An ultrasonic microfluidic integrated device and an ultrasonic chemiluminescence detection method

Abstract

The application discloses an ultrasonic micro-fluidic integrated device and an ultrasonic chemiluminescence detection method. The device comprises a wireless acoustic control flow chip, a first layer provided with a gas inlet and a sample inlet, a second layer provided with a pushing area, a third layer and a fourth layer provided with a sample storage area, a reaction area is formed on the first layer, the second layer and the third layer, an ultrasonic module, a first ultrasonic sheet and a second ultrasonic sheet, the first ultrasonic sheet is arranged on the surface of the third layer and located in the pushing area of the second layer, the second ultrasonic sheet is arranged on the fourth layer and located in the reaction area, the first ultrasonic sheet is connected with the sample storage area through a first channel, the sample storage area is connected with the second ultrasonic sheet through a second channel, a wireless module connected with the ultrasonic control module and a battery. The application realizes real-time monitoring of transient chemiluminescence signals under ultrasonic conditions and quantitative and timed control of a multi-step integrated system in a closed environment by wirelessly controlling ultrasonic power and frequency.

Description

Technical Field

[0001] This invention relates to the field of ultrasonic microfluidics, specifically to an integrated ultrasonic microfluidic device and an ultrasonic chemiluminescence detection method. Background Technology

[0002] Chemiluminescent immunoassay (CLIA) is an analytical method that combines a chemiluminescence system and an immunoassay. It uses a chemical reaction to generate light to label antibody probes and determine the concentration of the analyte. In recent decades, CLIA has been widely used in life sciences, clinical diagnostics, environmental monitoring, food safety, and pharmaceutical analysis due to its advantages such as high analytical sensitivity, wide linear range, absence of scattered light interference, no radioactive contaminants, and simple equipment.

[0003] Currently, CLIA has reached a higher level thanks to the help of novel nanomaterials (such as gold nanoparticles, quantum dots, and magnetic materials) and its coupling with other advanced technologies (such as immunochromatography and microfluidic chips), resulting in significant breakthroughs in its effectiveness in recent years. According to Google Scholar data from 2010 to 2018, the number of publications on CLIA containing terms such as "enhanced chemiluminescence," "nanomaterials," "magnetic particles," and "microfluidic chips" is increasing. Therefore, although CLIA only began developing a few decades ago, it is now a research hotspot in analytical chemistry. Especially in recent years, more and more researchers are keen to enhance CLIA with new materials and technologies.

[0004] Since its initial introduction in 2001, microarrays have demonstrated tremendous potential for revolutionary analyte detection. (CKDixit, GRAguirre, Protein microarrays with novel microfluidic methods: current advances, Microarrays 3 (2014) 180e202) Today, microfluidic chips are also being introduced as an emerging technology. A microfluidic chip is a set of microchannels etched or molded onto a material (glass, silicon, or polymer) that are interconnected to achieve the desired function. Microfluidic chips are a potentially powerful method for performing high-throughput multiplexing analysis and miniaturized experiments. Recently, CLIA has seen an increase in research on multiplexing analysis using microfluidic chips. For example, the detection of ochratoxin A (OTA), aflatoxin B1 (AFB1), and deoxynivalenol (DON) proposed by RRG Soares, DR Santos, V et al., has begun to utilize microfluidic chips extensively. Furthermore, S. Guo, R. Ishimatsu et al. proposed that centrifugal force can be effectively utilized to drive microfluidics through flow control, and designed a CD-type microfluidic chip for this purpose, realizing a fully integrated and automated CLIA system. Sample solution, washing solution, and luminol solution are placed in a reservoir, and the CD-type microchip is rotated according to a predetermined rotation program, sequentially delivering the samples to the detection area. The microfluidic chip provides a rapid and robust method to replace traditional microarray multi-analysis. All of these demonstrate the enormous potential of microfluidic chemiluminescence in immunosensing and its promising application in clinical testing.

[0005] The innovative development of microfluidic chips is moving towards portability, integration, and automation, aligning with the trend of chemiluminescence immunoassay applications in on-site rapid screening and portable home use. However, existing commercially available chemiluminescence spectrometers, multifunctional microplate readers with sample injectors, and other instruments using chemiluminescence signals typically cannot meet the requirements of ultrasonic chemiluminescence detection. Specific shortcomings include:

[0006] 1. Commonly used spectrometers usually lack ultrasonic control functions, making it impossible to control ultrasonic power and frequency in the detection area, and also impossible to monitor chemiluminescence signals under ultrasonic conditions in real time.

[0007] 2. Microfluidic chips designed based on traditional chemiluminescence detection methods can be adapted to some spectrometers, but they cannot achieve integrated microfluidic control, and therefore cannot provide simple and efficient chip detection.

[0008] 3. Traditional chemiluminescence instruments often lack closed-environment control functions and are difficult to achieve quantitative and timing control of multi-step reactions. They are easily affected by external environmental interference, which affects the accuracy of detection results. Summary of the Invention

[0009] The purpose of this invention is to provide an integrated ultrasonic microfluidic device and an ultrasonic chemiluminescence detection method to improve the above-mentioned problems.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows:

[0011] An ultrasonic microfluidic integrated device, comprising:

[0012] A wireless voice-controlled flow chip, comprising a first layer with an air inlet and a sample inlet, a second layer with an air inlet and a pushing area, and a third and fourth layers with a sample storage area; reaction areas are correspondingly formed on the first, second, and third layers.

[0013] An ultrasound module includes an ultrasound control module, a first ultrasound plate, and a second ultrasound plate. The first ultrasound plate is disposed on the surface of the third layer and located within the pushing area of ​​the second layer. The second ultrasound plate is disposed on the fourth layer and located within the reaction area. The ultrasound control module is electrically connected to the first ultrasound plate and the second ultrasound plate. The first ultrasound plate is connected to the sample storage area through a first channel, and the sample storage area is connected to the second ultrasound plate through a second channel.

[0014] The wireless module is connected to the ultrasonic control module;

[0015] The battery is electrically connected to the wireless module, the wireless voice-controlled flow chip, and the ultrasonic module.

[0016] Preferably, the wireless voice-controlled flow chip is generated by micro-machining and hot-pressing a multi-layer PMMA template.

[0017] Preferably, the ultrasonic strip is fixed to the wireless voice-controlled flow chip by PMMA thermal bonding.

[0018] Preferably, the wireless module is a Bluetooth module.

[0019] This invention provides an ultrasonic chemiluminescence detection method based on the above-described ultrasonic microfluidic integrated device, characterized by comprising the following steps:

[0020] S1, add the reaction substrate liquid required for ultrasonic chemiluminescence to the reaction region of the wireless acoustic fluid control chip;

[0021] S2, add the sample to be tested into the injection port and seal it, so that the sample to be tested and the reaction solution can undergo a recognition reaction in the sample storage area;

[0022] S3. After the recognition reaction is complete, add ultrasonic atomizing propellant to the propellant zone;

[0023] S4. Place the ultrasonic microfluidic integrated device into the dark field reading cavity of the photosensitive instrument and set the photosensitive instrument to real-time monitoring mode.

[0024] S5 activates the ultrasonic plate in the driving zone via a wireless module, runs for a set time, and then activates the ultrasonic plate in the reaction zone. The intensity of ultrasonic chemiluminescence in the reaction zone is monitored by a photosensitive instrument.

[0025] Preferably, in step S1, the reaction substrate solution is luminol-sodium alginate, luminol-sodium alginate-cobalt(II), or luminol-hydrogen peroxide; the volume range of the reaction substrate solution is 100 μL to 140 μL; and the buffer solution used is carbonate buffer, PBS buffer, or Tris-HCl buffer.

[0026] Preferably, the volume range of the sample to be tested in the sample storage area in step S2 is 60μL to 100μL, and the identification reaction conditions set in the sample storage area include: direct ultrasonic flow without identification, ultrasonic flow after full identification reaction, and ultrasonic flow after multi-step reaction.

[0027] Preferably, in step S3, the volume of the ultrasonic atomizing propellant is 200 μL, and the buffer solution used is carbonate buffer, PBS buffer, or Tris-HCl buffer.

[0028] Preferably, in step S4, the cavity of the photosensitive instrument includes an ELISA reader, a mobile phone-adapted dark box, and a DSLR camera-adapted dark box; the real-time monitoring modes include the ELISA reader's chemiluminescence dynamics detection mode, the mobile phone's fixed-focus camera mode, and the DSLR camera's fixed-focus camera mode.

[0029] In summary, the ultrasonic microfluidic integrated device based on this embodiment can control the reaction time and dosage of different reagents wirelessly in a closed environment. It is also compatible with ultrasonic chemiluminescence reactions. By wirelessly controlling the ultrasonic power and frequency, it can realize real-time monitoring of transient chemiluminescence signals under ultrasonic conditions and quantitative and timing control of multi-step integrated systems in a closed environment, which has important application value. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the ultrasonic microfluidic integrated device provided in the first embodiment of the present invention.

[0032] Figure 2 for Figure 1 A schematic diagram of the structure of a wireless voice-controlled stream chip.

[0033] Figure 3 This is a schematic flowchart of the ultrasonic chemiluminescence detection method provided in an embodiment of the present invention.

[0034] Figure 4(a) is a schematic diagram of selective analysis of the ultrasonic microfluidic integrated device for AA sensing.

[0035] Figure 4(b) shows the standard working curve between the intensity of sonic chemiluminescence and the concentration of AA. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0037] Please see Figure 1 and Figure 2 The first embodiment of the present invention provides an integrated ultrasonic microfluidic device, which includes:

[0038] The wireless voice-controlled flow chip 100 includes a first layer 110 with an air inlet 111 and a sample inlet 112, a second layer 120 with an air inlet 111 and a push area 121, a third layer 130 with a sample storage area 131, and a fourth layer 140; a reaction area 141 is correspondingly formed on the first layer 110, the second layer 120, and the third layer 130.

[0039] The air inlet 111 is used to detect the airtightness of the ultrasonic microfluidic integrated device.

[0040] The ultrasound module 200 includes an ultrasound control module, a first ultrasound plate 210, and a second ultrasound plate 220. The first ultrasound plate 210 is disposed on the surface of the third layer 130 and located within the pushing area 121 of the second layer 120. The second ultrasound plate 220 is disposed on the fourth layer 140 and located within the reaction area 14. The ultrasound control module is electrically connected to the first ultrasound plate 210 and the second ultrasound plate 220. The first ultrasound plate 210 is connected to the sample storage area 131 through a first channel 132, and the sample storage area 131 is connected to the second ultrasound plate 220 through a second channel 133.

[0041] In this embodiment, the wireless voice-controlled flow chip 100 can be generated by micromachining a multi-layer PMMA template. After micromachining, a first ultrasonic strip 210 and a second ultrasonic strip 220 are placed in the template. Then, the PMMA template stack is placed in a vulcanizing hot press for pressing. The temperature of the upper and lower plates of the hot press is 104°C, the pressing time is 6 minutes, and the pressure is 190 kPa. The pressed PMMA template stack yields the wireless voice-controlled flow chip 100. The positioning adhesive is then removed, and the chip is cleaned with ethanol.

[0042] The wireless module 300 is connected to the ultrasonic control module 210.

[0043] In this embodiment, the wireless module 300 can be a Bluetooth module, a WiFi module, or a 2 / 3 / 4 / 5G module. The present invention does not impose a specific limitation; however, the wireless module 300 is specifically a Bluetooth module. The wireless module 300 can connect to an external user terminal, such as a smartphone. Thus, the smartphone can send control commands to the wireless module 300 via an app or mini-program, thereby controlling the ultrasound control module, the first ultrasound chip 210, and the second ultrasound chip 220.

[0044] Battery 400 is electrically connected to the wireless module 300, the wireless voice-controlled flow chip 100, and the ultrasonic module 200.

[0045] In this embodiment, specifically, the battery 400 is a lithium battery, which is electrically connected to the wireless module 300, the wireless voice-controlled flow chip 100, and the ultrasonic module 200, thereby providing the electrical energy required for these modules to operate.

[0046] The following details the ultrasonic chemiluminescence detection method using the ultrasonic microfluidic integrated device according to embodiments of the present invention, such as... Figure 3 As shown:

[0047] S1, add the reaction substrate liquid required for ultrasonic chemiluminescence to the reaction zone 141 of the wireless acoustic fluid control chip 100.

[0048] Specifically, the reaction substrate solution is typically a chemiluminescent mixture such as luminol-sodium alginate, luminol-sodium alginate-cobalt(II), or luminol-hydrogen peroxide, with an optimal volume range of 100 μL to 140 μL.

[0049] Specifically, step S1 is performed in a buffer solution, such as carbonate buffer, PBS buffer, or Tris-HCl buffer.

[0050] S2, the sample to be tested is added to the inlet 112 and sealed, so that the sample to be tested and the reaction substrate solution can undergo a recognition reaction in the sample storage area 131.

[0051] Specifically, in step S2, the optimal volume range of the sample to be tested in the sample storage area 131 is 60 μL to 100 μL. The sample storage area 131 is set with different identification reaction conditions according to different systems: such as direct ultrasonic flow without identification, ultrasonic flow after full identification reaction, ultrasonic flow after multi-step reaction (including washing, redispersion, etc.), etc. The present invention does not make specific limitations.

[0052] S3. After the recognition reaction is complete, add ultrasonic atomizing propellant to the propellant zone 121.

[0053] Specifically, the optimal volume of the ultrasonic atomization propellant is 200 μL, and it should be consistent with the liquid buffer system of the substrate reaction solution, namely, carbonate buffer, PBS buffer or Tris-HCl buffer.

[0054] S4. Place the ultrasonic microfluidic integrated device into the dark field reading cavity of the photosensitive instrument and set the real-time monitoring mode of the photosensitive instrument.

[0055] The cavity of the photosensitive instrument includes an ELISA reader, a mobile phone-adapted darkroom, and a DSLR camera-adapted darkroom; the real-time monitoring modes include the ELISA reader's chemiluminescence dynamics detection mode, the mobile phone's fixed-focus camera mode, and the DSLR camera's fixed-focus camera mode. The specific real-time detection mode is selected according to actual needs.

[0056] S5, the first ultrasonic plate 210 of the driving zone 121 is started through the wireless module 300, and after running for a set time, the second ultrasonic plate 220 of the reaction zone 141 is started, and the ultrasonic chemiluminescence intensity of the reaction zone 141 is monitored by a photosensitive instrument.

[0057] In step S5, the sequential activation order of the two ultrasonic transducers must be strictly followed. The set time can be 6 seconds, which is obtained by optimizing the system based on the set channel length, ultrasonic transducer power, and optimal volume of the propellant fluid. Of course, it can also be set according to actual needs, and this invention does not impose specific limitations.

[0058] In summary, the ultrasonic microfluidic integrated device based on this embodiment can wirelessly control the reaction time and dosage of different reagents in a closed environment. It is also compatible with ultrasonic chemiluminescence reactions. By wirelessly controlling the ultrasonic power and frequency, it can realize real-time monitoring of transient chemiluminescence signals under ultrasonic conditions, and achieve quantitative and timing control of multi-step integrated systems in a closed environment, which has important application value.

[0059] To facilitate understanding of the present invention, some practical examples will be used below to illustrate its application.

[0060] (1) Used for quantitative detection of cobalt ions

[0061] At this point, the specific steps are as follows:

[0062] a. Add luminol-sodium alginate mixture to the reaction zone as the reaction substrate for sonochemiluminescence.

[0063] The concentration of luminol added was 600 μM and the volume was 40 μL; the concentration of sodium alginate added was 5 mg / mL and the volume was 30 μL.

[0064] The buffer system used was a carbonate buffer with a pH of 12.5.

[0065] b. Add a solution containing cobalt ions to the sample storage area as the sample to be tested.

[0066] c. Place the entire ultrasonic microfluidic integrated device into the dark field reading cavity of the photosensitive instrument, and set the real-time monitoring mode of the photosensitive instrument.

[0067] d. The first ultrasonic plate 210 of the driving zone is activated via the Bluetooth module on the mobile phone. After 6 seconds of operation, the second ultrasonic plate 220 of the reaction zone is activated. The intensity of ultrasonic chemiluminescence in the reaction zone is monitored by a photosensitive instrument throughout the process.

[0068] (2) Quantitative detection of ascorbic acid

[0069] At this point, the specific steps are as follows:

[0070] a. Add luminol-sodium alginate-cobalt(II) as a reaction substrate in the reaction zone to serve as the chemiluminescent reaction substrate.

[0071] The concentrations of luminol (600 μM), cobalt ions (0.1 mM), and sodium alginate (5 mg / mL) were specified. The volumes of luminol, cobalt ions, and sodium alginate were 40 μL, 10 μL, and 20 μL respectively.

[0072] The buffer system used was a carbonate buffer with a pH of 12.5.

[0073] b. Add a sample containing ascorbic acid (AA) to the sample storage area as the test liquid.

[0074] c. Place the ultrasonic microfluidic integrated device into the dark field reading cavity of the photosensitive instrument and set the real-time monitoring mode of the photosensitive instrument.

[0075] d. The first ultrasonic plate 210 of the driving zone is activated via the Bluetooth module on the mobile phone. After 6 seconds of operation, the second ultrasonic plate 220 of the reaction zone is activated. The intensity of ultrasonic chemiluminescence in the reaction zone is monitored by a photosensitive instrument throughout the process.

[0076] As shown in Figures 4(a)-(b), the ultrasonic chemiluminescence microfluidic detection device in Figure 4(a) is used for selective analysis of AA sensing. The buffer solution consists of [Cys] = [Glu] = [Phe] = [His] = 300 μM, [AA] = 50 μM, [Co(NO3)2] = 1.0 mM, [luminol] = 100 μM, and 0.1 M carbonate buffer at pH 12.5.

[0077] Figure 4(b) shows the standard working curve between sonochemiluminescence intensity and AA concentration. The error bars represent the calculation results of three parallel measurements. Cys represents cysteine, Glu represents glucose, Phe represents phenylalanine, and His represents histidine.

[0078] (3) Quantitative detection of carcinoembryonic antigen based on nucleic acid aptamer competitive recognition

[0079] At this point, the specific steps are as follows:

[0080] a. Mix 2 μL of 100 μM CEA aptamer (3' end modified with thiol) with 4 μL of 5 mM TCEP, incubate at 4 °C in the dark for 1 hour, and then dilute with 10 mM PBS to a final volume of 500 μL.

[0081] b. Add 50 μL of coupling solution to the sample storage area of ​​the wireless acoustic fluid control chip and soak for 30 min. Then wash three times with MES buffer. Add 50 μL of CEA aptamer solution to the sample storage area of ​​the chip and incubate for 4 hours. Then wash the surface with 10 mM PBS to form the aptamer nucleic acid modified sample storage area surface.

[0082] c. Prepare 30 μL of 0.0012 mg / mL CoFe2O4 nanoparticle (CoFe-NP) reagent with MES buffer, add 10 μL of coupling solution and react for 0.5 h, then add 10 μL of cDNA (100 μM) (3' end modified with amino group) and react for 2 h. Centrifuge to obtain cDNA-CoFe-NP chain, wash three times with hybridization buffer and disperse in 50 μL of hybridization buffer for later use.

[0083] The coupling solution contains 10 mM EDC and 10 mM NHS, and the hybridization buffer is 50 mM Tris-HCl containing 100 mM NaCl, 40 mM MgCl2, 100 mM KCl, and pH = 7.4.

[0084] d. Add 50 μL of cDNA-CoFe-NP strand to the nucleic acid storage area and incubate for 2 h. Then wash three times with PBS to form dsDNA-CoFe-NP probe on the surface of the storage area.

[0085] e. Add 100 μL of CEA-containing sample to the sample storage area and perform a specific recognition reaction for 1 hour.

[0086] f. After the reaction in the sample storage area is complete, add 100 μL of reaction substrate solution to the reaction area, place the ultrasonic microfluidic integrated device into the dark field reading cavity of the photosensitive instrument, and set the real-time monitoring mode of the photosensitive instrument.

[0087] The reaction substrate solution contained 600 mM luminol and 500 mM H2O2.

[0088] In particular, considering the long nucleic acid recognition reaction in the sample storage area, the reaction substrate solution was added before ultrasonic diversion.

[0089] g. The first ultrasonic plate 210 of the driving zone is activated via the Bluetooth module on the mobile phone. After 6 seconds of operation, the second ultrasonic plate 220 of the reaction zone is activated. The intensity of ultrasonic chemiluminescence in the reaction zone is monitored by a photosensitive instrument throughout the process.

[0090] (4) Quantitative detection of carcinoembryonic antigen based on double antibody sandwich immune recognition

[0091] At this point, the specific steps are as follows:

[0092] a. Add 50 μL of coupling solution to the reaction area of ​​the wireless acoustic fluid control chip and soak for 30 min. Then wash three times with MES buffer. Add 50 μL of CEA capture antibody solution with a concentration of 1 μg / mL to the sample storage area and incubate for 2 hours. Then incubate the reaction area with 50 μL of BSA with a concentration of 1% for 30 min to block unbound sites. Finally, wash the surface with 10 mM PBS to form the reaction area surface modified with CEA capture antibody.

[0093] The coupling solution contains 10 mM EDC and 10 mM NHS.

[0094] b. Add 50 μL of CEA-containing sample and 50 μL of CEA biotinylated antibody at a concentration of 200 ng / mL to the capture antibody-modified reaction zone and incubate for 1 h, then wash three times with PBS.

[0095] c. Add 50 μL of avidinized HRP (200 ng / mL) and incubate for 1 h, then wash three times with PBS to form a double-antibody sandwich complex on the surface of the reaction zone.

[0096] d. Add 140 μL of reaction substrate solution to the sample storage area, place the ultrasonic microfluidic integrated device into the dark field reading chamber of the photosensitive instrument, and set the real-time monitoring mode of the photosensitive instrument.

[0097] The reaction substrate solution contained 200 mM luminol and 300 mM H2O2.

[0098] e. The first ultrasonic plate 210 of the driving zone is activated via the Bluetooth module on the mobile phone. After 6 seconds of operation, the second ultrasonic plate 220 of the reaction zone is activated. The intensity of ultrasonic chemiluminescence in the reaction zone is monitored by a photosensitive instrument throughout the process.

[0099] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. An ultrasonic microfluidic integrated device, characterized by, The application relates to an ultrasonic microfluidic integrated device, which comprises the following parts: a wireless ultrasonic microfluidic chip, an ultrasonic module, a wireless module and a battery. The wireless ultrasonic microfluidic chip comprises a first layer provided with an air inlet and a sample inlet, a second layer provided with an air inlet and a pushing area, a third layer provided with a sample storage area and a fourth layer; reaction areas are correspondingly arranged on the first layer, the second layer and the third layer; The ultrasonic module comprises an ultrasonic control module, a first ultrasonic sheet and a second ultrasonic sheet; the first ultrasonic sheet is arranged on the surface of the third layer and located in the pushing area of the second layer; the second ultrasonic sheet is arranged on the fourth layer and located in the reaction area; the ultrasonic control module is electrically connected with the first ultrasonic sheet and the second ultrasonic sheet; the first ultrasonic sheet is connected with the sample storage area through a first channel, and the sample storage area is connected with the second ultrasonic sheet through a second channel; The wireless module is connected with the ultrasonic control module; The battery is electrically connected with the wireless module, the wireless ultrasonic microfluidic chip and the ultrasonic module. The ultrasonic microfluidic integrated device can be used for realizing an ultrasonic chemiluminescence detection method, which comprises the following steps: S1, adding a reaction substrate liquid required by ultrasonic chemiluminescence into a reaction area of the wireless ultrasonic microfluidic chip; S2, adding a sample to be detected into a sample inlet and sealing, so that the sample to be detected and the reaction substrate liquid perform an identification reaction in a sample storage area; S3, after the identification reaction is completed, adding an ultrasonic atomization propellant into a pushing area; S4, placing the ultrasonic microfluidic integrated device into a dark field reading cavity of a photosensitive instrument, and setting a real-time monitoring mode of the photosensitive instrument; S5, starting the ultrasonic sheet of the pushing area through the wireless module, starting the ultrasonic sheet of the reaction area after running for a set time, and monitoring the ultrasonic chemiluminescence intensity of the reaction area by using the photosensitive instrument.

2. The ultrasonic microfluidic integrated device of claim 1, wherein, The wireless ultrasonic microfluidic chip is generated by microprocessing and hot pressing a multilayer PMMA template.

3. The ultrasonic microfluidic integrated device of claim 1, wherein, The ultrasonic sheet is fixed on the wireless ultrasonic microfluidic chip through thermal bonding.

4. The ultrasonic microfluidic integrated device of claim 1, wherein, The wireless module is a Bluetooth module.

5. The ultrasonic microfluidic integrated device of claim 1, wherein, In step S1, the reaction substrate liquid is luminol-sodium alginate, luminol-sodium alginate-cobalt (II) or luminol-hydrogen peroxide; the volume of the reaction substrate liquid ranges from 100 muL to 140 muL; and the buffer solution used is a carbonate buffer solution, a PBS buffer solution or a Tris-HCl buffer solution.

6. The ultrasonic microfluidic integrated device of claim 1, wherein, In step S2, the volume of the sample to be detected in the sample storage area ranges from 60 muL to 100 muL, and the identification reaction conditions set for the sample storage area include direct ultrasonic conduction without identification, ultrasonic conduction after sufficient identification reaction and ultrasonic conduction after multi-step reaction.

7. The ultrasonic microfluidic integrated device of claim 1, wherein, In step S3, the volume of the ultrasonic atomization propellant is 200 muL, and the buffer solution used is a carbonate buffer solution, a PBS buffer solution or a Tris-HCl buffer solution.

8. The ultrasonic microfluidic integrated device of claim 1, wherein, In step S4, the cavity of the photosensitive instrument includes an enzyme marker instrument, a mobile phone adaptive dark box and a single-lens reflex camera adaptive dark box; and the real-time monitoring mode includes an enzyme marker instrument chemiluminescence kinetics detection mode, a mobile phone fixed focus camera mode and a single-lens reflex camera fixed focus camera mode.

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