Micro-fluidic chip for realizing classified counting of white blood cells and inflammation detection and use method of micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip for realizing classified counting of white blood cells and inflammation detection and a use method, the micro-fluidic chip comprises a sample adding port, a sample storage pool, a hemolytic agent liquid bag, a diluent liquid bag and a mixing pool, the sample adding port is connected with the hemolytic agent liquid bag through a channel, the sample adding port is connected with the sample storage pool through a channel, and the diluent liquid bag is connected with the mixing pool through a channel. The mixing pool is connected with the sample storage pool through a channel, the diluent sac is connected with the mixing pool through a channel, the mixing pool is connected with the probe pool through a channel, the air sac I is connected with the probe pool through a channel, and the probe pool is connected with the SAA antibody pool, the CRP antibody pool and the leucocyte sample pool through three channels. The SAA antibody pool is connected with the SAA detection pool, the air bag II is connected with the SAA detection pool through a channel, the CRP antibody pool is connected with the CRP detection pool, and the air bag III is connected with the CRP detection pool through a channel. According to the invention, blood routine examination and CRP and SAA verification index detection can be realized at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection equipment, and in particular relates to a microfluidic chip for realizing white blood cell classification counting and inflammation detection and a use method thereof. Background Art

[0002] White blood cells are an important part of the human immune system. Their number and classification play a key role in the diagnosis of many diseases and are an indispensable characteristic indicator for clinical diagnosis. Doctors can accurately assist in judging the patient's condition based on the results of white blood cell testing, thereby providing a strong basis for the formulation of subsequent treatment plans. CRP and SAA are important markers of inflammatory response. Their level changes can timely reflect the body's inflammatory state and play a key role in the early diagnosis, disease monitoring and treatment effect evaluation of infectious diseases. The birth of microfluidic chips has brought revolutionary changes to the field of traditional biochemical testing and brought it into a new era of the microscopic world. Microfluidic chips have many significant advantages, such as the extremely small amount of sample required, only a small amount of sample is needed to complete the test, which greatly reduces the burden of sample collection on the patient's body; the detection speed is extremely fast, and accurate results can be obtained in a short time, which saves precious time for the timely diagnosis of the disease; the low cost makes large-scale testing possible and reduces medical costs; high portability, easy to carry and operate, and can be tested anytime and anywhere. These advantages together realize the early diagnosis of the disease and effective prognosis evaluation, providing an extremely important means to improve the cure rate of the disease.

[0003] It is of great significance to realize the verification indicators of CRP (C-reactive protein), SAA (serum amyloid protein A) and the classification and counting of white blood cells in routine blood tests on microfluidic chips. Rapid and accurate detection of CRP and SAA through microfluidic chips can capture inflammatory signals at the early stage of the disease, provide timely diagnostic basis for clinicians, and enable patients to receive targeted treatment as soon as possible, thereby effectively controlling the development of inflammation, alleviating patients' pain, shortening the course of disease, and reducing medical expenses. At the same time, the classification and counting of white blood cells in routine blood tests is an important means to evaluate the body's immune function and diagnose blood system diseases. The simultaneous implementation of this test on a microfluidic chip can not only quickly and accurately obtain the classification and counting results of white blood cells, but also combine the test results of CRP and SAA to provide more comprehensive and accurate information for the comprehensive diagnosis of the disease. For example, in infectious diseases, by analyzing the changes in the classification and counting of white blood cells and the levels of CRP and SAA, the type of infection (bacterial infection or viral infection), the severity of the infection, and the state of the body's immune response can be more accurately judged, thereby providing strong support for the formulation of personalized treatment plans.

[0004] Technical solution of prior art 1 Latex turbidimetry: Latex turbidimetry is an immunoturbidimetric technique based on antigen-antibody reaction. When the antigen (CRP or SAA) binds to the corresponding antibody, antigen-antibody complexes are formed, and these complexes will cause the turbidity of the reaction system to change. Specifically, in the latex reagent, the antibody is adsorbed on the surface of the latex particles. When the antigen in the sample (such as CRP or SAA) is added, the antigen specifically binds to the antibody on the surface of the latex particles to form a latex-antibody-antigen complex. As the antigen concentration increases, the number of complexes formed increases, and the turbidity of the solution also increases. By detecting the turbidity change of the reaction system, the concentration of the antigen (CRP or SAA) in the sample can be indirectly reflected.

[0005] Disadvantages of the prior art 1 There are some disadvantages in the latex turbidimetry for the determination of CRP and SAA. When the CRP concentration is less than a certain concentration, the correlation between the latex enhanced transmission immunoturbidimetry and rate scattering immunoturbidimetry for the detection of CRP is low. In addition, although the linear range of the latex turbidimetry is wide, in some cases, especially at high or low concentration extremes, deviations in the test results may occur. Common factors such as hemolysis, lipemia, jaundice, and autoantibodies such as rheumatoid factor (RF), M protein, heterophilic antibodies, etc. can interfere with the immunoturbidimetry, and these interfering factors can cause a false increase or decrease in the results of the test object. The latex turbidimetry method also relies on large-scale biochemical analyzers, and requires a high-precision optical detection system to monitor the turbidity changes of the reaction system in real time. The equipment cost and maintenance cost are high and it cannot be miniaturized.

[0006] Technical solution of prior art 2 Enzyme-linked immunosorbent assay (ELISA) ELISA determination of CRP and SAA is based on the specific binding reaction of antigen-antibody and the efficient catalytic action of enzymes. Anti-CRP or SAA antibodies are pre-coated on the microplate. When CRP or SAA is added to the sample, they will specifically bind to the coated antibodies to form an antigen-antibody complex. Then, enzyme-labeled antibodies are added. This enzyme-labeled antibody can also bind to the antigen to form a sandwich complex. After the color developer is added, the enzyme catalyzes the chemical reaction of the color developer to produce a colored product. The absorbance is related to the amount of the colored product, which is proportional to the amount of CRP or SAA in the sample, so CRP or SAA can be quantitatively detected by measuring the absorbance.

[0007] Disadvantages of the second prior art The ELISA test process has many steps, including incubation, washing and other links. It usually takes 3-4 hours or even longer from sample processing to the final result. It is not suitable for clinical situations where test results are urgently needed. In addition, this method needs to be operated strictly according to the steps. Improper control of temperature, time, etc. will affect the test results. In addition, it is easily interfered by endogenous enzymes and has high technical requirements for operators. Finally, for low concentrations of CRP or SAA detection, its sensitivity may not be high enough. Summary of the invention

[0008] The present invention aims to make up for the deficiency of the existing technology that can only measure cell types or single SAA / CRP indicators separately, and proposes a method for using a microfluidic chip to achieve white blood cell classification and counting and inflammation detection. At the same time, the implementation method is different from the commonly used methods on the market. It uses long afterglow fluorescence technology to achieve the detection of CRP and SAA indicators. The entire measurement process is fast, low-cost and highly reliable.

[0009] The present invention can simultaneously realize white blood cell classification and counting and CRP and SAA detection, adopt the impedance method to realize the distinction of cell types, and use the long afterglow fluorescence technology to realize the measurement of two inflammatory indicators, CRP and SAA, providing powerful assistance for the accurate diagnosis of diseases in clinical practice.

[0010] The present invention adopts the following technical solution: The microfluidic chip for realizing leukocyte classification counting and inflammation detection comprises a sample loading port, a sample storage pool, a hemolytic agent sac, a diluent sac, a mixing pool, a probe pool, an SAA antibody pool, an SAA detection pool, a CRP antibody pool, a CRP detection pool, a leukocyte sample pool, a leukocyte counting pool and an air bag, wherein the hemolytic agent sac and the diluent sac are both aluminum foil sacs, the sample loading port and the hemolytic agent sac are connected to each other by a channel, the sample loading port and the sample storage pool are connected to each other by a channel, the mixing pool and the sample storage pool are connected to each other by a channel, and the diluent sac and the mixing pool are connected by a channel. The mixing pool and the probe pool are connected to each other by a channel, the air bag I and the probe pool are connected to each other by a channel, the probe pool is connected to the SAA antibody pool, the CRP antibody pool, and the white blood cell sample pool through three channels respectively, the SAA antibody pool is connected to the SAA detection pool, the air bag II and the SAA detection pool are connected to each other by a channel, the CRP antibody pool is connected to the CRP detection pool, the air bag III and the CRP detection pool are connected to each other by a channel, the white blood cell sample pool and the white blood cell counting pool are connected to each other by a channel, and the air bag IV and the white blood cell counting pool are connected to each other by a channel.

[0011] Furthermore, a deceleration valve is installed on the channel connecting the probe pool and the white blood cell sample pool.

[0012] Furthermore, a small hole is provided on the channel connecting the white blood cell sample pool and the white blood cell counting pool.

[0013] Furthermore, the diluent required for the test is pre-placed in the diluent liquid capsule, and the hemolytic agent is pre-placed in the hemolytic agent liquid capsule in the chip. The long afterglow probe "long afterglow probe-SAA-MAb1" labeled with SAA monoclonal antibody and the long afterglow probe "long afterglow probe-CRP-MAb1" labeled with CRP monoclonal antibody are pre-placed in the probe pool, and the SAA monoclonal antibody and the CRP monoclonal antibody are pre-placed in the SAA antibody pool and the CRP antibody pool, respectively.

[0014] Furthermore, the detection area and reference area in the SAA detection pool and the CRP detection pool were pre-coated with SAA-MAb2, goat anti-mouse IgG, CRP-MAb2, and goat anti-mouse IgG.

[0015] The method for using the microfluidic chip for realizing white blood cell classification counting and inflammation detection includes: Step 1: Add 10ul of whole blood sample to the sample injection port. Under the action of siphon, the liquid will automatically flow into the sample storage pool, and the excess volume sample will remain in the channel. At this time, press the hemolytic agent capsule, and the hemolytic agent will flow into the mixing pool with the sample in the channel under pressure to achieve quantitative volume mixing.

[0016] Step 2: Press the diluent capsule, and the diluent will flow into the mixing pool and mix with the sample droplets and hemolytic agent. When the airbag I is pressed and released, the liquid in the mixing pool will flow into the probe pool under negative pressure. When pressed again, the airbag I rebounds to generate pressure, driving part of the liquid back to the mixing pool. This is repeated many times, so that the sample liquid, hemolytic agent, diluent and antigen-antibody are fully mixed to dissolve the red blood cells.

[0017] Step 3. After the red blood cells are dissolved, press airbags II to IV in batches and at different times. The mixed sample liquid flows into the corresponding pools through three channels, namely the SAA antibody pool, the CRP antibody pool and the white blood cell sample pool.

[0018] Further, the method further comprises step 4 of detecting SAA antigen: S401, airbag II is pressed, and the "long afterglow probe-SAA-MAb1-Ag" sample liquid in the SAA antibody pool combines with the SAA-MAb3 antibody in the SAA antibody pool to form a "long afterglow probe-SAA-MAb1-Ag-MAb3" complex.

[0019] S402, the mixture containing "long afterglow probe-SAA-MAb1-Ag" and "long afterglow probe-SAA-MAb1-Ag-MAb3" flows to the SAA detection pool, in which SAA-MAb2 and goat anti-mouse IgG capture SAA antigen and mouse MAb3 antibody respectively to form "long afterglow probe-SAA-MAb1-Ag-MAb2" sandwich complex and "long afterglow probe-SAA-MAb1-Ag-MAb3-goat anti-mouse IgG" complex, and the remaining excess liquid finally flows into the airbag II below.

[0020] S403, a strong fluorescent signal is induced on the SAA detection line, and the signal intensity is analyzed to quantify the SAA level; when the sample to be tested is SAA negative, the reference line signal intensity in the sheep anti-mouse area is very weak or even absent.

[0021] Furthermore, the method further includes step 5 when detecting CRP: S501. The sample liquid flows into the CRP detection pool, and the "long afterglow probe-CRP-MAb1-Ag" will combine with the CRP-MAb2 in the detection pool to form a sandwich complex of "long afterglow probe-CRP-MAb1-Ag-MAb2".

[0022] S502. The sandwich complex will be fixed in the CRP detection pool and can no longer flow into air sac III. Mouse MAb3 is also captured and fixed by the secondary antibody goat anti-mouse IgG and cannot move.

[0023] S503. Use a 365nm monochromatic light source to irradiate the CRP detection area and the CRP reference area, and use time-sharing and fractional laser excitation. When the monochromatic light source continuously irradiates the CRP detection pool for 5 seconds, the laser source is turned off. At this time, the long afterglow probes in the complex captured on the CRP detection area and the CRP reference area will be excited, and the SAA detection pool will emit monochromatic fluorescence with a wavelength of 549nm for 300ms. Use CCD to shoot a 3-second fluorescence video.

[0024] S504. The whole process is recorded by a high-speed camera placed above the optical filter. The MCU locates the laser spot and then crops it into an image of specified pixel size. After grayscale conversion, the final frame-by-frame speed limit traversal is obtained, and the weighted average of the sum of the grayscale values ​​of each pixel will be recorded as the fluorescence intensity at this time.

[0025] S504. By identifying all signal peaks and integrating the effective area, the fluorescence intensity of the CRP detection area and the CRP reference area is calculated, and the fluorescence intensity value G is obtained by the ratio of CRP reference area / CRP detection area.

[0026] The fluorescence intensity of the S505.CRP reference area is not affected by the CRP concentration. The reference line area is used as the background value, and the intensity ratio obtained is related to the concentration of the CRP antibody in the sample.

[0027] Furthermore, smoothing algorithms and noise filtering algorithms are embedded in the MCU.

[0028] Furthermore, the method further comprises step 6 of counting white blood cells: S601. The blood sample is diluted in the mixing pool to form a cell suspension. Press the airbag IV, and the liquid slowly flows out from the front inlet of the chip in the white blood cell sample pool, flows into the pipe on the back of the chip, passes through the small holes in the pipe one by one, and then flows into the front inlet of the chip through the back pipe and flows into the white blood cell counting pool.

[0029] S602. Detection electrodes are led out from both ends of the small hole. During the detection process, when the current is turned on, the electrodes located on both sides of the small hole tube will generate a stable current; S603. When the cells pass through the small hole, the number and volume of blood cells in the blood are obtained. The pulse signal is amplified, identified and counted to obtain the cell counting result. Different types of cells have different volumes and generate different pulse amplitudes. The cells are classified and counted according to the pulse amplitude.

[0030] Beneficial effects of the present invention: The realization of CRP, SAA verification indicators and blood routine white blood cell classification and counting detection on the microfluidic chip not only fully utilizes the advantages of the microfluidic chip and improves the detection efficiency and accuracy, but also provides a more comprehensive and accurate detection method for clinical diagnosis. It has immeasurable value for the early detection and timely treatment of diseases, and is expected to play an increasingly important role in the field of medical testing in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the planar structure diagram of the microfluidic chip; Figure 2 The graphs are CRP and SAA excitation brightness and frame number. (a) is the CRP excitation brightness and frame number graph, and (b) is the SAA excitation brightness and frame number graph. Figure 3 The graphs are the relationship between the concentrations of CRP and SAA and the fluorescence intensity G. (a) is the relationship between the concentration of CRP and the fluorescence intensity G. (b) is the relationship between the concentration of CRP and the fluorescence intensity. Figure 4 Comparison charts of CRP and SAA test results with medical equipment. (a) is the comparison chart of CRP test results with medical equipment, and (b) is the comparison chart of SAA test results with medical equipment. Figure 5 It is the waveform diagram of impedance of white blood cells; Figure 6 This is a three-dimensional diagram of the chip structure.

[0032] In the figure, 1-sample loading port, 2-sample storage pool, 3-hemolytic agent sac, 4-dilution liquid sac, 5-mixing pool, 6-probe pool, 7-SAA antibody pool, 8-SAA detection pool, 9-CRP antibody pool, 10-CRP detection pool, 11-leukocyte sample pool, 12-leukocyte counting pool, 13-air bag I, 14-air bag II, 15-air bag III, 16-air bag IV. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example like Figure 1 , Figure 6 As shown, a microfluidic chip for realizing white blood cell classification and counting and inflammation detection includes a sample addition port 1, a sample storage pool 2, a hemolytic agent capsule 3, a diluent capsule 4, a mixing pool 5, a probe pool 6, a SAA antibody pool 7, a SAA detection pool 8, a CRP antibody pool 9, a CRP detection pool 10, a white blood cell sample pool 11, a white blood cell counting pool 12 and an air bag, wherein the hemolytic agent capsule 3 and the diluent capsule 4 are both aluminum foil capsules. The sample adding port 1 and the hemolytic agent sac 3 are connected to each other by a channel, the sample adding port 1 and the sample storage pool 2 are connected to each other by a channel, the mixing pool 5 and the sample storage pool 2 are connected to each other by a channel, the diluent sac 4 and the mixing pool 5 are connected to each other by a channel, the mixing pool 5 and the probe pool 6 are connected to each other by a channel, the air bag I 13 and the probe pool 6 are connected to each other by a channel, the probe pool 6 is connected to the SAA antibody pool 7, the CRP antibody pool 9, and the white blood cell sample pool 11 through three channels, the SAA antibody pool 7 is connected to the SAA detection pool 8, the air bag II 14 and the SAA detection pool 8 are connected to each other by a channel, the CRP antibody pool 9 is connected to the CRP detection pool 10, the air bag III 15 and the CRP detection pool 10 are connected to each other by a channel, the white blood cell sample pool 11 and the white blood cell counting pool 12 are connected to each other by a channel, and the air bag IV 16 and the white blood cell counting pool 12 are connected to each other by a channel. A deceleration valve is installed on the channel connecting the probe pool 6 and the white blood cell sample pool 11. The channel connecting the white blood cell sample pool 11 and the white blood cell counting pool 12 has a small hole.

[0035] The detection units are interconnected by channels to enable the inflow and outflow of sample droplets as needed.

[0036] Among them, the diluent required for detection is pre-placed in the diluent liquid capsule 4, and the hemolytic agent is pre-placed in the hemolytic agent liquid capsule 3 in the chip. The long afterglow probe "long afterglow probe-SAA-MAb1" labeled with SAA monoclonal antibody 1 (rabbit source) and the long afterglow probe "long afterglow probe-CRP-MAb1" labeled with CRP monoclonal antibody 1 (rabbit source) are pre-placed in the probe pool 6, and SAA monoclonal antibody 3 (mouse source) and CRP monoclonal antibody 3 (mouse source) are pre-placed in SAA antibody pool 7 and CRP antibody pool 9, respectively. In addition, the detection area and reference area in the SAA detection pool 8 and the CRP detection pool 10 are pre-coated with SAA-MAb2 (SAA monoclonal antibody 2), goat anti-mouse IgG, CRP-MAb2 (CRP monoclonal antibody 2), and goat anti-mouse IgG. It should be noted that goat anti-mouse IgG will only bind to mouse monoclonal antibodies, not rabbit antibodies. At the same time, the long afterglow probe can emit another fluorescence with a different wavelength from the excitation light under the excitation of external excitation light, and its intensity is proportional to the concentration of the target in the sample. The four air bags in the chip are used for pressing to achieve directional flow of the sample liquid.

[0037] The method for using the microfluidic chip for realizing white blood cell classification counting and inflammation detection includes: First, add 10ul of whole blood sample at the sample injection port 1. Under the action of siphon, the liquid will automatically flow into the sample storage pool 2, and the excess volume sample will remain in the channel. At this time, press the aluminum foil liquid bag of the hemolytic agent (hemolytic agent liquid bag 3), and the hemolytic agent will flow into the mixing pool 5 with the sample in the channel under the action of pressure to achieve quantitative volume mixing.

[0038] Once again, press the diluent aluminum foil capsule (diluent capsule 4), and the diluent will flow into the mixing pool 5 and mix with the sample droplets and the hemolytic agent. Since the aluminum foil capsule is non-elastic and will not rebound after being pressed, an air bag Ⅰ13 made of elastic material is also connected next to the probe pool 6. When the air bag Ⅰ13 is pressed and then released, the liquid in the mixing pool 5 will flow into the probe pool 6 under the drive of negative pressure. When pressed again, the air bag Ⅰ13 rebounds to generate pressure, driving part of the liquid back to the mixing pool 5. This is repeated many times, so that the sample liquid, hemolytic agent, diluent and antigen-antibody are fully mixed (the sample solution contains SAA antigen and CRP antigen), and the red blood cells are dissolved to avoid affecting the subsequent cell count and the measurement of CRP and SAA fluorescence intensity.

[0039] After the red blood cells are dissolved, the airbags II 14 to IV 16 are pressed down in time and in batches, and the mixed sample liquid flows into the corresponding pools through three channels, namely, the SAA antibody pool 7, the CRP antibody pool 9 and the white blood cell sample pool 11. Since SAA and CRP antigens have more than two antigenic sites, the sandwich mode in immunity is used for detection, based on the specific binding of SAA antibody-antigen (Ab-Ag). There are three corresponding detection pools and airbags under the three pools.

[0040] Taking SAA antigen as an example, when airbag II 14 is pressed, the "long afterglow probe-SAA-MAb1-Ag" sample liquid that has been formed by the combination of antigen and antibody in the SAA antibody pool 7 will first combine with the SAA-MAb3 (mouse source) antibody in the SAA antibody pool 7 under the action of negative pressure to form a "long afterglow probe-SAA-MAb1-Ag-MAb3" complex.

[0041] Then, the liquid containing the mixture of "long afterglow probe-SAA-MAb1-Ag" and "long afterglow probe-SAA-MAb1-Ag-MAb3" will continue to flow to the SAA detection pool 8 under the action of negative pressure and gravity. In the SAA detection pool 8, the pre-coated SAA-MAb2 will capture the SAA antigen in the complex, and the goat anti-mouse IgG will capture the mouse MAb3 antibody in the complex, and specific binding will occur to form the "long afterglow probe-SAA-MAb1-Ag-MAb2" sandwich complex and the "long afterglow probe-SAA-MAb1-Ag-MAb3-goat anti-mouse IgG" complex, and the remaining excess liquid will eventually flow into the airbag II 14 below. If a strong fluorescent signal is induced on the SAA detection line. Since the signal intensity is positively correlated with the SAA concentration, the signal intensity can be analyzed to quantify the SAA level. However, when the sample to be tested is SAA-negative, due to the absence of SAA antigen Ag, the "long afterglow probe-SAA-MAb1" cannot be sandwiched with SAA-MAb2, resulting in a very weak or even absent reference signal intensity in the sheep anti-mouse area.

[0042] CRP detection uses the same principle as SAA. When the sample liquid flows into the CRP detection pool 10, the "long afterglow probe-CRP-MAb1-Ag" will combine with the CRP-MAb2 (CRP monoclonal antibody 2) in the detection pool to form a sandwich complex of "long afterglow probe-CRP-MAb1-Ag-MAb2". Since the MAb2 of CRP is pre-planted in the CRP detection pool 10, and the secondary antibody goat anti-mouse IgG is also pre-planted in the CRP detection pool 10, the sandwich complex will be fixed in the CRP detection pool 10 and can no longer flow into the airbag III 15. The mouse MAb3 is also captured and fixed by the secondary antibody goat anti-mouse IgG and cannot move. A 365nm monochromatic light source is used to illuminate the CRP detection area and the CRP reference area in the CRP detection pool 10. In order to avoid reflected fluorescence aliasing, time-sharing and multiple-time laser excitation is used. When the monochromatic light source continuously irradiates the CRP detection pool for 5 seconds and then turns off the laser source, the long afterglow probe in the captured complex on the CRP detection area and the CRP reference area will be excited, and the SAA detection pool 10 will emit a monochromatic fluorescence with a wavelength of 549nm for 300ms, and a 3-second fluorescence video will be taken using CCD. The entire process is recorded by a high-speed camera placed above the optical filter. The core algorithm in the MCU will quickly locate the laser spot in each frame and then crop it to a specified pixel size image. After graying, the final frame will be traversed at a limited speed, and the weighted average of the sum of the grayscale values ​​of each pixel will be recorded as the fluorescence intensity at this time.

[0043] In order to obtain better repeatability, smoothing algorithm and noise filtering algorithm are embedded in MCU. The smoothing algorithm is used to smooth the signal curve, making integration easier. In the noise filtering algorithm, a noise baseline is set, and the three values ​​of the signal above the baseline will be considered as effective fluorescence signals.

[0044] Finally, by identifying all signal peaks and integrating the effective area below, the fluorescence intensity of the CRP detection area and the CRP reference area can be calculated, and the fluorescence intensity value G can be obtained by the CRP reference area / CRP detection area ratio. Since goat anti-mouse IgG can always bind to the labeled mouse MAb3 to produce a stable signal, the fluorescence intensity of the CRP reference area is not affected by the CRP concentration, so the reference line area can be used as the background value, and the fluorescence intensity of the CRP detection area can be compared with it to reduce the error, and the obtained intensity ratio is related to the concentration of CRP antibodies in the sample.

[0045] like Figure 2As shown, the detection curves of 0.1 ng / mL CRP and 0.1 ng / mL SAA are shown. The horizontal axis represents the image frame, and the vertical axis represents the excitation brightness. In order to test the effectiveness of the present invention, a series of CRP and SAA antigen standard samples with different concentrations were used, ranging from 0.05 ng / mL to 50 ng / mL. Each solution was tested 5 times to obtain a set of G values ​​corresponding to these concentrations, as shown in FIG. Figure 3 As shown. The standard CRP and SAA concentrations were logarithmically calculated and then linearly fitted. Finally, the linear fitting correlation curve between the stable fluorescence intensity ratio G and the concentration was as shown in Figure 4 (a) shows: CRP (slope, -0.3862; intercept, 1.6736; correlation coefficient, 0.9826) and Figure 4 Middle (b): SAA (slope, -0.5979; intercept, 0.5060; correlation coefficient, 0.9703). There is a good linear relationship between the steady-state fluorescence intensity ratio and the concentration of the analyte, indicating that the detection system of the present invention has high reliability.

[0046] The detection accuracy of the present invention in testing real clinical samples was verified by collecting multiple whole blood samples with known CRP concentrations, and 10 μL of each blood sample was dripped into the chip. Figure 4 As shown, the results prove that the test values ​​given by the present invention are highly consistent with the test values ​​of the medical instrument, showing good performance, with a correlation coefficient greater than 0.97 and a sensitivity as high as 0.98.

[0047] This principle is used to carry out specific experimental operations on three types of cells with different particle sizes.

[0048] Unlike CRP and SAA detection, the method used for white blood cell classification and counting is the classic impedance method, the core of which is to measure the resistance change caused by cells passing through the micropores to determine the number and size of cells, so as to distinguish and count different types of cells. The blood sample has been diluted in the mixing pool 5 to form a cell suspension. Next, press the air bag IV 16, and the liquid slowly flows out from the front entrance of the chip in the white blood cell sample pool 11, flows into the pipe on the back of the chip, and passes through the small holes in the pipe one by one. This small hole tube is the key part of the impedance method cell counting, and finally flows into the front entrance of the chip through the back pipe and flows into the white blood cell counting pool. Detection electrodes are led out at both ends of the small hole for collecting voltage pulses when cells pass through the small hole. During the detection process, when the current is turned on, the electrodes on both sides of the small hole tube will generate a stable current. If the supply current I and the impedance Z are stable, according to Ohm's law, the voltage E through the small hole is also constant (E=IZ).

[0049] When a cell passes through the small hole, the conductivity of the cell is lower than that of the diluent, so the resistance in the sensing area of ​​the small hole in the circuit will increase, causing the voltage to change and a pulse signal to appear. The amplitude of this pulse signal is proportional to the cell volume, that is, the larger the cell volume, the higher the pulse caused; the more pulses there are, the more cells there are. From this, the number and volume of blood cells in the blood can be obtained. The pulse signal is amplified, identified and counted to obtain the cell counting result. Because different types of cells have different volumes, the pulse amplitudes generated are also different. Figure 5 It is the pulse waveform diagram corresponding to different types of cells, so the cells can be classified and counted according to the pulse amplitude.

[0050] The present invention realizes the detection of inflammatory indicators CRP, SAA concentration and white blood cell classification and counting of routine blood tests on the chip at the same time, which makes up for the deficiency that the traditional detection method can only detect one of the two, and provides the function of one-time blood sampling and multi-index detection. At the same time, the long afterglow fluorescence technology is used for the detection of verification indicators, which is different from the traditional immunochromatography technology, and the fluorescence afterglow is used for concentration correlation. Compared with the microscope detection method, the impedance blood cell analyzer is easy to operate, has a short detection time, greatly improves the work efficiency, and has reliable results. The present invention is simple and practical, can quickly and accurately realize multi-index detection, and the instrument can be miniaturized.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic chip for realizing white blood cell classification and counting and inflammation detection, characterized in that: The invention comprises a sample adding port, a sample storage pool, a hemolytic agent sac, a diluent sac, a mixing pool, a probe pool, an antibody pool, a detection pool, a leukocyte sample pool, a leukocyte counting pool and an air bag. The sample adding port and the hemolytic agent sac are connected to each other by a channel, the sample adding port and the sample storage pool are connected to each other by a channel, the mixing pool and the sample storage pool are connected to each other by a channel, the diluent sac and the mixing pool are connected to each other by a channel, the mixing pool and the probe pool are connected to each other by a channel, the air bag I and the probe pool are connected to each other by a channel, the probe pool is connected to the SAA antibody pool, the CRP antibody pool and the leukocyte sample pool respectively through three channels, the SAA antibody pool is connected to the SAA detection pool, the air bag II and the SAA detection pool are connected to each other by a channel, the CRP antibody pool is connected to the CRP detection pool, the air bag III and the CRP detection pool are connected to each other by a channel, the leukocyte sample pool and the leukocyte counting pool are connected to each other by a channel, and the air bag IV and the leukocyte counting pool are connected to each other by a channel.

2. The chip according to claim 1, characterized in that: The hemolytic agent sac and the diluent sac are both aluminum foil sacs.

3. The chip according to claim 1, characterized in that: A deceleration valve is installed on the channel connecting the probe pool and the white blood cell sample pool.

4. The chip according to claim 1, characterized in that: A small hole is provided on the channel connecting the white blood cell sample pool and the white blood cell counting pool.

5. The chip according to claim 1, characterized in that: The diluent is placed in the diluent sac, the hemolytic agent is placed in the hemolytic agent sac, the long afterglow probe "long afterglow probe-SAA-MAb1" labeled with SAA monoclonal antibody and the long afterglow probe "long afterglow probe-CRP-MAb1" labeled with CRP monoclonal antibody are placed in the probe pool, and the SAA monoclonal antibody and the CRP monoclonal antibody are placed in the SAA antibody pool and the CRP antibody pool respectively.

6. The chip according to claim 1, characterized in that: The detection area and reference area in the SAA detection pool and the CRP detection pool are coated with SAA-MAb2, goat anti-mouse IgG, and CRP-MAb2, goat anti-mouse IgG, respectively.

7. A method for using a microfluidic chip for realizing white blood cell classification and counting and inflammation detection, characterized in that: include: Step 1, add 10ul whole blood sample at the sample injection port. Under the action of siphon, the liquid will automatically flow into the sample storage pool, and the excess volume sample will remain in the channel. At this time, press the hemolytic agent sac, and the hemolytic agent will flow into the mixing pool with the sample in the channel under pressure to achieve quantitative volume mixing; Step 2: Press the diluent capsule, and the diluent will flow into the mixing pool and mix with the sample droplets and the hemolytic agent. When the airbag I is pressed and released, the liquid in the mixing pool will flow into the probe pool under the negative pressure. When pressed again, the airbag I rebounds to generate pressure, driving part of the liquid to flow back to the mixing pool. This is repeated many times, so that the sample liquid, hemolytic agent, diluent and antigen-antibody are fully mixed to dissolve the red blood cells. Step 3. After the red blood cells are dissolved, press airbags II to IV in batches and at different times. The mixed sample liquid flows into the corresponding pools through three channels, namely the SAA antibody pool, the CRP antibody pool and the white blood cell sample pool.

8. The method according to claim 7, characterized in that Also includes step 4 when detecting SAA antigen: S401, airbag II is pressed, and the "long afterglow probe-SAA-MAb1-Ag" sample liquid in the SAA antibody pool combines with the SAA-MAb3 antibody in the SAA antibody pool to form a "long afterglow probe-SAA-MAb1-Ag-MAb3" complex; S402, the mixture containing "long afterglow probe-SAA-MAb1-Ag" and "long afterglow probe-SAA-MAb1-Ag-MAb3" flows to the SAA detection pool, in which SAA-MAb2 and goat anti-mouse IgG capture SAA antigen and mouse MAb3 antibody respectively to form "long afterglow probe-SAA-MAb1-Ag-MAb2" sandwich complex and "long afterglow probe-SAA-MAb1-Ag-MAb3-goat anti-mouse IgG" complex, and the remaining excess liquid finally flows into the airbag II below; S403, a strong fluorescent signal is induced on the SAA detection line, and the signal intensity is analyzed to quantify the SAA level. When the sample to be tested is SAA negative, the reference line signal intensity in the sheep anti-mouse area is very weak or even absent.

9. The method according to claim 8, characterized in that Also includes step 5 when testing CRP: S501. The sample liquid flows into the CRP detection pool, and the "long afterglow probe-CRP-MAb1-Ag" combines with the CRP-MAb2 in the detection pool to form a sandwich complex of "long afterglow probe-CRP-MAb1-Ag-MAb2"; S502. The sandwich complex will be fixed in the CRP detection pool and can no longer flow into the air sac III. The mouse MAb3 is also captured and fixed by the secondary antibody goat anti-mouse IgG and cannot move; S503. Use a 365nm monochromatic light source to illuminate the CRP detection area and the CRP reference area, and use time-sharing and fractional laser excitation. After the monochromatic light source continuously illuminates the CRP detection pool for 5 seconds, the laser source is turned off. At this time, the long afterglow probe in the complex captured on the CRP detection area and the CRP reference area will be excited, and the SAA detection pool will emit a monochromatic fluorescence with a wavelength of 549nm for 300ms. Use CCD to shoot a 3-second fluorescence video; S504. The whole process is recorded by a high-speed camera placed above the optical filter. The MCU locates the laser spot and then crops it into a specified pixel size image. After grayscale conversion, the final frame-by-frame speed limit traversal is obtained. The weighted average of the sum of the grayscale values ​​of each pixel will be recorded as the fluorescence intensity at this time. S504. Calculate the fluorescence intensity of the CRP detection area and the CRP reference area by identifying all signal peaks and integrating the effective area, and obtain the fluorescence intensity value G by the ratio of CRP reference area / CRP detection area; The fluorescence intensity of the S505.CRP reference area is not affected by the CRP concentration. The reference line area is used as the background value, and the obtained intensity ratio is related to the concentration of CRP antibodies in the sample.

10. The method according to claim 9, characterized in that Also includes step 6 for counting white blood cells: S601. The blood sample is diluted in the mixing pool to form a cell suspension. The airbag IV is pressed, and the liquid slowly flows out from the front inlet of the chip in the white blood cell sample pool, flows into the pipeline on the back of the chip, and passes through the small holes in the pipeline one by one, and then flows into the front inlet of the chip through the back pipeline, and flows into the white blood cell counting pool; S602. Detection electrodes are led out from both ends of the small hole. During the detection process, when the current is turned on, the electrodes located on both sides of the small hole tube will generate a stable current; S603. When cells pass through the small hole, the number and volume of blood cells in the blood are obtained. The pulse signal is amplified, identified and counted to obtain the cell counting result. Different types of cells have different volumes and generate different pulse amplitudes. The cells are classified and counted according to the pulse amplitude.

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