flow patterns. Through the synergistic cooperation of the above settings, this application can detect irregular antibodies with a titer of 1:128, effectively improving detection sensitivity.

[0038] Specifically, such as Figure 3 , 5As shown in Figure 6, both the washing channel 43 and the sample loading chamber 41 are formed by a downward recess of a predetermined depth on the surface of the chip body 2. The recess depth of the washing channel 43 is less than the recess depth of the sample loading chamber 41. The inlet end of the washing channel 43 is connected to the third region 413. The sidewall of the third region 413 is inclined at a predetermined angle relative to the central axis of the chip body 2 towards the washing channel 43. It should be noted that... Figure 6 The dashed line Y is parallel to the central axis of the chip body 2. By setting the depth of the washing channel 43 to be less than the depth of the sample loading chamber 41, the deeper sample loading chamber 41 provides sufficient volume to accommodate the sample to be tested and allows the sample fluid to flow at a lower average flow rate, which helps to reduce shear force, protect the fragile antigen-antibody complex, and because the red blood cell antigen and its complex with irregular antibodies have a large specific gravity, this setting prolongs the reaction contact time, further promotes the binding degree of red blood cell antigen and irregular antibody, and can also effectively reduce the probability of unbound or partially bound red blood cell antigen prematurely entering the washing channel 43. The shallower washing channel 43 helps to establish a stable washing interface under centrifugal force, because the velocity gradient of the shallow fluid in the depth direction is smaller, which can reduce shear disturbance at the interface, thereby maintaining the stability of the washing liquid interface. Secondly, the flow rate change caused by the depth difference creates a certain negative pressure at the inlet of the washing channel 43, which can attract the red blood cell antigen-antibody complex at the bottom of the sample loading chamber 41 and reduce its retention in the sample loading chamber 41; further through coordination Compared to the chip's central axis, the sidewall of the third zone 413 is inclined towards the washing channel 43, which serves as a guide. In the centrifugal force field, this inclined wall decomposes the radial centrifugal force into components perpendicular to and parallel to the wall. The parallel component causes the heavier erythrocyte antigen-antibody complex to slide along the wall towards the washing channel 43, thereby further reducing the amount of complex retained in the sample loading chamber 41. This ensures that more formed complexes can enter the subsequent washing steps, improving the utilization rate of effective complexes. Furthermore, the inclined wall guides the complexes to enter at a smaller angle to the channel axis, avoiding violent collisions between the complexes and the upper edge or sidewall of the washing channel 43 inlet, reducing the risk of mechanical damage to the complexes, and thus ensuring the effectiveness of the complexes. Therefore, through the above settings, problems such as complex retention, complex damage, and washing solution interface disturbance caused by abrupt changes in cross-section and flow direction during the transfer of erythrocyte antigens and their complexes from the sample loading chamber 41 to the washing channel 43 are effectively reduced, further ensuring the accuracy of detection.

[0039] Specifically, such as Figure 2-5As shown, a drainage section 44 is provided at the proximal end of the washing channel 43. The drainage section 44 extends obliquely downward and connects to the third zone 413. The drainage section 44 is composed of an inclined plane 441 and arc-shaped sidewalls 442 located on both sides of the inclined plane 441. The inclined plane 441 gradually tapers inward from the third zone 413 toward the washing channel 43. The inclined plane 441 is connected to the third zone 413 and the washing channel 43 in an arc transition. The bottom edge 4411 of the inclined plane 441 connected to the third zone 413 is set to be arc-shaped toward the washing channel 43.This design firstly solves the problem of flow separation vortices easily generated at the connection between the sample loading chamber 41 and the washing channel 43. When the sample fluid flows from the third zone 413 of the larger sample loading chamber 41 to the smaller washing channel 43, the guide section 44, as a smooth and gradually narrowing guide structure, guides the sample fluid to accelerate smoothly, with the streamlines closely adhering to the wall surface, thus suppressing the generation of separation vortices to the greatest extent and ensuring the continuity of flow and the high efficiency of energy transfer. Secondly, the inclined plane 441 is further coordinated with the third zone 413 and the washing channel 43, all connected by an arc transition, and the bottom... The edge 4411 is set to be arc-shaped towards the washing channel 43, which solves the problem of red blood cell antigen-antibody complexes being damaged or randomly bouncing due to collisions with sharp edges at the entrance of the washing channel 43. The full arc transition eliminates all sharp edges, providing a non-impact contact surface for the complex. The complex slides in along the arc-shaped wall, and the momentum direction is continuously and gently changed, thereby greatly reducing the risk of mechanical damage to the complex before entering the washing channel 43. Finally, because small-sized or weakly agglutinated red blood cell antigen-antibody complexes have low individual kinetic energy under the same centrifugal acceleration due to their small mass, they are very prone to failure to penetrate effectively. At the washing fluid interface, fluid trapped at the interface or carried back by the wash fluid can lead to false negatives. This application addresses this issue by using a gradually inward-curving inclined plane 441 and arc-shaped sidewalls 442 to form a three-dimensional constricted flow channel. The sample fluid is forcibly accelerated as it passes through, and small-sized or weakly aggregated complexes completely follow the fluid movement, thus passively increasing their velocity to match the local flow velocity. This results in significantly higher kinetic energy than within the sample loading chamber 41. The arc-shaped sidewalls 442 then act as a focusing mechanism, converging the sample fluid from the sample loading chamber 41 horizontally towards the centerline. This allows small-sized or weakly aggregated complexes, which might otherwise be dispersed, including those in low-velocity areas at the edges, to be concentrated. Weakly aggregated complexes are concentrated in the central high-speed region and collectively accelerated, avoiding the problem of insufficient kinetic energy for some complexes located in the low-speed region at the edge of the flow field. When the aggregated, accelerated, and oriented complexes impact the liquid-liquid interface in a high-speed, collinear jet form, not only does the individual kinetic energy of the complexes increase, but also the interfacial tension impedance per unit area is effectively reduced due to the aggregation of the complexes. This greatly improves the overall penetration success rate and effectively overcomes the problem of complex retention or backflow caused by small size and weak aggregation. As a result, the false negative rate of the automatic washing microfluidic chip described in this application is ≤0.5% when performing detection.

[0040] Specifically, such as Figure 4 , 5As shown, the arc-shaped sidewall 442 is an inward-facing arc, which connects to the bottom edge 4411 of the inclined plane 441 to form a pointed auxiliary flow channel 443. The auxiliary flow channel 443 is a locally refined design in the bottom region where the inclined plane 441 intersects the arc-shaped sidewall 442. First, it can generate a guiding boundary layer flow effect because the sample fluid near the pointed wall forms a boundary layer due to viscosity. The pointed auxiliary flow channel 443 can adsorb and guide this boundary layer, thereby stabilizing it, preventing premature separation, reducing flow resistance and energy loss caused by boundary layer separation, and minimizing shear stress pulsations generated by a stable boundary layer. This effectively reduces the disturbance energy transferred to the washing liquid interface. Furthermore, the auxiliary flow channel 443 clearly guides the edge flow, avoiding interference with the sample fluid. The problem of random eddies impacting the wall surface at the edge of the sample fluid is addressed. Secondly, guided by the auxiliary drainage channel 443, the movement direction of the erythrocyte antigen-antibody complex is adjusted to have a certain angle between it and the interface of the washing channel 43, allowing it to obliquely cut into the washing solution interface. Under the premise of ensuring that the complex has sufficient kinetic energy, the oblique impact can avoid the complex directly hitting the washing solution interface, greatly reducing the instantaneous impact pressure on the macroscopic morphology of the interface, reducing the risk of overall interface fluctuation, and avoiding the non-specific interferences being mixed into the washing solution due to interface rupture and local mixing of sample solution and washing solution, which would affect the sensitivity and accuracy of detection.

[0041] Specifically, such as Figure 6As shown, the angle β between the inclined plane 441 and the central axis of the chip body 2 is 1.4-1.8 times the inclination angle α2 of the sidewall of the third region 413 relative to the central axis of the chip body 2, and the inclination angle α2 satisfies: 15°≤α2≤20°. It should be noted that the included angle β is the angle between the contour line of the inclined plane 441 on the radial section of the center of the detection component 4 and the central axis of the chip body 2; the included angle α2 is the angle between the contour line of the third region 413 on the radial section of the center of the detection component 4 and the central axis of the chip body 2. With this setting, the inclination of the sidewall of the third region 413 provides initial guidance, initially deflecting the radial movement of the sample fluid to be tested towards the direction of the washing channel 43. Then, the inclined plane 441 of the drainage part 44, based on this, completes a more abrupt turning and acceleration, accurately introducing the red blood cell antigen antibody complex into the narrow washing channel 43, effectively limiting the occurrence of harmful flow separation, ensuring the smooth passage of the complex in the critical transition area, directly reducing the risk of retention and loss. It can avoid the formation of a vortex area at or below the inclination plane 441, thereby avoiding the problem of the complex getting stuck in the vortex area and being retained, and also ensures a stable wall pressure distribution, without extreme low-pressure areas, reducing the risk of pressure sudden changes. This reduces the possibility of complex trajectory disorder; it also allows the kinetic energy of the main stream of the sample to be tested to be more effectively transferred to the near-wall fluid, driving all complexes forward; if the included angle β is less than 1.4 times the included angle α2, the inclined plane 441 is too gentle and cannot complete sufficient deflection and acceleration within the limited length of the drainage section 44. The complexes contained in the sample fluid are very likely to partially collide with the upper edge of the inlet of the washing channel 43 due to insufficient turning, or lack kinetic energy due to insufficient acceleration, affecting subsequent penetration of the washing liquid interface; if the included angle β is greater than 1.8 times the included angle α2, the inclined plane 441 is too steep, and the sample fluid is very likely to experience flow separation problems, causing the fluid to be unable to follow the wall curvature and detach from the wall, forming a low-pressure vortex zone behind the wall, which not only causes energy loss, but also captures and retains the complexes. Preferably, in order to further ensure the dynamic stability of the washing liquid interface, after the washing liquid fills the detection cavity 42 and the washing channel 43 under centrifugal action, its interface is located in the region of the inclined plane 441.

[0042] Specifically, such as Figure 6As shown, the horizontal projection length H1 of the inclined plane 441 on the central axis of the chip body 2, the depth H2 of the washing channel 43, and the depth H3 of the sample loading chamber 41 satisfy H1:H2:H3=(0.35-0.45):(0.1-0.2):1. Because the complex that penetrates the washing liquid interface needs to undergo a swimming-like movement process in the washing liquid channel, it must maintain a stable laminar flow state during this process to avoid a decrease in the probability of contact with anti-human immunoglobulin or inconsistent reaction time due to turbulence, tumbling, or uneven velocity distribution. This application limits the washing channel 43 to a shallow depth H2, which is beneficial for forming a stable liquid-liquid interface, reducing the velocity gradient and disturbance in the depth direction, effectively ensuring the dynamic stability of the interface, and making it easy for the washing channel 43 to maintain a laminar flow state. Its shallow depth greatly inhibits... The velocity gradient and secondary flow that may form perpendicular to the flow direction ensure that the local flow velocity and direction are highly consistent when the complex moves in the washing channel 43. This provides each complex with a nearly identical hydrodynamic microenvironment for contact with the anti-human immunoglobulin in the surrounding washing solution, effectively promoting the reaction between the erythrocyte antigen-antibody complex and the anti-human immunoglobulin, thereby further ensuring detection accuracy. If the depth H2 of the washing channel 43 is less than 0.1H3, the washing channel 43 is easily blocked by tiny impurities or air bubbles, and the flow resistance is too high, requiring excessive centrifugal force to drive it. If the depth H2 of the washing channel 43 is greater than 0.2H3, the channel cross-section increases, the flow velocity decreases, which is not conducive to maintaining a stable laminar interface and weakens the effect of swimming-style washing. Furthermore, by limiting the horizontal projection length H1 of the inclined plane 441, which works in conjunction with the inclination angle β, sufficient spatial distance is ensured to complete the reshaping and calibration of the test sample fluid with a certain velocity and direction distribution. This allows the erythrocyte antigen-antibody complex to be sufficiently accelerated, and the motion state of the complex is adjusted to be faster horizontally and with a higher flow direction when entering the washing channel 43. The vertical velocity component is extremely small, ensuring that the complex clusters entering the washing channel 43 are in a stable laminar flow state that is almost parallel to the bottom of the washing channel 43. This allows the kinetic energy of the complex to be used efficiently to overcome interfacial tension without generating strong disturbances in the depth direction of the washing channel 43. This further promotes the reaction between the erythrocyte antigen antibody complex and the anti-human immunoglobulin. Through this setting, this application ensures the flow stability and reaction uniformity from dynamic penetration to static reaction, thereby ensuring that the complexes that successfully penetrate the washing liquid cross-section barrier can react with the anti-human immunoglobulin under optimal fluid conditions.

[0043] Specifically, such as Figure 5 , 6As shown, the relationship between the length S of the washing channel 43, the width K of the washing channel 43, and the angle β between the inclined plane 441 and the central axis of the chip body 2 satisfies In the formula, This is a correction factor, with a value of 1.0; The standard included angle is 90°, with S in mm and K ranging from 0.8 mm to 1 mm. This invention utilizes the synergy of these three parameters. When the length S of the washing channel 43 increases, under the same centrifugation conditions, it leads to increased flow resistance. If the width K remains constant or decreases, a significant increase in centrifugal force is required to drive the erythrocyte antigen-antibody complex through the longer washing channel 43. However, this increases the shear force on the fragile antigen-antibody complex, thus reducing detection accuracy. By simultaneously increasing the width K of the washing channel 43, the cross-sectional area of ​​the channel is increased, significantly reducing flow resistance. This offsets the increased resistance caused by the increased length S of the washing channel 43, ensuring that the flow resistance remains within a reasonable range of centrifugal force. While maintaining a stable detection process, the wider inlet at this point results in a wider lateral distribution of the impact interface of the erythrocyte antigen-antibody complex, leading to a decrease in the dynamic stability of the liquid-liquid interface. When the complex penetrates the interface laterally, the weak dynamic stability of the interface makes it easy for non-specific interfering substances to be entrained and mixed into the washing solution, thereby affecting the sensitivity and accuracy of the detection. By further reducing the angle β between the inclined plane 441 and the central axis of the chip body 2, the movement direction of the erythrocyte antigen-antibody complex tends to be more parallel to the axis of the washing channel 43, that is, more parallel to the liquid-liquid interface. This enhances the effect of oblique entry, reduces the vertical impact component of the complex on the interface, and can effectively compensate for the interface disturbance risk caused by the widening of the washing channel 43.

[0044] Example 2

[0045] The fabrication method of the microfluidic chip described in Embodiment 1 of this application is as follows:

[0046] S1. Adhere and fix the film 1 and the chip body 2 together;

[0047] S2. Prepare the washing solution: Add 10ml of glycerol, 10g of PEG (polyethylene glycol) with a molecular weight of 800, 10ml of gelatin, preservative (0.01% proclin 300), and 2ul of anti-human globulin antibody to every 100ml of aqueous solution.

[0048] S3. Add the prepared washing solution to the sample chamber of the microfluidic chip, centrifuge at 3000-3500 rpm for 30-40 seconds, and set aside.

[0049] The washing solution prepared by this method not only meets the specific gravity requirements, but also stabilizes the liquid-liquid interface through the thickening properties of gelatin. At the same time, polyethylene glycol can promote the immune response, solving the problems of easy interface damage and lack of immune enhancement effect caused by traditional physiological saline washing solutions.

[0050] Example 3

[0051] The detection method using the microfluidic chip described in Embodiments 1 and 2 of this application is as follows:

[0052] S1. Sample processing: Collect 2-3 mL of venous blood and centrifuge to separate the serum sample;

[0053] S2. Preparation of reagent addition: Take 30 μl of sample, add 15 μl of commercial red blood cell antigen reagent, mix and incubate at 37°C for 15 minutes;

[0054] S3. Add the mixed sample to the sample loading chamber, centrifuge at 3000-3500 rpm for 120-150 seconds, and observe the results after standing for 15 minutes after centrifugation.

[0055] Comparative Example 1

[0056] Screening for irregular antibodies using the test tube method:

[0057] Sample processing: Collect 2-3 mL of venous blood and centrifuge to separate serum;

[0058] Take 30 μl of sample, add 15 μl of commercial red blood cell antigen reagent, mix and incubate at 37°C for 15 minutes, then centrifuge at 1000g for 3-5 minutes, discard the supernatant and retain the red blood cell precipitate;

[0059] Add an equal volume of physiological saline to the red blood cell pellet, shake well and centrifuge again. Repeat this step 2-3 times to thoroughly remove unbound antibodies, plasma proteins or fibrin and other interfering substances.

[0060] The washed red blood cells were mixed with anti-human globulin antibody reagent, incubated at 37°C for 15 minutes, and then centrifuged at 1000g for 3-5 minutes to observe the agglutination reaction and interpret the results.

[0061] Example 3 and Comparative Example 1 are compared as shown in Table 1 below:

[0062]

[0063] The comparison shows that the microfluidic chip described in this application effectively improves detection efficiency, detection sensitivity, and detection accuracy compared to the traditional test tube method when performing irregular antibody detection.

[0064] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are exhaustively listed. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0065] For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. An automated washing microfluidic chip for irregular antibody detection, comprising a chip body with a mounting hole at its center, wherein a film is attached and fixed to the surface of the chip body, characterized in that, Also includes: The detection component has several groups arranged in a ring around the rotation center of the chip body, including sample application chambers and detection chambers arranged sequentially away from the rotation center. The sample application chambers and the detection chambers are connected by a washing channel. The membrane has a sample application hole corresponding to the position of the sample application chamber. The detection component is pre-filled with washing liquid that can fill the detection chamber and washing channel under centrifugation. The washing liquid contains anti-human immunoglobulin that can react with the red blood cell antigen-antibody complex. The specific gravity of the washing liquid is configured to be greater than the specific gravity of the sample to be tested and less than the specific gravity of the red blood cell antigen. The centerline of the sample loading chamber, the centerline of the washing channel, and the centerline of the detection chamber are collinear with the same radial line passing through the center of rotation; The sample loading chamber includes a first zone, a second zone, and a third zone from near to far from the rotation center. The sidewalls of the first zone and the third zone are both arc-shaped, and the radius of curvature of the sidewall of the first zone is smaller than that of the sidewall of the third zone. The sidewall of the second zone is straight. The second zone sidewall gradually moves away from the radial line collinear with the center of rotation from near to far, and the angle α1 between the second zone sidewall and the radial line collinear with the center of the sample dispensing cavity ranges from 6° to 11°.

2. The automated washing microfluidic chip for irregular antibody detection according to claim 1, characterized in that, The relationship between the horizontal projection length L1 from the first region to the center line of the sample dispensing cavity, the horizontal projection length L2 from the second region to the center line of the sample dispensing cavity, and the horizontal projection length L3 from the bottom edge contour of the third region to the center line of the sample dispensing cavity satisfies L1:L2:L3=1:(1.9-2.2):(1.55-1.75), and the sample dispensing hole is connected to the first region.

3. An automated washing microfluidic chip for irregular antibody detection according to any one of claims 1-2, characterized in that, Both the washing channel and the sample dispensing chamber are formed by a predetermined depth of downward indentation on the surface of the chip body, and the indentation depth of the washing channel is less than that of the sample dispensing chamber. The inlet end of the washing channel is connected to the third region, and the sidewall of the third region is inclined at a predetermined angle relative to the central axis of the chip body towards the washing channel.

4. An automated washing microfluidic chip for irregular antibody detection according to claim 3, characterized in that, The washing channel is provided with a drainage section at its proximal end. The drainage section extends obliquely downward and connects to the third zone. The drainage section is composed of an inclined plane and arc-shaped sidewalls located on both sides of the inclined plane. The inclined plane gradually tapers inward from the third zone toward the washing channel. The inclined plane is connected to both the third zone and the washing channel in an arc transition, and its bottom edge connected to the third zone is set to be an arc facing the washing channel.

5. An automated washing microfluidic chip for irregular antibody detection according to claim 4, characterized in that, The arc-shaped sidewall is an inward-facing arc, which connects with the bottom edge of the inclined plane to form a pointed auxiliary drainage channel.

6. An automated washing microfluidic chip for irregular antibody detection according to claim 5, characterized in that, The angle β between the inclined plane and the central axis of the chip body is 1.4-1.8 times the inclination angle α2 of the third region sidewall relative to the central axis of the chip body, and the inclination angle α2 satisfies: 15°≤α2≤20°.

7. An automated washing microfluidic chip for irregular antibody detection according to claim 6, characterized in that, The relationship between the horizontal projection length H1 of the inclined plane on the central axis of the chip body, the washing channel depth H2, and the sample dispensing chamber depth H3 satisfies H1:H2:H3=(0.35-0.45):(0.1-0.2):1.

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