Centrifugal micro-fluidic chip for automatic detection of nano-enzyme test strip and application of centrifugal micro-fluidic chip

Abstract

The invention relates to a centrifugal micro-fluidic chip for automatic detection of a nano-enzyme test strip and application of the centrifugal micro-fluidic chip. The centrifugal micro-fluidic chip integrates the steps of sample adding, chromatography, color development and the like of the nano-enzyme test strip, and full automation of nano-enzyme experiments on the micro-fluidic chip is realized. Comprising a reaction structure main body, a bottom runner layer, a vertical through hole layer, a pressing layer, a top runner layer and a sealing body. The closed structure of the chip effectively reduces the interference of external environmental factors on the experiment, reduces the risk of sample pollution, and ensures the high reliability of the detection result.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices, and specifically relates to a centrifugal microfluidic chip for automated detection of nanoenzyme test strips and its application. Background Art

[0002] Nanoenzyme immunochromatography technology has been widely used in various detection scenarios, especially in the rapid detection of biomarkers, with the advantages of simplicity, speed, and high sensitivity. Traditional immunochromatographic test strips generally require manual operation, including steps such as sample addition and strip reading. This not only increases the time and complexity of detection, but is also easily affected by the subjective factors of the operator, resulting in inconsistent and inaccurate results. Therefore, how to achieve automated detection of nanoenzyme immunochromatographic test strips has become a problem that needs to be solved urgently in current technology.

[0003] Microfluidics technology integrates bioanalysis processes such as sample pretreatment, immune response, and signal detection into microchips, creating portable "lab-on-a-chip" systems. Centrifugal microfluidic chips, in particular, utilize centrifugal force to drive fluid movement, eliminating the reliance of traditional microfluidic systems on complex pump and valve structures. Their high throughput and ease of parallelization make them ideal vehicles for rapid multi-target detection.

[0004] Compared with traditional centrifugal microfluidic chips that use surface-treated valves as the core of fluid control, chips based on soluble membrane valves achieve precise fluid control through the intrinsic properties of the material rather than surface energy modification, which significantly reduces environmental sensitivity and process complexity. Traditional chips rely on hydrophilic / hydrophobic modification or physical valve structure to control fluid flow, which is prone to surface energy decay due to temperature and humidity fluctuations or storage aging, causing fluid leakage or valve control failure, increasing manufacturing costs and the risk of system failure. In addition, traditional valves are difficult to coordinate timing logic in multi-step detection. For example, key links such as the sequential mixing of samples and reagents and reaction incubation time control often lead to deviations in detection results due to fluid lag or asynchronous triggering. More importantly, traditional chips rely solely on a single motor speed adjustment to achieve full process control, requiring frequent manual intervention in fixed speed mode, and the level of automation is insufficient.

[0005] In view of the above description, it is essential to combine nanozyme immunochromatographic test strips with centrifugal microfluidic chips to realize automatic loading of samples, flow control and automatic reading of results, while meeting the needs of high-throughput and multi-target detection, and improving its detection sensitivity and automation level, to promote the development of nanozyme test strip detection technology.

[0006] Based on this, the present invention is proposed. Summary of the Invention

[0007] The present invention first relates to an automatic detection centrifugal microfluidic chip, wherein the microfluidic chip is loaded with a ruthenium-loaded peroxide nanozyme probe and an immunochromatographic test strip for high-sensitivity pathogen detection; preferably, the microfluidic chip can detect a sample volume of 10-100 μL; the chip comprises a chip body (29) having a reaction structure, a bottom flow channel layer (36), a vertical through-hole layer (42) equipped with a soluble membrane, a compression layer (48), a top flow channel layer (54) and a sealing body (64) ( Figure 1-6 );in,

[0008] The sealing body (64) includes: a first exhaust hole (55), a first liquid injection hole (56), a second liquid injection hole (57), a second exhaust hole (58), a third liquid injection hole (59), a fourth liquid injection hole (60), a fifth liquid injection hole (61), a positioning mechanism (30), a sixth positioning hole (62) and a seventh positioning hole (63).

[0009] The bottom flow channel layer (36) includes: a color developing liquid air hole (31), a sample liquid soluble membrane filling and releasing hole (32), and a color developing liquid soluble membrane filling and releasing hole (33).

[0010] The reaction structure of the chip body (29) Figure 1 The apparatus comprises: a first storage chamber (13) for storing a ruthenium nanozyme probe, a second storage chamber (12) for storing a sample liquid, a third storage chamber (1) for storing a DAB buffer solution, a fourth storage chamber (2) for storing H2O2, a fifth storage chamber (3) for storing a DAB substrate, a first mixing chamber (16) for mixing a sample liquid and a ruthenium nanozyme probe, a second mixing chamber (7) for mixing a DAB substrate, H2O2, and a DAB buffer solution, a test strip storage chamber (11) for storing a ruthenium nanozyme test strip, a first mixing chamber (16) and a second mixing chamber (7), a dispensing unit (8) connected to the second mixing chamber (7), a ventilation pipe, and various fluid valves;

[0011] Preferably, the volume of the first storage chamber (13) for storing the ruthenium nanozyme probe is 2-10 μL, the volume of the second storage chamber (12) for storing the sample liquid is 10-100 μL, the volume of the third storage chamber (1) for storing the DAB buffer is 40-60 μL, the volume of the fourth storage chamber (2) for storing H2O2 is 4-60 μL, and the volume of the fifth storage chamber (3) for storing the DAB substrate is 2-4 μL.

[0012] The centrifugal microfluidic chip interconnects the functional cavities in the ventilation structure to form a self-closed loop, comprising:

[0013] The first exhaust hole (55) and the second exhaust hole (58) located on the sealing body (64) are connected to the internal structure ( Figure 2 The first communicating channel (18), the second communicating channel (19), the third communicating channel (20), the fourth communicating channel (21), the fifth communicating channel (22), the sixth communicating channel (23), the seventh communicating channel (24), the capillary blocking valve (25), the eighth communicating channel (26), the exhaust channel (17), the internal circulation vent (37), the sample liquid vent (38), and the color developing liquid vent (39) are provided on the apparatus.

[0014] The first communication channel (18), the second communication channel (19), the third communication channel (20), the fourth communication channel (21), the fifth communication channel (22), the sixth communication channel (23), and the seventh communication channel (24) are connected to the eighth communication channel (26) via a capillary blocking valve (25).

[0015] The internal circulation vent (37), the sample liquid vent (38), and the color developing liquid vent (39) are connected through the exhaust channel (17).

[0016] The first liquid injection hole (56) is located directly above the first storage chamber (13).

[0017] During the injection process, part of the air in the first storage chamber (13) is discharged from the first exhaust hole (55) to the outside atmosphere via the first communicating channel (18) and the second communicating channel (19); and part of the air enters the downstream chamber via the first capillary valve (14).

[0018] The second liquid injection hole (57) is located directly above the second storage chamber (12).

[0019] During the injection process, part of the air in the second storage chamber (12) is discharged from the first exhaust hole (55) to the outside atmosphere via the first communication channel (18); and part of the air enters the downstream chamber via the second capillary valve (15).

[0020] The third liquid injection hole (59) is located directly above the fourth storage chamber (2).

[0021] During the injection process, part of the air in the fourth storage chamber (2) is discharged from the second exhaust hole (58) to the outside atmosphere via the fifth communication channel (22); and part of the air enters the downstream chamber via the fourth capillary valve (5).

[0022] The fourth liquid injection hole (60) is located directly above the fifth storage chamber (3).

[0023] During the injection process, part of the air in the fifth storage chamber (3) is discharged from the second exhaust hole (58) to the outside atmosphere via the sixth communication channel (23) and the seventh communication channel (24); and part of the air enters the downstream chamber via the third capillary valve (4).

[0024] The fifth liquid injection hole (61) is located directly above the third storage chamber (1).

[0025] During the injection process, part of the air in the third storage chamber (1) is discharged from the second exhaust hole (58) to the outside atmosphere via the fourth communication channel (21); and part of the air enters the downstream chamber via the fifth capillary valve (6).

[0026] The first storage chamber (13) and the first mixing chamber (16) are communicated with each other through a first capillary valve (14).

[0027] At a certain rotational speed, the liquid in the first storage chamber (13) breaks through the first capillary valve (14) and enters the first mixing chamber (16); the gas in the first mixing chamber (16) can flow to the outside atmosphere through the third connecting channel (20) via the capillary blocking valve (25), and can also flow into the second storage chamber (12) through the second capillary valve (15).

[0028] The second storage chamber (12) is communicated with the first mixing chamber (16) via a second capillary valve (15).

[0029] At a certain rotation speed, the liquid in the second storage chamber (12) breaks through the second capillary valve (15) and enters the first mixing chamber (16).

[0030] The first capillary valve (14) is located between the first storage chamber (13) and the first mixing chamber (16), and the second capillary valve (15) is located between the second storage chamber (12) and the first mixing chamber (16).

[0031] The third storage chamber (1) is communicated with the second mixing chamber (7) via a fifth capillary valve (6).

[0032] At a certain rotational speed, the liquid in the third storage chamber (1) breaks through the fifth capillary valve (6) and enters the second mixing chamber (7); the gas in the third storage chamber (1) can flow to the outside atmosphere through the fourth communication channel (21) and the capillary blocking valve (25).

[0033] The fourth storage chamber (2) is communicated with the second mixing chamber (7) via a fourth capillary valve (5).

[0034] At a certain rotational speed, the liquid in the fourth storage chamber (2) breaks through the fourth capillary valve (5) and enters the second mixing chamber (7); the gas in the second mixing chamber (7) can flow to the outside atmosphere through the seventh communication channel (24) via the capillary blocking valve (25), and the gas in the fourth storage chamber (2) can flow to the outside atmosphere through the fifth communication channel (22) via the capillary blocking valve (25).

[0035] The fifth storage chamber (3) is communicated with the second mixing chamber (7) via the third capillary valve (4).

[0036] At a certain rotational speed, the liquid in the fifth storage chamber (3) breaks through the third capillary valve (4) and enters the second mixing chamber (7); the gas in the second mixing chamber (7) can flow to the outside atmosphere through the seventh communication channel (24) via the capillary blocking valve (25), and the gas in the fifth storage chamber (3) can flow to the outside atmosphere through the sixth communication channel (23) via the capillary blocking valve (25).

[0037] The third capillary valve (4) is located between the fifth storage chamber (3) and the second mixing chamber (7), the fourth capillary valve (5) is located between the fourth storage chamber (2) and the second mixing chamber (7), and the fifth capillary valve (6) is located between the third storage chamber (1) and the second mixing chamber (7).

[0038] After the liquid in the first mixing chamber (16) passes through the first siphon channel (49), the liquid is retained in the first siphon channel (49) under the action of the first dissolvable membrane valve (44);

[0039] After the liquid in the second mixing chamber (7) passes through the air pressure channel (51), the liquid is retained in the air pressure channel (51) under the action of the second soluble membrane valve (45) and the air pressure hole (43).

[0040] The size of the first mixing chamber (16) is smaller than the sum of the sizes of the first storage chamber (13) and the second storage chamber (12), and the distance between the first mixing chamber (16) and the rotation center is greater than the distance between the first storage chamber (13) and the second storage chamber (12) and the rotation center. The volume of the sample mixture can just fill or partially fill the first mixing chamber (16).

[0041] The size of the second mixing chamber (7) is smaller than the sum of the sizes of the third storage chamber (1), the fourth storage chamber (2) and the fifth storage chamber (3), and the distance between the second mixing chamber (7) and the rotation center is greater than the distance between the third storage chamber (1), the fourth storage chamber (2) and the fifth storage chamber (3) and the rotation center. The volume of the developer solution mixture can just fill or partially fill the second mixing chamber (7).

[0042] Optionally, for viscous reagents (such as H1N1 sample solution and color development solution), the reagents in the first mixing chamber (16) and the second mixing chamber (7) can be stirred during amplification by the Euler force generated by the motor acceleration and deceleration process.

[0043] The distance between the siphon peak of the first siphon channel (49) and the rotation center is smaller than the distance between the inner end of the first mixing chamber (16) close to the second capillary valve (15) and the rotation center.

[0044] The distance between the siphon peak of the air pressure channel (51) and the rotation center is greater than or equal to the distance between the inner end of the second mixing chamber (7) close to the fifth capillary valve (6) and the rotation center.

[0045] The first siphon channel (49) and the air pressure channel (51) are connected through the exhaust channel (17) to balance the air pressure, the air pressure channel (51) and the test strip storage chamber (11) are connected through the second liquid inlet channel (9), and the first siphon channel (49) and the test strip storage chamber (11) are connected through the first liquid inlet channel (50).

[0046] Optionally, the sample ruthenium nanozyme premix in the first mixing chamber (16) and the color development premix in the second mixing chamber (7) can be fully mixed by the Euler force generated during the acceleration and deceleration process of the motor.

[0047] The liquid in the first mixing chamber (16) passes through the first siphon channel (49) through the sample liquid soluble membrane filling and releasing hole (32) to dissolve the soluble membrane, and then flows through the first liquid inlet channel (50) into the paper strip chromatography pad of the test strip storage chamber (11), and the color developing liquid air hole (31) is opened. The liquid in the second mixing chamber (7) passes through the color developing liquid soluble membrane filling and releasing hole (33) through the air pressure channel (51) to dissolve the soluble membrane, and then flows through the second liquid inlet channel (9) into the paper strip middle detection area of ​​the test strip storage chamber (11).

[0048] Optionally, for viscous reagents such as colorimetric reagents, the reagents in the second liquid inlet channel (9) can achieve sufficient contact between the reagent components by slowly flowing in the S-shaped flow channel, thereby improving the detection sensitivity.

[0049] To facilitate sealing, the chip body (29) has a first positioning hole (27) and a second positioning hole (28), wherein the first positioning hole (27) matches the sixth positioning hole (62) in the sealing body (64) and the ninth positioning hole (35) in the bottom flow channel layer (36), and the second positioning hole (28) matches the seventh positioning hole (63) in the sealing body (64) and the eighth positioning hole (34) in the bottom flow channel layer (36).

[0050] The chip body (29), the bottom flow channel layer (36), the vertical through-hole layer (42), the pressing layer (48), the top flow channel layer (54) and the sealing body (64) are provided with a positioning mechanism (30) capable of fixing the chip on the carrier.

[0051] The ruthenium-loaded peroxide nanozyme probe is obtained by coupling ruthenium-loaded microsphere peroxide nanozyme with an antibody. The coupling method comprises the following steps:

[0052] (1) The ruthenium-loaded microsphere peroxidase nanozyme was dissolved in 50 mM MES buffer at pH 5.0, and then appropriate amounts of EDC and NHS were added in sequence. After shaking at room temperature, the mixture was centrifuged and the supernatant was discarded;

[0053] (2) mixing the antibody with the solution, centrifuging and discarding the supernatant after coupling is completed, adding Tris-HCl solution for washing, and then centrifuging and discarding the supernatant to obtain the ruthenium nanozyme antibody probe;

[0054] and optionally:

[0055] (3) Add BSA blocking solution to block nonspecific binding sites;

[0056] (4) After centrifugation, discard the supernatant and resuspend in pH 7.4 PBS (optionally containing 0.1% Tween-20).

[0057] The preparation method of the ruthenium-loaded microsphere peroxidase nanozyme comprises the following steps:

[0058] (1) adding polystyrene microspheres to pure water at a concentration of 0.1-0.15 mg / mL and stirring at a rate of 300-500 rpm / min until completely dissolved and clarified to prepare a polystyrene microsphere solution;

[0059] (2) adding a solution containing a ruthenium salt and a reducing agent to a polystyrene microsphere solution to obtain a mixed solution, stirring the mixed solution and / or performing a reduction reaction under ultrasound, separating and discarding the supernatant after the reaction is completed to obtain the ruthenium-loaded microsphere peroxidase nanozyme;

[0060] Preferably,

[0061] Dissolve RuCl3·nH2O in pure water, with the mass ratio of ruthenium salt to polystyrene microspheres in the mixture being 1:1.25-10, preferably 1:2.5-5; dissolve the reducing agent and ruthenium salt separately, and add them to pure water at the same time to a concentration of 0.04-0.08 mg / mL;

[0062] The solution containing the ruthenium salt and the reducing agent is added at a rate of 50-100 μL / min, and the reducing agent is sodium borohydride;

[0063] The stirring rate of the mixed solution is 300-500 rpm / min; the ultrasonic frequency is 35-50 Hz; the ultrasonic time is 30-60 min; the reaction temperature is 20-25° C.; and the separation is centrifugal separation.

[0064] And optionally, (3) washing the ruthenium-loaded microsphere peroxidase nanozyme; preferably, the washing liquid is water.

[0065] The present invention also relates to a method for realizing automatic detection of a nanoenzyme immunochromatographic test strip in a centrifugal microfluidic chip, wherein the method comprises the following steps:

[0066] (1) Liquid injection: a ruthenium nanozyme probe solution is injected into the first storage chamber (13) through the first injection hole (56), wherein the ruthenium nanozyme is a ruthenium-loaded microsphere peroxidase nanozyme having peroxidase-like activity; a sample liquid to be detected is injected into the second storage chamber (12) through the second injection hole (57), a DAB buffer solution is injected into the third storage chamber (1) through the fifth injection hole (61), an H2O2 solution is injected into the fourth storage chamber (2) through the third injection hole (59), and a DAB substrate solution is injected into the fifth storage chamber (3) through the fourth injection hole (60).

[0067] (2) Sample flow and immune response: Through the micropump drive mechanism built into the centrifugal microfluidic chip, the sample is controlled to flow along the microchannel inside the chip at a certain rotation speed to ensure that the sample can be evenly mixed and fully contacted with the reaction area of ​​the ruthenium nanoenzyme immunochromatographic test strip.

[0068] (3) Result determination: When the sample passes through the immunochromatographic test strip, an antigen-antibody reaction occurs, and a detectable ruthenium nanozyme marker is formed on the T line of the ruthenium nanozyme test strip. The ruthenium nanozyme catalyzes the oxidation of the chromogenic substrate, thereby amplifying the detection signal and further improving the detection sensitivity.

[0069] Further,

[0070] In step (1),

[0071] The injection volume of the ruthenium nanozyme probe solution is 3-5 μL. The ruthenium nanozyme is a ruthenium-loaded microsphere peroxidase nanozyme. The antibody probe is obtained by coupling the ruthenium-loaded microsphere peroxidase nanozyme with an antibody. The injection volume of the sample solution is 70-100 μL, the injection volume of the DAB buffer is 40-60 μL, the injection volume of the H2O2 is 4-60 μL, and the injection volume of the DAB substrate solution is 2-4 μL.

[0072] In step (2),

[0073] Part of the air in the first storage chamber (13) is discharged from the first exhaust hole (55) to the outside atmosphere via the first communication channel (18) and the second communication channel (19); and part of the air enters the downstream chamber via the first capillary valve (14).

[0074] A portion of the air in the second storage chamber (12) is discharged from the first exhaust hole (55) to the outside atmosphere via the first communication channel (18); and a portion of the air enters the downstream chamber via the second capillary valve (15).

[0075] A portion of the air in the fourth storage chamber (2) is discharged from the second exhaust hole (58) to the outside atmosphere via the fifth communication channel (22); and a portion of the air enters the downstream chamber via the fourth capillary valve (5).

[0076] A portion of the air in the fifth storage chamber (3) is discharged from the second exhaust hole (58) to the outside atmosphere via the sixth communication channel (23) and the seventh communication channel (24); and a portion of the air enters the downstream chamber via the third capillary valve (4).

[0077] A portion of the air in the third storage chamber (1) is discharged from the second exhaust hole (58) to the outside atmosphere via the fourth communication channel (21); and a portion of the air enters the downstream chamber via the fifth capillary valve (6).

[0078] In step (3),

[0079] The ruthenium nanozyme marker can catalyze the oxidation of a chromogenic substrate in hydrogen peroxide (H2O2). Preferably, the chromogenic substrate is dimethylbenzidine (DAB).

[0080] The present invention also relates to the use of the centrifugal microfluidic chip in the preparation of a pathogen detection device based on immunochromatography. Preferably, the detection device is an immunochromatography detection device, the immunochromatography uses the nanozyme-loaded colorimetric probe, and the pathogen is a virus; more preferably, the pathogen is influenza A virus.

[0081] The beneficial effects of the present invention are:

[0082] 1. This invention designs a centrifugal microfluidic chip with an integrated ruthenium nanozyme test strip, achieving fully automated detection of ruthenium nanozyme test strips on the microfluidic chip. The entire detection process requires no human intervention. After sample addition, fluid distribution, chromatography reaction, and color development are automatically completed, significantly improving the convenience and repeatability of the experiment, while reducing human error and enhancing detection reliability.

[0083] 2. This invention proposes a precise fluid timing control solution based on a dissolvable membrane valve, avoiding the hydrophilic / hydrophobic surface treatment techniques commonly used in traditional microfluidic chips, ensuring stable and timely fluid control. Compared to hydrophilic / hydrophobic treatment methods, dissolvable membrane valves are free from issues such as material aging, humidity influence, and uneven surface treatment, thereby improving chip production consistency and reducing manufacturing costs.

[0084] 3. The centrifugal microfluidic chip of the present invention can realize parallel detection of multiple samples. Multiple samples can be analyzed simultaneously on a single chip, thereby improving detection throughput and being suitable for large-scale screening and high-throughput detection needs.

[0085] 4. The microfluidic chip of the present invention adopts a high-sensitivity detection strategy. By precisely controlling the fluid reaction sequence and the ruthenium nanozyme catalytic signal amplification mechanism, the detection signal intensity is improved, achieving the goal of detecting trace pathogens, such as the efficient detection of H1N1 virus nucleoprotein. The chip can maximize the use of the original antigen signal and improve the detection sensitivity. Suitable for low disease Early screening of toxic load samples .

[0086] 5. The microfluidic chip of this invention significantly shortens testing time and reduces testing costs. Thanks to precise automated fluid control and efficient enzyme-catalyzed reactions, the chip can complete immunoassays for trace pathogens in a relatively short period of time. Compared to traditional laboratory testing methods, the chip eliminates the need for complex equipment and time-consuming sample pretreatment, significantly improving detection efficiency and providing a viable solution for point-of-care testing (POCT). BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1-Figure 7 A centrifugal microfluidic chip with four sets of four-sample combination integrated nanozyme test strips for detection microfluidic structures is set on a circular chip. In actual work, it can be set as a single-sample chip, a double-sample combination chip, or a three-sample to N-sample combination chip as needed.

[0088] Figure 1 This is a schematic diagram of a partial structure of a chip body (29). In the figure: 1, third storage chamber, 2, fourth storage chamber, 3, fifth storage chamber, 4, third capillary valve, 5, fourth capillary valve, 6, fifth capillary valve, 7, second mixing chamber, 8, distribution unit, 9, second liquid inlet channel, 10, blocking plate, 11, test strip storage chamber, 12, second storage chamber, 13, first storage chamber, 14, first capillary valve, 15, second capillary valve, 16, first mixing chamber, 17, exhaust channel, 29, chip body, 30, positioning mechanism.

[0089] Figure 2 This is a schematic diagram of the internal structure of the chip body (29). In the figure: 18, first communication channel, 19, second communication channel, 20, third communication channel, 21, fourth communication channel, 22, fifth communication channel, 23, sixth communication channel, 24, seventh communication channel, 25, capillary blocking valve, 26, eighth communication channel, 27, first positioning hole, 28, second positioning hole, 29, chip body, 30, positioning mechanism.

[0090] Figure 3 Schematic diagram of the bottom flow channel layer (36). In the figure: 31, color liquid air hole, 32, sample liquid dissolvable membrane filling and releasing hole, 33, color liquid dissolvable membrane filling and releasing hole, 34, eighth positioning hole, 35, ninth positioning hole, 36, bottom flow channel layer.

[0091] Figure 4 Schematic diagram of the vertical through hole layer (42). In the figure: 37, internal circulation vent, 38, sample liquid vent, 39, color development liquid vent, 40, third positioning hole, 41, fourth positioning hole, 42, vertical through hole layer.

[0092] Figure 5 The diagram is a schematic diagram of the compression layer (48). In the diagram: 43, air pressure hole, 44, first dissolvable membrane valve, 45, second dissolvable membrane valve, 46, fifth positioning hole, 47, tenth positioning hole, 48, compression layer.

[0093] Figure 6 The top flow channel layer (54) is a schematic diagram. In the figure: 49, the first siphon channel, 50, the first liquid inlet channel, 51, the air pressure channel, 52, the eleventh positioning hole, 53, the twelfth positioning hole, 54, the top flow channel layer.

[0094] Figure 7 The figure is a schematic diagram of a sealing body (64). In the figure: 55, first exhaust hole, 56, first liquid injection hole, 57, second liquid injection hole, 58, second exhaust hole, 59, third liquid injection hole, 60, fourth liquid injection hole, 61, fifth liquid injection hole, 62, sixth positioning hole, 63, seventh positioning hole, 64, sealing body.

[0095] Figure 8 Schematic diagram of the structure of the nanozyme test strip.

[0096] Figure 9 Schematic diagram for explaining the test results.

[0097] Figure 10 This figure shows the test results of the centrifugal microfluidic chip for automated detection of influenza A antigens at different concentrations. DETAILED DESCRIPTION

[0098] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0099] Unless otherwise specifically stated, the relative arrangement of components and steps, the numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention.

[0100] At the same time, it should be understood that for the convenience of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship.

[0101] Technologies, methods and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, such technologies, methods and equipment should be considered part of the authorization specification.

[0102] Example 1: Centrifugal microfluidic chip for automated detection of nanozyme test strips

[0103] The preparation process of the nanozyme probe solution used in this example is as follows:

[0104] 1) After centrifugation of the nanozyme aqueous solution, 50 mM pH 5.0 MES buffer was added, mixed evenly, and then 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 25 mM N-hydroxysuccinimide (NHS) were added in sequence. After shaking at room temperature, centrifugation was performed and the supernatant was discarded.

[0105] 2) Mix 1 mg / mL influenza A virus monoclonal antibody with the solution and shake at 4°C overnight;

[0106] 3) Centrifuge and discard the supernatant, add 50 mM Tris-HCl solution, shake at room temperature for 30 minutes, and then centrifuge and discard the supernatant;

[0107] 4) Add BSA blocking solution and shake at room temperature for 2 hours to block nonspecific binding sites;

[0108] 5) Centrifuge and discard the supernatant, resuspend in pH 7.4 PBS (0.1% TWEEN20) to obtain the nanozyme probe solution, and store it at 4°C.

[0109] This embodiment provides a centrifugal microfluidic chip for automated detection of nanozyme test strips. The chip can be glued together before injection. Figure 1-2 The chip body (29) shown Figure 3 The bottom channel layer (36) shown, Figure 4 The vertical through hole layer (42) shown Figure 5 The compression layer (48) shown Figure 6 The top channel layer (54) and Figure 7 The sealing body (64) shown is bonded together through the positioning holes and is ready for use.

[0110] like Figure 2 and 7As shown, the nanozyme probe solution, sample solution, DAB buffer, DAB substrate solution and H2O2 solution are respectively injected into the corresponding first storage chamber (13), second storage chamber (12), third storage chamber (1), fifth storage chamber (3) and fourth storage chamber (2) through the first injection hole (56), second injection hole (57), fifth injection hole (61), fourth injection hole (60) and third injection hole (59) using a pipette. Subsequently, a sealing member is used to seal the first injection hole (56), second injection hole (57), fifth injection hole (61), third injection hole (59), fourth injection hole (60) and the first exhaust hole (55) and the second exhaust hole (58). The sealing member can be a 3M single-sided tape or a PET film.

[0111] The chip positioning mechanism (30) is then matched with its corresponding carrier to perform subsequent fluid control and chromatography color development reactions.

[0112] (1) The liquid in the first storage chamber (13) and the second storage chamber (12) sequentially breaks through the first capillary valve (14) and the second capillary valve (15) at a certain rotation speed and enters the first mixing chamber (16); the liquid in the third storage chamber (1), the fifth storage chamber (3) and the fourth storage chamber (2) sequentially breaks through the third capillary valve (6), the fourth capillary valve (5) and the fifth capillary valve (4) at a certain rotation speed and enters the second mixing chamber (7).

[0113] (2) Subsequently, part of the liquid in the chamber passes through the first siphon channel (49) and the air pressure channel (51) under capillary action and stops after the peak. Then, the sample mixture begins to dissolve the soluble membrane to open the first soluble membrane valve (44) and the air pressure hole (43).

[0114] (3) After the first soluble membrane valve (44) is opened, at a certain rotation speed, the sample mixture in the first mixing chamber (16) enters the test strip storage chamber (11) through the first liquid inlet channel (50) to perform a chromatography reaction in the paper strip chromatography pad, and the color development liquid mixture in the second mixing chamber (7) dissolves and breaks through the second soluble membrane valve (45). The sample liquid is precisely controlled in the second liquid inlet channel (9) for 7 minutes to wait for the sample liquid chromatography to be complete, and enters the middle detection area of ​​the test strip storage chamber (11) through the second liquid inlet channel (9). The two liquids undergo a color development reaction in the middle detection area of ​​the test strip storage chamber (11). After the reaction is complete, the detection signal will be greatly enhanced.

[0115] In order to prevent the color-developing liquid from entering the test strip storage chamber (11) and not being spread out in the middle detection area according to the preset state for color development reaction, a barrier plate (10) is added to prevent the color-developing liquid from entering the absorbent pad under the action of centrifugation and failing to fully develop the color, thereby ensuring reaction stability.

[0116] To facilitate chip sealing, the chip body (29), the bottom flow channel layer (36), the vertical through-hole layer (42), the compression layer (48), the top flow channel layer (54) and the sealing body (64) can be sealed and connected by gluing, hot pressing, laser welding and ultrasonic welding.

[0117] Example 2: Method for assembling nanoenzyme test strips on a centrifugal microfluidic chip for automated detection

[0118] In this embodiment, the nanozyme test strip is installed in the test strip storage cavity (11) of the microfluidic chip described in Example 1.

[0119] The nanozyme test strip has a conventional structure, specifically:

[0120] The test strip includes a PVC base, a sample pad, a nitrocellulose membrane and an absorbent pad. The nitrocellulose membrane is adhered to the PVC base, and then the absorbent pad is adhered to the PVC base in a manner that covers 2 mm of the upper edge of the nitrocellulose membrane. The sample pad is also adhered to the PVC base in a manner that covers 2 mm of the lower edge of the nitrocellulose membrane. The quality control line on the nitrocellulose membrane is close to the absorbent pad, and the detection line on the nitrocellulose membrane is close to the sample pad. The complete nano enzyme immunochromatography test strip is assembled and then cut into test strips with a width of 4 mm using a strip cutter.

[0121] This example uses the detection of influenza A virus as an example to detect the NP protein of influenza A virus. In the detection system of this example, the dosage of the detection antibody for influenza A virus is 2 mg / mL; the line concentrations of other detection antibodies for different pathogens can be adjusted according to actual conditions.

[0122] In this embodiment, the influenza A virus detection line is influenza A virus monoclonal antibody (5.7 mg / mL, item number FLUA-REAB-G1-019), the quality control line antibody is goat anti-mouse IgG antibody (2 mg / mL, item number BF02001C), and the standard sample for detection is influenza A virus antigen standard (item number FA01). Goat anti-mouse IgG was purchased from Suzhou Biolong Technology Co., Ltd., influenza A virus monoclonal antibody was purchased from Hangzhou Feipeng Biological Co., Ltd., and influenza A virus antigen was purchased from Hangzhou Qitai Biological Co., Ltd.

[0123] The test strip test line and quality control line are prepared as follows:

[0124] 1) Preparation of test and control lines: Dilute influenza A virus monoclonal antibody (test antibody) to 2 mg / mL in 0.02 M PB and streak a nitrocellulose membrane using a streak marker to serve as the test line. Dilute goat anti-mouse IgG antibody (control antibody) to 2 mg / mL in 0.02 M PB and streak a nitrocellulose membrane using a streak marker to serve as the control line. Use a streak setting of 0.1 μL / mm and a 7 mm distance between the control and test lines. Dry the streaked nitrocellulose membrane at 37°C overnight to obtain the treated nitrocellulose membrane.

[0125] 2) Assemble the test strips: First, adhere the nitrocellulose membrane to the PVC base plate, then adhere the absorption pad to the PVC base plate in a way that covers 2 mm of the upper edge of the nitrocellulose membrane, and also adhere the sample pad to the PVC base plate in a way that covers 2 mm of the lower edge of the nitrocellulose membrane. Place the quality control line on the nitrocellulose membrane close to the absorption pad, and place the detection line on the nitrocellulose membrane close to the sample pad; the absorption pad is absorbent filter paper and the sample pad is glass fiber membrane; assemble into a complete nano enzyme immunochromatography test strip, and then use a strip cutter to cut into test strips with a width of 4 mm, as shown in the figure. Figure 8 .

[0126] 3) Installing a test strip: Install the above-mentioned nanozyme test strip in the centrifugal microfluidic chip of Example 1, and further install it in the test strip storage cavity (11).

[0127] Example 3: Method for using a centrifugal microfluidic chip containing a nanozyme test strip

[0128] This embodiment provides a method for realizing automatic detection using a centrifugal microfluidic chip containing a nanozyme test strip prepared in Example 1-2. The test strip used in this embodiment is the nanozyme immunochromatographic test strip prepared in Example 2. The antigen detected is an influenza A virus antigen. The monoclonal antibody used to detect the antigen is (FLUA-REAB-G1-019). When preparing the test line of the test strip, the concentration of the line is 2 mg / ml.

[0129] The detection method includes the following steps:

[0130] 4 μL of ruthenium nanozyme probe solution is injected into the first storage chamber (13) through the first injection hole (56), 80 μL of the sample solution to be detected is injected into the second storage chamber (12) through the second injection hole (57), 50 μL of DAB buffer solution is injected into the third storage chamber (1) through the fifth injection hole (61), 5 μL of H2O2 solution is injected into the fourth storage chamber (2) through the third injection hole (59), and 3 μL of DAB substrate solution is injected into the fifth storage chamber (3) through the fourth injection hole (60). Subsequently, a sealing member is used to seal the first injection hole (56), the second injection hole (57), the fifth injection hole (61), the fourth injection hole (60), the third injection hole (59), the first exhaust hole (55), and the second exhaust hole (58).

[0131] The chip positioning mechanism (30) is matched with its corresponding carrier to perform subsequent fluid control, and each liquid begins to flow along the microchannel in the chip at a certain rotation speed, thereby realizing the chromatography and color development reaction of the ruthenium nanozyme test strip.

[0132] The test results are judged as follows Figure 9 :

[0133] If the control line and the test line appear on the ruthenium nanoenzyme immunochromatographic test strip at the same time, the test result is positive, that is, influenza A virus is present in the sample to be tested;

[0134] If only the quality control line appears on the influenza A virus ruthenium nanozyme immunochromatographic test strip, the test result is negative, which means that there is no influenza A virus in the sample to be tested;

[0135] If only a test line appears on the influenza A virus ruthenium nanozyme immunochromatographic test strip, the test result is invalid and needs to be retested.

[0136] 2. Influenza A virus standard sample detection

[0137] (1) Purchase influenza A virus antigen and dilute it with chromatography buffer (50 mM TBS + 1% BSA + 0.5% NP40) to obtain influenza A virus antigen solutions of different concentrations for later use.

[0138] (2) 80 μL of influenza A virus antigens of different concentrations (7 different concentrations of influenza A virus antigens to be tested, 5000, 2500, 1000, 250, 50, 10 and 0 pg / mL) were injected into the second storage chamber (12) through the second injection hole (57), and the nanozyme probe solution, DAB buffer, DAB substrate solution and H2O2 solution were injected into the corresponding first storage chamber (13), third storage chamber (1), fifth storage chamber (3) and fourth storage chamber (2) through the first injection hole (56), fifth injection hole (61), fourth injection hole (60) and third injection hole (59) using a pipette. After the setting program is completed, the color development results are observed. In the color development mode, the T zone of the positive and low-concentration positive test paper appears brownish red.

[0139] The test results are as follows Figure 10 In seven influenza A virus antigen concentration tests, the lowest antigen detection concentration of the ruthenium nanozyme immunochromatographic test strip in the centrifugal microfluidic chip was 50 pg / mL, while the lowest antigen detection concentration of the commercial colloidal gold test strip was 2500 pg / mL. The centrifugal microfluidic chip for automated detection of the ruthenium nanozyme test strips involved in the present invention has increased the detection sensitivity of influenza A virus antigens by about 50 times.

[0140] The above drawings and specific embodiments are intended only to illustrate the present invention and are not intended to limit the present invention. Minor modifications to the present invention within the spirit and scope of the invention as defined by the claims fall within the scope of protection of the present invention. These modifications include, for example, the shape and size of the chamber, the structure and size of various valves, and so on.

[0141] Those skilled in the art will understand that all or part of the steps to implement the above embodiments may be accomplished by hardware, or by a program to instruct the relevant hardware, and the program may be stored in a computer-readable storage medium, which may be a read-only memory, a disk, or an optical disk, etc.

[0142] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the scope of protection of the present invention.

Claims

1. A centrifugal microfluidic chip, characterized in that: The microfluidic chip is loaded with ruthenium-loaded peroxide nanozyme probes and immunochromatographic test strips for detecting trace pathogens; preferably, the microfluidic chip can detect a sample volume of 10-100 μL; The chip is composed of a chip body (29) with a reaction structure, a bottom flow channel layer (36), a vertical through-hole layer (42) with a soluble film, a compression layer (48), a top flow channel layer (54) and a sealing body (64) (Figures 1-6); wherein, The reaction structure of the chip body (29) includes: a first storage chamber (13) for storing the ruthenium nanozyme probe, a second storage chamber (12) for storing the sample liquid, a third storage chamber (1) for storing the DAB buffer solution, a fourth storage chamber (2) for storing H2O2, a fifth storage chamber (3) for storing the DAB substrate, a first mixing chamber (16) for mixing the sample liquid and the ruthenium nanozyme probe, a second mixing chamber (7) for mixing the DAB substrate, H2O2, and the DAB buffer solution, a test strip storage chamber (11) for storing the ruthenium nanozyme test strip, the first mixing chamber (16) and the second mixing chamber (7), a distribution unit (8) connected to the second mixing chamber (7), a ventilation pipe, and various fluid valves; Preferably, the volume of the first storage chamber (13) for storing the ruthenium nanozyme probe is 2-10 μL, the volume of the second storage chamber (12) for storing the sample liquid is 10-100 μL, the volume of the third storage chamber (1) for storing the DAB buffer is 40-60 μL, the volume of the fourth storage chamber (2) for storing H2O2 is 4-60 μL, and the volume of the fifth storage chamber (3) for storing the DAB substrate is 2-4 μL.

2. The centrifugal microfluidic chip according to claim 1, characterized in that: The sealing body (64) includes: a first exhaust hole (55), a first liquid injection hole (56), a second liquid injection hole (57), a second exhaust hole (58), a third liquid injection hole (59), a fourth liquid injection hole (60), a fifth liquid injection hole (61), a positioning mechanism (30), a sixth positioning hole (62) and a seventh positioning hole (63); The bottom flow channel layer (36) includes: a color developing liquid air hole (31), a sample liquid soluble membrane filling and releasing hole (32), and a color developing liquid soluble membrane filling and releasing hole (33); The centrifugal microfluidic chip interconnects the functional cavities in the ventilation structure to form a self-closed loop, comprising: The first exhaust hole (55) and the second exhaust hole (58) located on the sealing body (64) are connected to the first communication channel (18), the second communication channel (19), the third communication channel (20), the fourth communication channel (21), the fifth communication channel (22), the sixth communication channel (23), the seventh communication channel (24), the capillary blocking valve (25), the eighth communication channel (26), the exhaust channel (17), the internal circulation vent (37), the sample liquid vent (38), and the color development liquid vent (39) located on the internal structure of the chip body (29); in, A. The first communication channel (18), the second communication channel (19), the third communication channel (20), the fourth communication channel (21), the fifth communication channel (22), the sixth communication channel (23), and the seventh communication channel (24) are connected to the eighth communication channel (26) via the capillary blocking valve (25); The internal circulation vent (37), the sample liquid vent (38), and the color developing liquid vent (39) are connected through the exhaust channel (17); B. The first liquid injection hole (56) is located directly above the first storage chamber (13); during the liquid injection process, a portion of the air in the first storage chamber (13) is discharged from the first exhaust hole (55) to the outside atmosphere via the first communication channel (18) and the second communication channel (19); a portion of the air enters the downstream chamber via the first capillary valve (14); C. The second liquid injection hole (57) is located directly above the second storage chamber (12); during the liquid injection process, a portion of the air in the second storage chamber (12) is discharged from the first exhaust hole (55) to the outside atmosphere via the first communication channel (18); and a portion of the air enters the downstream chamber via the second capillary valve (15); D. The third liquid injection hole (59) is located directly above the fourth storage chamber (2); during the liquid injection process, a portion of the air in the fourth storage chamber (2) is discharged from the second exhaust hole (58) to the outside atmosphere via the fifth communication channel (22); and a portion of the air enters the downstream chamber via the fourth capillary valve (5); E. The fourth liquid injection hole (60) is located directly above the fifth storage chamber (3); during the liquid injection process, a portion of the air in the fifth storage chamber (3) is discharged from the second exhaust hole (58) to the outside atmosphere via the sixth communication channel (23) and the seventh communication channel (24); a portion of the air enters the downstream chamber via the third capillary valve (4); F. The fifth liquid injection hole (61) is located directly above the third storage chamber (1); during the liquid injection process, a portion of the air in the third storage chamber (1) is discharged from the second exhaust hole (58) to the outside atmosphere via the fourth connecting channel (21); and a portion of the air enters the downstream chamber via the fifth capillary valve (6).

3. The centrifugal microfluidic chip according to claim 1 or 2, characterized in that: The first storage chamber (13) and the first mixing chamber (16) are connected via a first capillary valve (14); at a certain rotational speed, the liquid in the first storage chamber (13) breaks through the first capillary valve (14) and enters the first mixing chamber (16); the gas in the first mixing chamber (16) can flow to the outside atmosphere through the third communication channel (20) via the capillary blocking valve (25), or can flow into the second storage chamber (12) through the second capillary valve (15); The second storage chamber (12) is connected to the first mixing chamber (16) via a second capillary valve (15); at a certain rotation speed, the liquid in the second storage chamber (12) breaks through the second capillary valve (15) and enters the first mixing chamber (16); The first capillary valve (14) is located between the first storage chamber (13) and the first mixing chamber (16), and the second capillary valve (15) is located between the second storage chamber (12) and the first mixing chamber (16); The third storage chamber (1) is connected to the second mixing chamber (7) via a fifth capillary valve (6); at a certain rotational speed, the liquid in the third storage chamber (1) breaks through the fifth capillary valve (6) and enters the second mixing chamber (7); and the gas in the third storage chamber (1) can flow to the outside atmosphere through the fourth communication channel (21) and the capillary blocking valve (25); The fourth storage chamber (2) and the second mixing chamber (7) are connected via a fourth capillary valve (5); at a certain rotational speed, the liquid in the fourth storage chamber (2) breaks through the fourth capillary valve (5) and enters the second mixing chamber (7); the gas in the second mixing chamber (7) can flow to the outside atmosphere through the seventh communication channel (24) via the capillary blocking valve (25), and the gas in the fourth storage chamber (2) can flow to the outside atmosphere through the fifth communication channel (22) via the capillary blocking valve (25); The fifth storage chamber (3) is connected to the second mixing chamber (7) via the third capillary valve (4); At a certain rotational speed, the liquid in the fifth storage chamber (3) breaks through the third capillary valve (4) and enters the second mixing chamber (7); the gas in the second mixing chamber (7) can flow to the outside atmosphere through the seventh communication channel (24) via the capillary blocking valve (25), and the gas in the fifth storage chamber (3) can flow to the outside atmosphere through the sixth communication channel (23) via the capillary blocking valve (25); The third capillary valve (4) is located between the fifth storage chamber (3) and the second mixing chamber (7), the fourth capillary valve (5) is located between the fourth storage chamber (2) and the second mixing chamber (7), and the fifth capillary valve (6) is located between the third storage chamber (1) and the second mixing chamber (7).

4. The centrifugal microfluidic chip according to any one of claims 1 to 3, characterized in that: After the liquid in the first mixing chamber (16) passes through the first siphon channel (49), the liquid is retained in the first siphon channel (49) under the action of the first dissolvable membrane valve (44); After the liquid in the second mixing chamber (7) passes through the air pressure channel (51), the liquid is retained in the air pressure channel (51) under the action of the second dissolvable membrane valve (45) and the air pressure hole (43); The size of the first mixing chamber (16) is smaller than the sum of the sizes of the first storage chamber (13) and the second storage chamber (12), and the distance between the first mixing chamber (16) and the rotation center is greater than the distance between the first storage chamber (13) and the second storage chamber (12) and the rotation center. The volume of the sample mixture can just fill or partially fill the first mixing chamber (16); The size of the second mixing chamber (7) is smaller than the sum of the sizes of the third storage chamber (1), the fourth storage chamber (2) and the fifth storage chamber (3), and the distance between the second mixing chamber (7) and the rotation center is greater than the distance between the third storage chamber (1), the fourth storage chamber (2) and the fifth storage chamber (3) and the rotation center. The volume of the developer solution mixture can just fill or partially fill the second mixing chamber (7).

5. The centrifugal microfluidic chip according to any one of claims 1 to 4, characterized in that: For high-viscosity reagents, the reagents in the first mixing chamber (16) and the second mixing chamber (7) can be stirred during amplification by the Euler force generated by the motor acceleration and deceleration process.

6. The centrifugal microfluidic chip according to any one of claims 1 to 4, characterized in that: The distance between the siphon peak of the first siphon channel (49) and the rotation center is smaller than the distance between the inner end of the first mixing chamber (16) close to the second capillary valve (15) and the rotation center; The distance between the siphon peak of the air pressure channel (51) and the rotation center is greater than or equal to the distance between the inner end of the second mixing chamber (7) close to the fifth capillary valve (6) and the rotation center; The first siphon channel (49) and the air pressure channel (51) are connected through the exhaust channel (17) to balance the air pressure, the air pressure channel (51) and the test strip storage chamber (11) are connected through the second liquid inlet channel (9), and the first siphon channel (49) and the test strip storage chamber (11) are connected through the first liquid inlet channel (50); and Optionally, the sample ruthenium nanozyme premix in the first mixing chamber (16) and the color development premix in the second mixing chamber (7) can be fully mixed by the Euler force generated during the acceleration and deceleration process of the motor.

7. The centrifugal microfluidic chip according to any one of claims 1 to 4, characterized in that: The liquid in the first mixing chamber (16) passes through the first siphon channel (49) through the sample liquid soluble membrane filling and releasing hole (32) to dissolve the soluble membrane, and then flows through the first liquid inlet channel (50) into the paper strip chromatography pad of the test strip storage chamber (11), and the color developing liquid air hole (31) is opened. The liquid in the second mixing chamber (7) passes through the color developing liquid soluble membrane filling and releasing hole (33) to dissolve the soluble membrane, and then flows through the second liquid inlet channel (9) into the paper strip middle detection area of ​​the test strip storage chamber (11); as well as Optionally, for viscous reagents, the reagents in the second liquid inlet channel (9) can achieve sufficient contact between the reagent components by slowly flowing in the S-shaped flow channel, thereby improving the detection sensitivity.

8. The centrifugal microfluidic chip according to any one of claims 1 to 7, characterized in that: The ruthenium-loaded peroxide nanozyme probe is obtained by coupling ruthenium-loaded microsphere peroxide nanozyme with an antibody. The coupling method comprises the following steps: (1) The ruthenium-loaded microsphere peroxidase nanozyme was dissolved in 50 mM MES buffer at pH 5.0, and then appropriate amounts of EDC and NHS were added in sequence. After shaking at room temperature, the mixture was centrifuged and the supernatant was discarded; (2) mixing the antibody with the solution, centrifuging and discarding the supernatant after coupling is completed, adding Tris-HCl solution for washing, and then centrifuging and discarding the supernatant to obtain the ruthenium nanozyme antibody probe; and optionally: (3) Add BSA blocking solution to block nonspecific binding sites; (4) Centrifuge, discard the supernatant, and resuspend in pH 7.4 PBS (optionally, containing 0.1% Tween-20); The preparation method of the ruthenium-loaded microsphere peroxidase nanozyme comprises the following steps: (1) adding polystyrene microspheres at a concentration of 0.1-0.15 mg / mL into pure water, stirring at a rate of 300-500 rpm / min until completely dissolved and clarified, to prepare a polystyrene microsphere solution; (2) adding a solution containing a ruthenium salt and a reducing agent to a polystyrene microsphere solution to obtain a mixed solution, stirring the mixed solution and / or performing a reduction reaction under ultrasound, separating and discarding the supernatant after the reaction is completed to obtain the ruthenium-loaded microsphere peroxidase nanozyme; Preferably, Dissolve RuCl3·nH2O in pure water, with the mass ratio of ruthenium salt to polystyrene microspheres in the mixture being 1:1.25-10, preferably 1:2.5-5; dissolve the reducing agent and ruthenium salt separately, and add them to pure water at the same time to a concentration of 0.04-0.08 mg / mL; The solution containing the ruthenium salt and the reducing agent is added at a rate of 50-100 μL / min, and the reducing agent is sodium borohydride; The stirring rate of the mixed solution is 300-500 rpm / min; the ultrasonic frequency is 35-50 Hz; the ultrasonic time is 30-60 min; the reaction temperature is 20-25° C.; and the separation is centrifugal separation. And optionally, (3) washing the ruthenium-loaded microsphere peroxidase nanozyme; preferably, the washing liquid is water.

9. A method for realizing automatic detection using the centrifugal microfluidic chip according to any one of claims 1 to 8, the method comprising the following steps: (1) Liquid injection: a ruthenium nanozyme probe solution is injected into the first storage chamber (13) from the first injection hole (56), wherein the ruthenium nanozyme is a ruthenium-loaded microsphere peroxidase nanozyme having peroxidase-like activity; a sample liquid to be detected is injected into the second storage chamber (12) from the second injection hole (57), a DAB buffer solution is injected into the third storage chamber (1) from the fifth injection hole (61), an H2O2 solution is injected into the fourth storage chamber (2) from the third injection hole (59), and a DAB substrate solution is injected into the fifth storage chamber (3) from the fourth injection hole (60); (2) Sample flow and immune response: The micropump drive mechanism built into the centrifugal microfluidic chip controls the sample flow along the microchannel within the chip at a certain rotation speed, ensuring that the sample can be evenly mixed and fully contact the reaction area of ​​the ruthenium nanoenzyme immunochromatographic test strip; (3) Result determination: When the sample passes through the immunochromatographic test strip, an antigen-antibody reaction occurs, forming a detectable ruthenium nanozyme marker on the T line of the ruthenium nanozyme test strip. The ruthenium nanozyme catalyzes the oxidation of the chromogenic substrate, thereby amplifying the detection signal and further improving the detection sensitivity. Further, In step (1), The injection volume of the ruthenium nanozyme probe solution is 3-5 μL. The ruthenium nanozyme is a ruthenium-loaded microsphere peroxidase nanozyme. The antibody probe is obtained by coupling the ruthenium-loaded microsphere peroxidase nanozyme with an antibody. The injection volume of the sample solution is 70-100 μL, the injection volume of the DAB buffer is 40-60 μL, the injection volume of the H2O2 is 4-60 μL, and the injection volume of the DAB substrate solution is 2-4 μL. In step (2), A portion of the air in the first storage chamber (13) is discharged into the outside atmosphere from the first exhaust hole (55) via the first communication channel (18) and the second communication channel (19); A portion of the air enters the downstream chamber through the first capillary valve (14); A portion of the air in the second storage chamber (12) is discharged into the outside atmosphere from the first exhaust hole (55) via the first communication channel (18); A portion of the air enters the downstream chamber through the second capillary valve (15); A portion of the air in the fourth storage chamber (2) is discharged into the outside atmosphere from the second exhaust hole (58) via the fifth communication channel (22); A portion of the air enters the downstream chamber through the fourth capillary valve (5); A portion of the air in the fifth storage chamber (3) is discharged into the outside atmosphere from the second exhaust hole (58) via the sixth communication channel (23) and the seventh communication channel (24); A portion of the air enters the downstream chamber through the third capillary valve (4); A portion of the air in the third storage chamber (1) is discharged into the outside atmosphere from the second exhaust hole (58) via the fourth communication channel (21); A portion of the air enters the downstream chamber through the fifth capillary valve (6); In step (3), The ruthenium nanozyme marker can catalyze the oxidation of a chromogenic substrate in hydrogen peroxide (H2O2). Preferably, the chromogenic substrate is dimethylbenzidine (DAB).

10. Use of the centrifugal microfluidic chip according to any one of claims 1 to 8 in preparing a pathogen detection device. Preferably, the detection device is an immunochromatographic detection device, the immunochromatographic method uses the nanozyme loaded colorimetric probe, and the pathogen is a virus; More preferably, the pathogen is influenza A virus.

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