Centrifugal microfluidic chip for automatic detection of nanoscale enzyme test strip and application thereof

Abstract

The application relates to a centrifugal microfluidic chip for automatic detection of a nano-enzyme test strip and application. The centrifugal microfluidic chip integrates sample adding, chromatography, color development and other steps of the nano-enzyme test strip, and realizes full automation of the nano-enzyme experiment on the microfluidic chip. The centrifugal microfluidic chip comprises a reaction structure main body, a bottom flow channel layer, a vertical through-hole layer, a compression layer, a top flow channel 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] This invention belongs to the field of medical device technology, specifically relating to a centrifugal microfluidic chip for automated detection of nanozyme test strips and its application. Background Technology

[0002] Nanozyme immunochromatography has been widely applied in various detection scenarios, especially in the rapid detection of biomarkers, offering advantages such as simplicity, speed, and high sensitivity. Traditional immunochromatographic test strips generally require manual operation, including sample addition and strip reading. This not only increases the detection time and complexity but is also susceptible to the influence of operator subjectivity, leading to inconsistent and inaccurate results. Therefore, how to automate the detection process using nanozyme immunochromatographic test strips has become a pressing technological challenge.

[0003] Microfluidics technology integrates bioanalytical processes such as sample pretreatment, immune responses, and signal detection into microchips, creating portable "lab-on-a-chip" systems. Among them, centrifugal microfluidic chips utilize centrifugal force to drive fluid movement, eliminating the dependence on complex pump and valve structures in traditional microfluidic systems. With its high throughput and ease of parallelization, it has become an ideal carrier for rapid multi-target detection.

[0004] Compared to 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, significantly reducing environmental sensitivity and process complexity. Traditional chips rely on hydrophilic / hydrophobic modifications or physical valve structures to control fluid flow, which are susceptible to surface energy decay due to temperature and humidity fluctuations or storage aging, leading to fluid leakage or valve control failure, increasing manufacturing costs and system failure risks. Furthermore, traditional valves struggle to coordinate timing logic in multi-step detection processes; critical steps such as the sequential mixing of samples and reagents and the control of reaction incubation time often result in deviations in detection results due to fluid lag or asynchronous triggering. More importantly, traditional chips rely solely on single motor speed adjustment for full-process control, requiring frequent manual intervention in fixed-speed modes, resulting in insufficient automation.

[0005] Based on the above description, combining nanozyme immunochromatographic test strips with centrifugal microfluidic chips to achieve automatic sample loading, flow control, and automatic result reading, while meeting the needs of high-throughput and multi-target detection, is essential to improving detection sensitivity and automation level and promoting the development of nanozyme test strip detection technology.

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

[0007] This invention first relates to an automated detection centrifugal microfluidic chip, wherein the microfluidic chip is loaded with a ruthenium-loaded peroxide nanozyme probe and an immunochromatographic test strip for highly sensitive pathogen detection; preferably, the microfluidic chip can detect sample volumes of 10-100 μL; the chip consists of a chip body (29) with a reaction structure, a bottom channel layer (36), a vertical through-hole layer (42) with a soluble membrane, a compression layer (48), a top channel layer (54), and a sealing body (64). Figure 1-6 );in,

[0008] The sealing body (64) includes: a first vent hole (55), a first injection hole (56), a second injection hole (57), a second vent hole (58), a third injection hole (59), a fourth injection hole (60), a fifth 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: colorimetric liquid pores (31), sample liquid soluble membrane filling pores (32), and colorimetric liquid soluble membrane filling pores (33).

[0010] The reaction structure of the chip body (29) Figure 1 The device includes: a first storage chamber (13) for storing ruthenium nanozyme probes, a second storage chamber (12) for storing sample solution, a third storage chamber (1) for storing DAB buffer, a fourth storage chamber (2) for storing H2O2, a fifth storage chamber (3) for storing DAB substrate, a first mixing chamber (16) for mixing sample solution and ruthenium nanozyme probes, a second mixing chamber (7) for mixing DAB substrate, H2O2 and DAB buffer, a test strip storage chamber (11) for storing ruthenium nanozyme test strips, the first mixing chamber (16) and the second mixing chamber (7), a dispensing unit (8) connected to the second mixing chamber (7), a venting 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 solution 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 self-ventilating structure of the centrifugal microfluidic chip, in which the interconnected functional cavities form a self-closed loop, includes:

[0013] The first vent (55) and the second vent (58) located on the sealing body (64) are connected to the internal structure of the chip body (29). Figure 2 The first connecting channel (18), the second connecting channel (19), the third connecting channel (20), the fourth connecting channel (21), the fifth connecting channel (22), the sixth connecting channel (23), the seventh connecting channel (24), the capillary blocking valve (25), the eighth connecting channel (26), the exhaust channel (17), the internal circulation vent (37), the sample liquid vent (38), and the colorimetric liquid vent (39) are shown.

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

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

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

[0017] During the injection process, a portion of the air in the first storage chamber (13) is discharged into the outside atmosphere through the first vent (55) via the first connecting channel (18) and the second connecting channel (19); a portion of the air enters the downstream chamber via the first capillary valve (14).

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

[0019] During the injection process, a portion of the air in the second storage chamber (12) is discharged into the outside atmosphere through the first exhaust port (55) via the first connecting channel (18); a portion of the air enters the downstream chamber via the second capillary valve (15).

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

[0021] During the injection process, a portion of the air in the fourth storage chamber (2) is discharged into the outside atmosphere through the second exhaust port (58) via the fifth connecting channel (22); a portion of the air enters the downstream chamber via the fourth capillary valve (5).

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

[0023] During the injection process, a portion of the air in the fifth storage chamber (3) is discharged into the outside atmosphere through the second exhaust port (58) via the sixth connecting channel (23) and the seventh connecting channel (24); a portion of the air enters the downstream chamber via the third capillary valve (4).

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

[0025] During the injection process, a portion of the air in the third storage chamber (1) is discharged into the outside atmosphere through the second exhaust port (58) via the fourth connecting channel (21); a portion 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 connected by the 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), or it can flow into the second storage chamber (12) through the second capillary valve (15).

[0028] The second storage chamber (12) is connected to the first mixing chamber (16) through the second capillary valve (15).

[0029] At a certain rotational 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 connected to the second mixing chamber (7) through the 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 connecting channel (21) via the capillary blocking valve (25).

[0033] The fourth storage chamber (2) is connected to the second mixing chamber (7) through the 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 connecting 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 connecting channel (22) via the capillary blocking valve (25).

[0035] The fifth storage chamber (3) is connected to the second mixing chamber (7) through 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 connecting 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 connecting 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] The liquid in the first mixing chamber (16) passes through the first siphon channel (49) and is then trapped in the first siphon channel (49) by the action of the first soluble membrane valve (44);

[0039] The liquid in the second mixing chamber (7) passes through the air pressure channel (51) and is then trapped in the air pressure channel (51) by 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 center of rotation is greater than the distance between the first storage chamber (13) and the second storage chamber (12) and the center of rotation. 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 distances 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 colorimetric solution mixture can just fill or partially fill the second mixing chamber (7).

[0042] Optionally, for viscous reagents (such as H1N1 sample solution and colorimetric 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 during the acceleration and deceleration process of the motor.

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

[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) and 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 cavity (11) are connected through the second liquid inlet channel (9). The first siphon channel (49) and the test strip storage cavity (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 colorimetric 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) flows through the first siphon channel (49) through the sample solution soluble membrane filling hole (32) to dissolve the soluble membrane and flows through the first liquid inlet channel (50) into the paper strip chromatography pad of the test strip storage chamber (11). The colorimetric liquid vent (31) is opened. The liquid in the second mixing chamber (7) flows through the air pressure channel (51) through the colorimetric liquid soluble membrane filling hole (33) to dissolve the soluble membrane and flows through the second liquid inlet channel (9) into the middle detection area of ​​the test strip in the test strip storage chamber (11).

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

[0049] For ease of 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), bottom flow channel layer (36), vertical through hole layer (42), clamping layer (48), top flow channel layer (54) and sealing body (64) have a positioning mechanism (30) that can fix the chip to the carrier.

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

[0052] (1) Dissolve the ruthenium-loaded microsphere peroxide nanozyme in 50 mM pH 5.0 MES buffer, then add appropriate amounts of EDC and NHS in sequence, shake at room temperature, centrifuge and discard the supernatant;

[0053] (2) Mix the antibody with the solution, centrifuge and discard the supernatant after coupling, add Tris-HCl solution to wash, centrifuge and discard the supernatant to obtain the ruthenium nanozyme antibody probe;

[0054] And optional:

[0055] (3) Add BSA blocking solution to block non-specific binding sites;

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

[0057] The preparation method of the ruthenium-loaded microsphere peroxide nanozyme is as follows:

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

[0059] (2) A solution containing ruthenium salt and reducing agent is added to a polystyrene microsphere solution to obtain a mixed solution. The mixed solution is stirred and / or subjected to a reduction reaction under ultrasound. After the reaction is completed, the supernatant is separated and discarded to obtain the ruthenium-loaded microsphere peroxide nanozyme.

[0060] Preferred,

[0061] RuCl3·nH2O is dissolved in pure water, and the mass ratio of ruthenium salt to polystyrene microspheres in the mixture is 1:1.25-10, preferably 1:2.5-5; the reducing agent and ruthenium salt are dissolved separately and added to pure water at a concentration of 0.04-0.08 mg / mL.

[0062] The solution containing ruthenium salt and 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℃; and the separation is performed by centrifugation.

[0064] Optionally, (3) the ruthenium-loaded microsphere peroxide nanozyme is washed; preferably, the washing solution is water.

[0065] This invention also relates to a method for automated detection of nanozyme immunochromatographic test strips in a centrifugal microfluidic chip, the method comprising the following steps:

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

[0067] (2) Sample flow and immune reaction: The sample is controlled to flow along the microchannels in the chip at a certain rotation speed through the built-in micropump mechanism of the centrifugal microfluidic chip, ensuring that the sample can be mixed evenly and fully contact the reaction area of ​​the ruthenium nanozyme immunochromatographic test strip.

[0068] (3) Result determination: When the sample passes through the immunochromatographic test strip, an antigen-antibody reaction occurs, forming a detectable ruthenium nanozyme label 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] Furthermore,

[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 peroxide nanozyme, and the antibody probe is obtained by coupling the ruthenium-loaded microsphere peroxide 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 H2O2 is 4-60 μL, and the injection volume of the DAB substrate solution is 2-4 μL.

[0072] In step (2),

[0073] A portion of the air in the first storage chamber (13) is discharged into the outside atmosphere through the first exhaust port (55) via the first connecting channel (18) and the second connecting channel (19); a portion 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 into the outside atmosphere through the first exhaust port (55) via the first connecting channel (18); 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 into the outside atmosphere through the second exhaust port (58) via the fifth connecting channel (22); 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 into the outside atmosphere through the second exhaust port (58) via the sixth connecting channel (23) and the seventh connecting channel (24); 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 into the outside atmosphere through the second exhaust port (58) via the fourth connecting channel (21); a portion of the air enters the downstream chamber via the fifth capillary valve (6).

[0078] In step (3),

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

[0080] This 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 immunochromatographic detection device, wherein the immunochromatography uses the nanozyme-loaded chromogenic probe, and the pathogen is a virus; more preferably, the pathogen is influenza A virus.

[0081] The beneficial effects of this invention are as follows:

[0082] 1. This invention designs a centrifugal microfluidic chip integrating ruthenium nanozyme test strips, realizing fully automated detection of ruthenium nanozyme test strips on a microfluidic chip. The entire detection process requires no manual intervention; after sample addition, fluid distribution, chromatographic reaction, and colorimetric reaction are automatically completed, significantly improving the convenience and repeatability of the experiment, while reducing human error and improving detection reliability.

[0083] 2. This invention proposes a precise fluid timing control scheme based on a soluble membrane valve, avoiding the hydrophilic / hydrophobic surface treatment technology commonly used in traditional microfluidic chips, thus ensuring the stability and timeliness of fluid control. Compared to hydrophilic / hydrophobic treatment methods, the soluble membrane valve does not suffer from material aging, environmental humidity effects, or uneven surface treatment, thereby improving chip production consistency and reducing manufacturing costs.

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

[0085] 4. The microfluidic chip of this invention employs a high-sensitivity detection strategy. By precisely controlling the fluid reaction timing and the ruthenium nanozyme catalytic signal amplification mechanism, it enhances the detection signal intensity, achieving the goal of detecting trace pathogens, such as the efficient detection of H1N1 virus nucleoprotein. This chip maximizes the utilization of the original antigen signal, improving detection sensitivity. Suitable for low disease Early screening of toxic load samples .

[0086] 5. The microfluidic chip of this invention can significantly shorten detection time and reduce detection costs. Thanks to precise automated fluid control and efficient enzyme catalysis, this chip can complete the immunodetection of trace pathogens in a short time. Compared to traditional laboratory detection methods, this chip eliminates the need for complex equipment and time-consuming sample pretreatment, greatly improving detection efficiency and providing a feasible solution for point-of-care testing (POCT). Attached Figure Description

[0087] Figures 1-7 To develop a centrifugal microfluidic chip with four sets of detection microfluidic structures on a circular chip, which is a four-sample integrated nanozyme test strip, in actual work, it can be set as a single-sample chip, a dual-sample combined chip, or a three- to N-sample combined chip as needed.

[0088] Figure 1 A partial structural diagram of the 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, barrier 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 connecting channel; 19, second connecting channel; 20, third connecting channel; 21, fourth connecting channel; 22, fifth connecting channel; 23, sixth connecting channel; 24, seventh connecting channel; 25, capillary blocking valve; 26, eighth connecting channel; 27, first positioning hole; 28, second positioning hole; 29, chip body; 30, positioning mechanism.

[0090] Figure 3 This is a schematic diagram of the bottom flow channel layer (36). In the diagram: 31, chromogenic liquid pore, 32, sample liquid soluble membrane filling hole, 33, chromogenic liquid soluble membrane filling hole, 34, eighth positioning hole, 35, ninth positioning hole, 36, bottom flow channel layer.

[0091] Figure 4 This is a schematic diagram of the vertical through-hole layer (42). In the diagram: 37, internal circulation vent, 38, sample liquid vent, 39, colorimetric liquid vent, 40, third positioning hole, 41, fourth positioning hole, 42, vertical through-hole layer.

[0092] Figure 5 Schematic diagram of the pressing layer (48). In the figure: 43, air pressure hole, 44, first soluble membrane valve, 45, second soluble membrane valve, 46, fifth positioning hole, 47, tenth positioning hole, 48, pressing layer.

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

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

[0095] Figure 8 This is a schematic diagram of the structure of a nanozyme test strip.

[0096] Figure 9 A diagram illustrating the test results.

[0097] Figure 10 The image shows the detection results of different concentrations of influenza A antigen using a centrifugal microfluidic chip for automated detection. Detailed Implementation

[0098] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0100] At the same time, it should be understood that, for ease of description, the dimensions of the various parts shown in the accompanying drawings are not drawn according to actual scale.

[0101] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the 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 embodiment is as follows:

[0104] 1) After centrifuging the nanozyme aqueous solution, add 50mM pH5.0 MES buffer, mix well, and then add 25mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 25mM N-hydroxysuccinimide (NHS) in sequence. After shaking at room temperature, centrifuge and discard the supernatant.

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

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

[0107] 4) Add BSA blocking solution and shake at room temperature for 2 hours to block non-specific 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℃.

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

[0110] like Figure 2 and 7As shown, the nanozyme probe solution, sample solution, DAB buffer, DAB substrate solution, and H2O2 solution are 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 well (56), second injection well (57), fifth injection well (61), fourth injection well (60), and third injection well (59), respectively, using a pipette. Then, a sealing element is used to seal the first injection well (56), second injection well (57), fifth injection well (61), third injection well (59), fourth injection well (60), first vent (55), and second vent (58). The sealing element can be 3M single-sided adhesive or PET film.

[0111] The chip positioning mechanism (30) is then used in conjunction with its corresponding carrier for subsequent fluid control and chromatography color development reaction.

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

[0113] (2) Subsequently, some of the liquid inside the chamber stops after passing through the first siphon channel (49) and the pressure channel (51) under capillary action. Immediately afterwards, the sample mixture begins to dissolve the soluble membrane to open the first soluble membrane valve (44) and the pressure orifice (43).

[0114] (3) After the first soluble membrane valve (44) is opened, at a certain speed, the sample mixture in the first mixing chamber (16) enters the paper strip chromatography pad in the test strip storage chamber (11) through the first liquid inlet channel (50) to carry out the chromatography reaction. The color-developing solution mixture in the second mixing chamber (7) dissolves and breaks through the second soluble membrane valve (45). In the second liquid inlet channel (9), it is precisely controlled for 7 minutes to wait for the sample solution to be completely chromatographically analyzed. It 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 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] To prevent the color developing solution from not being laid flat in the middle detection area for color development after entering the test strip storage cavity (11) and undergoing the color development reaction as preset, a barrier plate (10) is added to prevent the color developing solution from entering the absorbent pad under centrifugal action and failing to fully develop the color reaction, thus ensuring the stability of the reaction.

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

[0117] Example 2: Method for assembling nanozyme 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] Nanozyme test strips have a conventional structure, specifically:

[0120] The test strip consists of a PVC base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane is adhered to the PVC base plate. Then, the absorbent pad is adhered to the PVC base plate so that it covers the top edge of the nitrocellulose membrane by 2 mm, and the sample pad is also adhered to the PVC base plate so that it covers the bottom edge of the nitrocellulose membrane by 2 mm. The control line on the nitrocellulose membrane is positioned close to the absorbent pad, and the detection line on the nitrocellulose membrane is positioned close to the sample pad. This complete nano-enzyme immunochromatographic test strip is assembled and then cut into 4 mm wide strips using a strip cutter.

[0121] This embodiment takes the detection of influenza A virus as an example to detect the NP protein of influenza A virus. In the detection system of this embodiment, the amount of detection antibody against influenza A virus is 2 mg / mL; the streak concentration of other detection antibodies against different pathogens can be adjusted according to the actual situation.

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

[0123] The preparation of the test strip's detection line and control line is as follows:

[0124] 1) Preparation of the test line and control line: Influenza A virus monoclonal antibody (detection antibody) was diluted to 2 mg / mL with 0.02 M PB and streaked onto a nitrocellulose membrane using a streak scribing apparatus to serve as the test line. Goat anti-mouse IgG antibody (control antibody) was diluted to 2 mg / mL with 0.02 M PB and streaked onto a nitrocellulose membrane using a streak scribing apparatus to serve as the control line. The streaking parameters were 0.1 μL / mm, and the distance between the control line and the test line was 7 mm. The streaked nitrocellulose membrane was then dried overnight at 37°C to obtain the treated nitrocellulose membrane.

[0125] 2) Assemble the test strip: First, adhere the nitrocellulose membrane to the PVC base plate. Then, adhere the absorbent pad to the PVC base plate, covering the top edge of the nitrocellulose membrane by 2 mm. Similarly, adhere the sample pad to the PVC base plate, covering the bottom edge of the nitrocellulose membrane by 2 mm. Ensure the 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 absorbent pad is absorbent filter paper, and the sample pad is a glass fiber membrane. Assemble the complete nano-enzyme immunochromatographic test strip, and then cut it into strips with a width of 4 mm using a strip cutter. Figure 8 .

[0126] 3) Installing test strips: The above-mentioned nanozyme test strips are installed in the centrifugal microfluidic chip of Example 1, and further, they are installed in the test strip storage cavity (11).

[0127] Example 3: Method of using a centrifugal microfluidic chip containing nanozyme test strips

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

[0129] The detection method includes the following steps:

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

[0131] The chip positioning mechanism (30) is used in conjunction with its corresponding carrier for subsequent fluid control. At a certain rotation speed, each liquid begins to flow along the microchannel inside the chip, realizing the chromatography and colorimetric reaction of the ruthenium nanoenzyme test strip.

[0132] The determination of test results is as follows Figure 9 :

[0133] If both the control line and the test line appear on the ruthenium nanozyme immunochromatographic test strip, the test result is positive, meaning that the sample contains influenza A virus.

[0134] If the ruthenium nanozyme immunochromatographic test strip for influenza A virus only shows a control line, the test result is negative, meaning that there is no influenza A virus in the sample being tested.

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

[0136] 2. Detection of standard samples of influenza A virus

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

[0138] (2) Inject 80 μL of different concentrations of influenza A virus antigen (7 different concentrations of influenza A virus antigen test solutions: 5000, 2500, 1000, 250, 50, 10, and 0 pg / mL) into the second storage chamber (12) through the second injection well (57). Inject the nanozyme probe solution, DAB buffer, DAB substrate solution, and H2O2 solution into the corresponding first storage chamber (13), third storage chamber (1), fifth storage chamber (3), and fourth storage chamber (2) through the first injection well (56), fifth injection well (61), fourth injection well (60), and third injection well (59), respectively, using a pipette. Observe the color development results after setting the program. In the color development mode, the T area of ​​the positive and low-concentration positive test strips appears brownish-red.

[0139] Test results are as follows Figure 10 In the detection of seven H1N1 virus antigen concentrations, 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 strip involved in this invention improves the detection sensitivity of H1N1 virus antigen by about 50 times.

[0140] The above figures and specific embodiments are for illustrative purposes only, and the invention is not limited thereto. Minor modifications to the invention within the scope and essence defined by the claims fall within the protection scope of the invention. Examples include the shape and size of the chambers, and the structure and dimensions of various valves.

[0141] Those skilled in the art will understand that all or part of the steps of the above embodiments can be implemented by hardware or by a program instructing related hardware. The program can be stored in a computer-readable storage medium, such as a read-only memory, a disk, or an optical disk.

[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 intended to limit the scope of protection of the present invention.

Claims

1. A centrifugal microfluidic chip, characterized in that, The microfluidic chip described herein is loaded with a ruthenium-loaded peroxide nanozyme probe and an immunochromatographic test strip for the detection of trace pathogens. The chip comprises a chip body (29) with a reactive structure, a bottom flow channel layer (36), a vertical through-hole layer (42) with a soluble membrane, a clamping layer (48), a top flow channel layer (54), and a sealing body (64); wherein, The reaction structure of the chip body (29) includes: a first storage chamber (13) for storing ruthenium nanozyme probes, a second storage chamber (12) for storing sample solution, a third storage chamber (1) for storing DAB buffer, a fourth storage chamber (2) for storing H2O2, a fifth storage chamber (3) for storing DAB substrate, a first mixing chamber (16) for mixing sample solution and ruthenium nanozyme probes, a second mixing chamber (7) for mixing DAB substrate, H2O2 and DAB buffer, a test strip storage chamber (11) for storing ruthenium nanozyme test strips, a distribution unit (8) connected to the second mixing chamber (7), a venting pipe and various fluid valves; The liquid in the first mixing chamber (16) flows through the first siphon channel (49) through the sample liquid soluble membrane filling hole (32) to dissolve the soluble membrane and flows through the first liquid inlet channel (50) into the paper strip chromatography pad of the test strip storage chamber (11), opening the colorimetric liquid vent (31). The liquid in the second mixing chamber (7) flows through the air pressure channel (51) through the colorimetric liquid soluble membrane filling hole (33) to dissolve the soluble membrane and flows through the second liquid inlet channel (9) into the middle detection area of ​​the test strip in the test strip storage chamber (11).

2. The centrifugal microfluidic chip according to claim 1, characterized in that, The microfluidic chip described above can detect sample volumes of 10-100 μL. The volume of the first storage chamber (13) for storing ruthenium nanozyme probe is 2-10 μL, the volume of the second storage chamber (12) for storing sample solution is 10-100 μL, the volume of the third storage chamber (1) for storing 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 DAB substrate is 2-4 μL.

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

4. The centrifugal microfluidic chip according to claim 3, characterized in that, The first storage chamber (13) and the first mixing chamber (16) are connected by the first capillary valve (14); at a certain rotation 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), or it 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) through the 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) through the fifth capillary valve (6); at a certain rotation 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 connecting channel (21) via the capillary blocking valve (25); The fourth storage chamber (2) is connected to the second mixing chamber (7) through the fourth capillary valve (5); at a certain rotation 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 connecting 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 connecting channel (22) via the capillary blocking valve (25); The fifth storage chamber (3) is connected to the second mixing chamber (7) through 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 connecting 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 connecting 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).

5. The centrifugal microfluidic chip according to claim 1, characterized in that, The liquid in the first mixing chamber (16) passes through the first siphon channel (49) and is then trapped in the first siphon channel (49) by the action of the first soluble membrane valve (44); The liquid in the second mixing chamber (7) passes through the air pressure channel (51) and is then trapped in the air pressure channel (51) by the action of the second soluble 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). 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 color developing solution mixture can just fill or partially fill the second mixing chamber (7).

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

7. The centrifugal microfluidic chip according to any one of claims 1-5, characterized in that, The distance between the siphon peak of the first siphon channel (49) and the rotation center is less than the distance between the inner end of the first mixing chamber (16) and the rotation center, which is close to the second capillary valve (15). 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) and 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 cavity (11) are connected through the second liquid inlet channel (9). The first siphon channel (49) and the test strip storage cavity (11) are connected through the first liquid inlet channel (50). The sample ruthenium nanozyme premix in the first mixing chamber (16) and the colorimetric 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.

8. The centrifugal microfluidic chip according to any one of claims 1-5, characterized in that, The ruthenium-loaded peroxide nanozyme probe is obtained by conjugating ruthenium-loaded microsphere peroxide nanozyme with an antibody. The conjugation method includes the following steps: (1) Dissolve the ruthenium-loaded microsphere peroxidase in 50 mM pH 5.0 MES buffer, then add appropriate amounts of EDC and NHS in sequence, shake at room temperature, centrifuge and discard the supernatant; (2) Mix the antibody with the solution, centrifuge and discard the supernatant after coupling, add Tris-HCl solution to wash, centrifuge and discard the supernatant to obtain the ruthenium nanozyme antibody probe; The preparation method of the ruthenium-loaded microsphere peroxide nanozyme includes the following steps: (1) Add polystyrene microspheres to pure water at a concentration of 0.1-0.15 mg / mL and stir at a rate of 300-500 rpm / min until completely dissolved and clear to obtain a polystyrene microsphere solution; (2) A solution containing ruthenium salt and reducing agent is added to a polystyrene microsphere solution to obtain a mixed solution. The mixed solution is stirred and / or subjected to a reduction reaction under ultrasound. After the reaction is completed, the supernatant is separated and discarded to obtain the ruthenium-loaded microsphere peroxide nanozyme.

9. The centrifugal microfluidic chip according to claim 8, characterized in that, The coupling method further includes: (3) Add BSA blocking solution to block non-specific binding sites; (4) After centrifugation and discarding the supernatant, resuspend in pH 7.4 PBS; In the preparation method of the ruthenium-loaded microsphere peroxide nanozyme, RuCl3·nH2O was dissolved in pure water, and the mass ratio of ruthenium salt to polystyrene microspheres in the mixture was 1:1.25~10; the reducing agent and ruthenium salt were dissolved separately and added to pure water at a concentration of 0.04-0.08 mg / mL. The solution containing ruthenium salt and 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℃; and the separation is performed by centrifugation. The method further includes step (3), washing the ruthenium-loaded microsphere peroxide nanozyme.

10. A method for achieving automatic detection using the centrifugal microfluidic chip according to any one of claims 1-9, the method comprising the following steps: (1) Liquid injection: The ruthenium nanozyme probe solution is injected into the first storage chamber (13) through the first injection hole (56). The ruthenium nanozyme is a ruthenium-loaded microsphere peroxidase nanozyme with peroxidase-like activity. The sample solution to be tested is injected into the second storage chamber (12) through the second injection hole (57). The DAB buffer solution is injected into the third storage chamber (1) through the fifth injection hole (61). The H2O2 solution is injected into the fourth storage chamber (2) through the third injection hole (59). The DAB substrate solution is injected into the fifth storage chamber (3) through the fourth injection hole (60). (2) Sample flow and immune reaction: The sample is controlled to flow along the microchannels in the chip at a certain rotation speed by the micropump driven mechanism built into the centrifugal microfluidic chip, ensuring that the sample can be mixed evenly and fully contact the reaction area of ​​the ruthenium nanozyme 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 label 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.

11. The method according to claim 10, characterized in that, In step (1), The injection volume of the ruthenium nanozyme probe solution is 3-5 μL. The ruthenium nanozyme is a ruthenium-loaded microsphere peroxide nanozyme. The antibody probe is obtained by coupling the ruthenium-loaded microsphere peroxide 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 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 through the first connecting channel (18) and the second connecting channel (19) from the first exhaust port (55); 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 through the first exhaust port (55) via the first connecting channel (18); a portion of the air enters the downstream chamber via the second capillary valve (15). A portion of the air in the fourth storage chamber (2) is discharged into the outside atmosphere through the second exhaust port (58) via the fifth connecting channel (22); a portion of the air enters the downstream chamber via the fourth capillary valve (5); A portion of the air in the fifth storage chamber (3) is discharged into the outside atmosphere through the second exhaust port (58) via the sixth connecting channel (23) and the seventh connecting channel (24); a portion of the air enters the downstream chamber via the third capillary valve (4); A portion of the air in the third storage chamber (1) is discharged into the outside atmosphere through the second exhaust port (58) via the fourth connecting channel (21); a portion of the air enters the downstream chamber via the fifth capillary valve (6); In step (3), The ruthenium nanozyme label can catalyze the oxidation of the chromogenic substrate in hydrogen peroxide (H2O2).

12. Use of the centrifugal microfluidic chip according to any one of claims 1-9 in the preparation of pathogen detection devices.

13. Use according to claim 12, characterized in that, The detection device is an immunochromatographic detection device, the immunochromatographic method uses the nanozyme-loaded colorimetric probe, and the pathogen is a virus.

14. Use according to claim 12 or 13, characterized in that, The pathogen mentioned is influenza A virus.

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