Micro-fluidic immunoassay chip integrating lateral flow and vertical flow and analysis method of micro-fluidic immunoassay chip

Abstract

The invention provides a micro-fluidic immunoassay chip integrating lateral flow and vertical flow and an analysis method of the micro-fluidic immunoassay chip. Cooperative optimization of lateral flow immunoassay and vertical flow immunoassay is achieved through a self-driven micro-fluidic technology. According to the chip, the porous anodic aluminum oxide film is adopted to replace a test line in a lateral flow immune test strip, and a surface enhanced Raman scattering technology is combined, so that a target biomarker can be subjected to rapid, high-sensitivity and repeatable naked eye / Raman dual-readout detection. And the chip has the characteristics of being convenient to carry, simple and convenient to operate, low in cost, high in expansibility and the like, and has important significance on primary health care screening and field detection of public health emergencies.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection, and more particularly to a microfluidic immunoassay chip integrating lateral and vertical flow and its analytical method. Background Technology

[0002] Point-of-care testing (POCT), as a key technology connecting centralized laboratory diagnostics with on-site medical services, has demonstrated enormous application potential in the field of clinical diagnostics due to its cost-effectiveness, user-friendly design, and rapid result feedback capabilities. Especially during public health emergencies, cost-effective, easy-to-use, and high-performance POCT platforms have become core tools for achieving early and rapid screening of disease biomarkers, securing a critical window of opportunity for clinical early warning and life-saving interventions.

[0003] In point-of-care testing (POCT) technology, lateral flow immunoassay (LFA) has been widely used due to its ease of operation, user-friendliness, and ability to deliver results within 15 minutes. However, traditional LFA has significant performance limitations. Its core defect stems from the nitrocellulose (NC) membrane used. This membrane has a disordered porous structure, which, while suitable for biomolecule recognition, suffers from inherent microenvironment heterogeneity, leading to uneven distribution of immune complexes, unstable flow dynamics, and enhanced non-specific adsorption. Furthermore, its high sensitivity to hook effect interference ultimately results in insufficient detection sensitivity and poor result consistency, severely limiting improvements in detection performance.

[0004] Currently, research on improving LFAs mainly focuses on signal amplification techniques based on nanomaterials, microfluidic system integration, and AI-assisted data analysis. However, these strategies primarily focus on improving single-dimensional performance (such as sensitivity) and fail to fundamentally address the core issues inherent in LFAs, such as the difficulty in controlling fluid flow and non-specific adsorption. Although some studies have attempted to optimize LFA performance through thin-film structure engineering, the process is complex and relies on micro / nano fabrication techniques, making large-scale application difficult. Summary of the Invention

[0005] This invention discloses a microfluidic immunoassay chip that integrates lateral and vertical flow, which can effectively solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention discloses a microfluidic immunoassay chip that integrates lateral and vertical flow, aiming to provide a rapid and highly sensitive "sample to answer" solution for point-of-care testing systems.

[0007] This chip employs a "CAD to 3D" manufacturing and integration design. It utilizes laser cutting technology to precisely process hydrophilic PET film and double-sided adhesive tape, followed by vertical assembly to achieve a stable bond between the interlayer structures. Its core innovation lies in utilizing a capillary-driven microfluidic chip to achieve synergistic optimization of lateral and vertical flow. It replaces the NC film test lines in traditional LFAs with porous anodic aluminum oxide (AAO) films and integrates surface-enhanced Raman scattering (SERS) technology to achieve rapid, sensitive, and repeatable marker detection.

[0008] The chip's functionality relies on the synergistic effect of lateral and vertical flow fields: the device uses lateral capillary forces to drive the analyte to migrate directionally within the flow channel, during which it specifically binds to the pre-coated SERS nanotags; when the liquid flows through the AAO membrane region, its orderly arranged vertical nanochannels and absorbent pads provide vertical capillary forces, which directionally guide the formed immune complex to the surface of the AAO membrane pre-modified with capture antibodies, achieving targeted capture.

[0009] like Figure 1 As shown, taking the detection of interleukin-6 as an example, the chip's workflow is as follows: After adding the sample solution at the chip inlet, the capillary force within the hydrophilic PET film drives the liquid to pass sequentially through the conjugate pad, releasing the SERS nanotag and initiating the reaction; after a 5-minute reaction cycle, a visible color change appears in the detection area, meeting the requirements for qualitative analysis; simultaneously, by acquiring surface-enhanced Raman spectroscopy in situ, the linear correlation between the SERS signal intensity and the density of immune complexes captured on the AAO membrane surface is utilized to achieve highly sensitive quantitative detection of the analyte.

[0010] This chip is constructed using a vertical lamination process involving laser cutting and hydrophilic PET film and double-sided adhesive tape. This simplified manufacturing process effectively reduces production costs while ensuring the device's portability and adaptability to various application scenarios. It innovatively combines the ease of operation of LFA, the high detection efficiency and anti-hook effect characteristics of VFA, with the ultra-high sensitivity of SERS technology and the excellent mass transfer performance of the ordered nanostructure of AAO membranes. Through optimized fluid channel design and interfacial reaction mechanisms, it solves the problems of low detection sensitivity and insufficient uniformity of traditional LFA and VFA due to the disordered porous structure of NC membranes, as well as the susceptibility of LFA to false negatives caused by the hook effect in high-concentration samples and the complex operation of VFA. The orthogonal design of the lateral and longitudinal flow architecture not only achieves dual reading modes of vision and spectroscopy but also reduces interference from uneven fluid distribution and non-specific adsorption, significantly improving the reproducibility of detection results over a wide dynamic range.

[0011] Specifically, it is a microfluidic immunoassay chip integrating lateral and vertical flow, the fabrication process of which includes the following steps: 1. Chip fabrication: AutoCAD was used to complete the parametric design of the microfluidic channel and construct the microfluidic channel; the hydrophilic PET film and double-sided tape were laser-cut to obtain the functional layer of the preset size; according to the designed interlayer positioning relationship, the pre-treated conjugate pad, AAO film, absorbent pad, hydrophilic PET film and double-sided tape were sequentially laminated through a vertical lamination process to form a complete chip structure.

[0012] Immerse the conjugate pad in pretreatment buffer (containing 50 mM PB, 5% sucrose, 3% trehalose, 1% BSA, and 0.5% Tween 20), incubate at 37°C for 1-3 hours, and then dry at 37°C for 1-3 hours. After drying, cut into 3 mm × 5 mm sizes, and use a micropipette to uniformly drop 1-20 μL of antibody-modified SERS nanotags onto their surface for later use.

[0013] 2. AAO surface modification: The AAO membrane was first washed with ethanol and deionized water, then subjected to oxygen plasma treatment for 1-5 minutes to enhance hydrophilicity. Next, it was immersed in a 1-5% (v / v) ethanol solution containing 3-aminopropyltriethoxysilane (APTES) at 50-70°C for 4-10 hours. The AAO membrane was then rinsed repeatedly with ethanol and deionized water to remove residual APTES and baked at 120°C for 30 minutes. Afterward, the AAO membrane was immersed in a 1-5% (v / v) PBS solution containing glutaraldehyde (GA) at 25-37°C for 1-3 hours to obtain a formyl-functionalized AAO membrane. Unreacted glutaraldehyde was removed using deionized water. Then, a 0.001-10 mg / mL marker capture antibody solution was dropped onto the activated AAO membrane surface and incubated at 4°C for 10-18 hours. After washing with PBS, the membrane was then subjected to a 1-10% PBS solution containing glutaraldehyde (GA). BSA was used to block nonspecific sites in PBS solution at 37°C for 0.5–2 hours; finally, the functionalized AAO membrane was rinsed with PBS and stored at 4°C for subsequent use.

[0014] 3. Synthesis of SERS nanotag Au@Ag-Au: (1) Synthesis of AuNPs: Gold nanoparticles (AuNPs) with a diameter of 15 nm were prepared by reducing HAuCl4 with citric acid. (2) Synthesis of Au@AgNPs: 4 mL of 100 mM CTAC, 339.6 mL of ultrapure water, 20 mL of AuNPs solution, and 0.4 mL of 100 mM NaOH were mixed and heated with magnetic stirring at 65 °C. 18 mL of 1 mM AgNO3 solution containing 20 mM CTAC, 2 mM L-ascorbic acid (AA), and 2 mM NaOH were simultaneously injected into the mixture at a rate of 50 μL / min using a syringe pump. After the reaction was complete, the mixture was cooled to room temperature and centrifuged at 7000-10000 r / min for 8-15 minutes. The precipitate was redispersed in 200 mL of 10 mM CTAC to obtain Au@Ag nanoparticles.

[0015] (3) Synthesis of Au@Ag-Au: Pre-preparation of gold growth solution: Mix 85.5 mL of ultrapure water, 2 mL of 25 mM HAuCl4・3H2O, 2.5 mL of 100 mM NaOH and 10 mL of 10 mM Na2SO3 and stir until colorless, then store at 4℃; Take 40 mL of ultrapure water, 5 mL of 100 mM CTAC, 5 mL of Au@Ag nanoparticle solution, 2 mL of 1 mM 4-MBA and 0.5 mL of 100 mM AA and inject 20 mL of gold growth solution at a rate of 2 mL / min under magnetic stirring. After reacting for 4 hours, centrifuge at 3000-5000 r / min for 8-15 minutes, and redisperse the precipitate in 5 mL of 10 mM CTAC to obtain Au@Ag-Au nanoparticles.

[0016] 4. Antibody functionalization of Au@Ag-Au nanotags (1) Take 200 μL of Au@Ag-Au nanotags and sonicate them in ultrapure water for 1-3 minutes until they are evenly dispersed. Replace the buffer with 500 μL of reaction buffer containing 0.4 mM CTAC, 30 mM Tris-HCl, and pH=8.75.

[0017] (2) Incubate with 1-5 μL of detection antibody at 37℃ for 0.5-2 hours. The concentration of detection antibody is 0.001-10 mg / mL. Then add blocking solution (containing 2% BSA, 0.1% casein, and 30 mM Tris-HCl) and block for 2-4 hours with gentle stirring.

[0018] (3) Centrifuge at 2500-4000 r / min for 8-15 minutes to remove free antibodies. Redisperse the precipitate in 10-400 μL of optimized reaction buffer containing 50 mM PB, 1% PVP, 1% mannitol and 1% BSA. Store at 4℃ for later use.

[0019] Advantages compared to existing technologies: In this invention, replacing the NC membrane with an AAO membrane, previously limited to the construction of a single vertical flow immunoassay (VFA) platform, effectively reduces non-specific adsorption and improves detection sensitivity and uniformity through nano-confinement effect and optimized mass transfer of ordered vertical nanofluidic channels. Compared with LFA, VFA further improves detection efficiency by shortening the fluid path, resisting the hook effect, and enhancing the scalability of multiplex detection. However, VFA requires several complex operation steps such as premixing and pre-incubation, limiting its application in simple scenarios.

[0020] Based on this, integrating the ease of operation of LFA with the advantages of VFA, such as rapid detection and resistance to hook effects, has become a key direction for breaking through the bottlenecks of existing point-of-care testing technologies. By integrating these technologies to address the inherent defects in the background technologies, the following effects have been achieved: Firstly, capillary-driven microfluidic design overcomes the limitations of traditional LFA and VFA. By precisely controlling fluid behavior through an ordered flow channel structure, it improves the problems of uneven flow, solution evaporation, and non-specific adsorption caused by disordered porous NC membranes, thereby enhancing detection uniformity.

[0021] Secondly, it integrates the advantages of LFA and VFA. It retains the ease of operation of LFA while integrating the high-efficiency detection capability and anti-hook effect advantage of VFA, thus reducing the risk of false negatives and enhancing the reliability of detection while ensuring detection speed.

[0022] Third, by using an AAO membrane instead of a traditional NC membrane as the detection line, the detection sensitivity and result uniformity can be further improved by leveraging its ordered nanochannel structure and excellent mass transfer performance.

[0023] Fourth, it possesses cost-effectiveness and manufacturing advantages. As a passive microfluidic device, it does not require complex drive components, making it suitable for resource-limited scenarios. Relying on the modular stacking process of hydrophilic polyester film and commercially available DSA tape, it avoids the complex manufacturing and surface modification processes relied upon by traditional passive microfluidics, simplifying production and facilitating large-scale promotion.

[0024] Fifth, it boasts strong technological scalability. The modular design supports flexible switching of detection modes; simply changing the nanomaterial of the connecting pad allows for adaptation to colloidal gold, fluorescence, and other modes, meeting the detection needs of different biomarkers. By modifying the chip flow channel design, superior mixing efficiency is achieved, further enhancing detection sensitivity. Adding detection sites to the AAO membrane and combining it with multifunctional SERS markers enables simultaneous detection of multiple biomarkers, ensuring diversified applications in complex clinical scenarios. This is of great significance for expanding the large-scale application of point-of-care testing technology. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of chip detection according to the present invention; Figure 2 This is a schematic diagram showing the specific parameters of the chip in this invention; Figure 3 This is a schematic diagram of the chip assembly of the present invention; Figure 4 This is a physical image of the chip used in this invention; Figure 5 This is a schematic diagram of the chip operation process of the present invention; Figure 6 The images show (a) a physical image, (b) a SERS spectrum, and (c) a linear regression curve for IL-6 detection according to the present invention. Figure 7 This is a schematic diagram of the chip-specific detection method of the present invention; Figure 8 This is a schematic diagram of the chip repeatability detection of the present invention; Figure 9 For the chip uniformity detection of the present invention, (a) 100 ng / mL; (b) 10 ng / mL; (c) 1 ng / mL; Figure 10 This is for the chip storage stability test of the present invention.

[0026] In the diagram: 101. Hydrophilic PET film, 102. Double-sided tape, 103. AAO membrane, 104. Absorbent pad, 105. Conjugate pad, 201. Chip inlet, 202. Serpentine channel, 203. Detection area, 301. Visual inspection, 302. SERS detection, 303. SERS nanotag, 304. Antigen, 305. Biomarker antibody, 401. Sample acquisition, 402. Sample addition, 403. Signal acquisition, 404. Data analysis, 30101. Positive visual inspection, 30102. Negative visual inspection. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0028] Example 1, refer to Appendix Figure 1-10 A microfluidic immunoassay chip integrating lateral and vertical flow, the fabrication process of which includes the following steps: 1. Chip fabrication: The chip is integrated using laser cutting and vertical lamination processes. The specific steps are as follows: The parametric design of the microfluidic channel was completed using AutoCAD, constructing a serpentine microfluidic channel. Design parameters are shown below. Figure 2 Laser cutting of hydrophilic PET film 101 and double-sided tape 102 to obtain a functional layer of preset size; (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 3 The interlayer positioning relationship involves sequentially laminating the pretreated conjugate pad, AAO film 103, absorbent pad 104, PET film, and double-sided adhesive tape 102 using a vertical lamination process to form a complete chip structure. See the physical image below. Figure 4 .

[0029] The conjugate pads were immersed in a pretreatment buffer containing 50 mM PB, 5% sucrose, 3% trehalose, 1% BSA, and 0.5% Tween 20. After incubation at 37°C for 2 hours, the pads were dried at 37°C for 2 hours. After drying, the pads were cut into 3 mm × 5 mm pieces. The antibody-modified SERS nanotags 303 were evenly dropped onto the surface of the pads at a rate of 5 μL / piece using a micropipette.

[0030] 2. AAO surface modification: AAO membrane 103 was first washed with ethanol and deionized water, then subjected to oxygen plasma treatment for 3 minutes to enhance hydrophilicity; it was then immersed in a 5% (v / v) ethanol solution containing APTES at 65°C for 6 hours; AAO membrane 103 was then rinsed multiple times with ethanol and deionized water to remove residual APTES, and baked at 120°C for 30 minutes; subsequently, AAO membrane 103 was immersed in a 5% (v / v) PBS solution containing GA at 25°C for 2 hours to obtain formyl-functionalized AAO membrane 103; unreacted glutaraldehyde was removed using deionized water; then, 1 mg / mL IL-6 capture antibody solution was dropped onto the surface of the activated AAO membrane 103 and incubated at 4°C for 12 hours; after washing with PBS, nonspecific sites were blocked with a 5% BSA PBS solution at 37°C for 1 hour; finally, the functionalized AAO membrane 103 was rinsed with PBS and stored at 4°C for subsequent use.

[0031] 3. Synthesis of SERS nanotag Au@Ag-Au (1) Synthesis of AuNPs: Gold nanoparticles (AuNPs) with a diameter of 15 nm were prepared by reducing HAuCl4 with citric acid.

[0032] (2) Synthesis of Au@AgNPs: 4 mL of 100 mM CTAC, 339.6 mL of ultrapure water, 20 mL of AuNPs solution, and 0.4 mL of 100 mM NaOH were mixed and heated with magnetic stirring at 65 °C. 18 mL of 1 mM AgNO3 solution and 18 mL of the mixture (containing 20 mM CTAC, 2 mM L-ascorbic acid (AA), and 2 mM NaOH) were simultaneously injected via a syringe pump at a rate of 50 μL / min. After the reaction was complete, the mixture was cooled to room temperature and centrifuged at 8000 r / min for 10 minutes. The precipitate was redispersed in 200 mL of 10 mM CTAC to obtain Au@Ag nanoparticles.

[0033] (3) Synthesis of Au@Ag-Au: Pre-preparation of gold growth solution: Mix 85.5 mL of ultrapure water, 2 mL of 25 mM HAuCl4・3H2O, 2.5 mL of 100 mM NaOH and 10 mL of 10 mM Na2SO3 and stir until colorless, then store at 4℃; Take 40 mL of ultrapure water, 5 mL of 100 mM CTAC, 5 mL of Au@Ag nanoparticle solution, 2 mL of 1 mM 4-MBA and 0.5 mL of 100 mM AA and inject 20 mL of gold growth solution at a rate of 2 mL / min under magnetic stirring. After reacting for 4 hours, centrifuge at 4500 r / min for 10 minutes, and redisperse the precipitate in 5 mL of 10 mM CTAC to obtain Au@Ag-Au nanoparticles.

[0034] 4. Antibody functionalization of Au@Ag-Au nanotags (1) Take 200 μL of Au@Ag-Au nanotags and sonicate them in ultrapure water for 2 minutes until they are evenly dispersed. Replace the buffer with 500 μL of reaction buffer (containing 0.4 mM CTAC, 30 mM Tris-HCl, pH=8.75).

[0035] (2) Incubate with 2 μL of detection antibody (1 mg / mL) at 37℃ for 1 hour, then add blocking solution (containing 2% BSA, 0.1% casein, and 30 mM Tris-HCl) and block for 3 hours with gentle stirring.

[0036] (3) Centrifuge at 3000 r / min for 15 minutes to remove free antibodies. The precipitate is redispersed in 25 μL of optimized reaction buffer (containing 50 mM PB, 1% PVP, 1% mannitol and 1% BSA) and stored at 4℃ for later use.

[0037] Example 2: The performance of the microfluidic immunoassay chip integrating lateral and vertical flow provided in Example 1 was evaluated as follows: 1. Sensitivity test: like Figure 5 As shown, 50 μL of IL-6 antigen solution (diluted with PBS) with a concentration ranging from 100 fg / mL to 10 μg / mL was added to the chip inlet 201. Driven by capillary action, the sample flowed through the conjugation pad 105, releasing the antibody-modified SERS nanotag 303 (i.e., the tag surface was modified with a marker antibody 305 specifically recognizing the analyte). The two were thoroughly mixed in the serpentine channel 202 to form an immune complex. The immune complex entered the AAO membrane 103 region with the fluid and was specifically captured by the capture antibody immobilized on the membrane surface. After 5 minutes of reaction, qualitative analysis was performed by visually observing the color change in the detection area 203, or quantitative analysis was performed by acquiring the SERS signal using a confocal Raman microscope (each sample was tested three times). The visually positive result 30101 is shown in the attached figure. Figure 1 As shown on the left side of the lower right area, the visual inspection result for 30102 is negative (see attached image). Figure 1 As shown on the right side of the lower right area.

[0038] Detection results of different concentrations of IL-6 are as follows Figure 6 As shown in Figure a, the SERS spectrum is as follows: Figure 6 As shown in b. The linear dynamic detection curve is as follows. Figure 6 As shown in c. The detection limit for visual inspection (301) was 100 pg / mL, and the detection limit for SERS (302) was 0.95 pg / mL. The calibration curves exhibited a linear dynamic range from 100 pg / mL to 1 μg / mL (correlation coefficient R0). 2 =0.972).

[0039] 2. Specificity test: Alpha-fetoprotein (AFP), C-reactive protein (CRP), SARS-CoV-2 N protein, respiratory syncytial virus (RSV), Tau 181, interleukin-β, influenza A protein, and matrix controls (10 mM PBS, human serum) were selected as interfering agents. All were used at a concentration of 10 ng / mL to replace IL-6 for detection. Specificity was assessed by comparing the SERS signal differences between positive and interfering samples. Figure 7 As shown, the presence of the other 8 substances did not trigger a significant signal, demonstrating the good specificity of this strategy.

[0040] 3. Repeatability testing: Eight parallel assays were performed using 10 ng / mL IL-6 samples. Repeatability was assessed by statistically analyzing the coefficient of variation of the signal. Figure 8 As shown, the RSD is 6.39%.

[0041] 4. Uniformity test: SERS mapping was performed on the surface of the AAO film, and the uniformity was detected by Raman characteristic peak intensity distribution analysis. For example... Figure 9 As shown, the chip surface uniformity detection under the 100 ng / mL-1 ng / mL IL-6 system is demonstrated, with RSDs of 5.11%, 6.88%, and 9.09%, respectively, all below 10%, indicating that the chip has good detection uniformity. 5. Storage stability test: like Figure 10 As shown, the chip in the 100 ng / mL IL-6 system was stored at room temperature for 6 weeks, and the change in SERS signal intensity was monitored. The results showed that the signal decreased by only 8.96%, which verified the long-term stability.

[0042] Example 3: Based on the microfluidic immunoassay chip integrating lateral and vertical flow provided in Example 1, this invention also provides an analysis method for the microfluidic immunoassay chip integrating lateral and vertical flow. See Appendix. Figure 5 This includes the following steps: 1. Sample Acquisition 401: The sample acquisition 401 step includes: Venous blood samples were collected from the subjects, centrifuged, and the supernatant was extracted as the test sample; the test sample was used to detect the concentration of the target analyte in vitro.

[0043] The sample acquisition method 401 includes the following steps: a) Blood collection: Collect 3-5 mL of venous blood from the subject using an anticoagulant vacuum blood collection tube; b) Centrifugation: Place the blood collection tube in a centrifuge and centrifuge at 1500-3000×g for 8-15 minutes; c) Layered extraction: Separating the supernatant layer of the sample after centrifugation, which contains serum or plasma; d) Sample storage: Transfer the extracted supernatant to sterile EP tubes and store at -20°C to -80°C or use it directly for subsequent in vitro testing.

[0044] 2. Add sample 402: Add 50 μL of the test sample containing target antigen 304 to the chip inlet 201.

[0045] Driven by capillary forces, the sample flows through the binding pad, releasing antibody-modified SERS nanotags 303. The two mix thoroughly in the serpentine channel 202 to form an immune complex. The immune complex enters the AAO membrane 103 region with the fluid and is specifically captured by the capture antibody immobilized on the membrane surface.

[0046] 3. Signal Acquisition 403: After 5 minutes of reaction, qualitative analysis is performed by visually observing the color change of the detection area 203, while quantitative analysis is performed by acquiring SERS signals using a confocal Raman microscope (each sample is tested 3 times).

[0047] 4. Data Analysis 404: The data analysis 404 method includes: The SERS signal spectrum of the sample is input into the quantitative detection model, which outputs the concentration value of the target analyte; wherein, the quantitative detection model establishes a mapping relationship between the intensity of SERS characteristic peaks and concentration through machine learning algorithms.

[0048] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0049] All parts not covered in this invention are implemented using existing technologies.

Claims

1. A microfluidic immunoassay chip integrating lateral and vertical flow, characterized in that: A hydrophilic PET film and double-sided tape are laser-cut to create fluid channels, with the inner wall of the hydrophilic film providing capillary force for spontaneous liquid drive. The pretreated conjugate pad, the surface-modified AAO film, the absorbent pad, the hydrophilic PET film, and the double-sided tape are sequentially laminated to form a complete chip structure. Surface-enhanced Raman scattering technology is integrated on the surfaces of the conjugate pad and the AAO film to achieve detection functionality.

2. The microfluidic immunoassay chip integrating lateral and vertical flow according to claim 1, characterized in that: The pretreatment process of the "pretreated conjugate pad" includes the following steps: immersing the conjugate pad in pretreatment buffer, incubating at 37°C for 1-3 hours, and drying at 37°C for 1-3 hours; cutting the dried conjugate pad into the same size, and uniformly adding antibody-modified SERS nanotags at a dosage of 1-20 μL / piece using a micropipette.

3. The microfluidic immunoassay chip integrating lateral and vertical flow according to claim 1, characterized in that: The "surface-modified AAO membrane" undergoes the following modification process: the AAO membrane is first cleaned with ethanol and deionized water, then subjected to oxygen plasma treatment for 1-5 minutes to enhance hydrophilicity; next, it is immersed in a 1-5% (v / v) ethanol solution containing 3-aminopropyltriethoxysilane (APTES) at 50-70°C for 4-10 hours; the AAO membrane is then rinsed multiple times with ethanol and deionized water to remove residual APTES, and baked at 120°C for 30 minutes; subsequently, the AAO membrane is immersed in a 1-5% (v / v) PBS solution containing glutaraldehyde (GA) at 25-37°C for 1-3 hours to obtain a formyl-functionalized AAO membrane; unreacted glutaraldehyde is removed using deionized water; then, 0.001-10 A mg / mL marker capture antibody solution was dropped onto the surface of the activated AAO membrane and incubated at 4°C for 10–18 h. After washing with PBS, nonspecific sites were blocked with PBS solution containing 1–10% BSA at 37°C for 0.5–2 h. Finally, the functionalized AAO membrane was rinsed with PBS and stored at 4°C for subsequent use.

4. The microfluidic immunoassay chip integrating lateral and vertical flow according to claim 2, characterized in that: The "same specification" refers to 3 mm × 5 mm.

5. The microfluidic immunoassay chip integrating lateral and vertical flow according to claim 2, characterized in that: The pretreatment buffer solution comprises 50 mM PB, 5% sucrose, 3% trehalose, 1% BSA, and 0.5% Tween 20.

6. The microfluidic immunoassay chip integrating lateral and vertical flow according to claim 2, characterized in that: The "SERS nanotag" includes Au@Ag-Au; the synthesis of Au@Ag-Au includes the following process: (1) Synthesis of AuNPs: Gold nanoparticles (AuNPs) with a diameter of 15 nm were prepared by reducing HAuCl4 with citric acid. (2) Synthesis of Au@AgNPs: 4 mL of 100 mM CTAC, 339.6 mL of ultrapure water, 20 mL of AuNPs solution, and 0.4 mL of 100 mM NaOH were mixed and heated with magnetic stirring at 65 °C. Simultaneously, 18 mL of 1 mM AgNO3 solution containing 20 mM CTAC, 2 mM L-ascorbic acid (AA), and 2 mM NaOH were injected into the mixture at a rate of 50 μL / min using a syringe pump. After the reaction was complete, the mixture was cooled to room temperature and centrifuged at 7000-10000 r / min for 8-15 minutes. The precipitate was redispersed in 200 mL of 10 mM CTAC to obtain Au@Ag nanoparticles. (3) Synthesis of Au@Ag-Au: Pre-preparation of gold growth solution: Mix 85.5 mL of ultrapure water, 2 mL of 25 mM HAuCl4・3H2O, 2.5 mL of 100 mM NaOH and 10 mL of 10 mM Na2SO3 and stir until colorless, then store at 4℃; Take 40 mL of ultrapure water, 5 mL of 100 mM CTAC, 5 mL of Au@Ag nanoparticle solution, 2 mL of 1 mM 4-MBA and 0.5 mL of 100 mM AA and inject 20 mL of gold growth solution at a rate of 2 mL / min under magnetic stirring. After reacting for 4 hours, centrifuge at 3000-5000 r / min for 8-15 minutes, and redisperse the precipitate in 5 mL of 10 mM CTAC to obtain Au@Ag-Au nanoparticles; (4) Antibody functionalization of Au@Ag-Au nanotags.

7. The microfluidic immunoassay chip integrating lateral and vertical flow according to claim 6, characterized in that: The antibody functionalization of the Au@Ag-Au nanotag includes the following steps: (1) Take 200 μL of Au@Ag-Au nanotags, sonicate them in ultrapure water for 1-3 minutes until they are evenly dispersed, and then replace the buffer with 500 μL of reaction buffer. (2) Incubate with 1-5 μL of detection antibody at 37℃ for 0.5-2 hours. The concentration of detection antibody is 0.001-10 mg / mL. Then add blocking solution containing 2% BSA, 0.1% casein and 30 mM Tris-HCl. Block for 2-4 hours with gentle stirring. (3) Centrifuge at 2500-4000 r / min for 8-15 minutes to remove free antibodies. Redisperse the precipitate in 10-400 μL of optimized reaction buffer and store at 4℃ for later use.

8. The microfluidic immunoassay chip integrating lateral and vertical flow according to claim 7, characterized in that: The "reaction buffer" comprises 0.4 mM CTAC, 30 mM Tris-HCl, and pH=8.

75.

9. A microfluidic immunoassay chip integrating lateral and vertical flow according to claim 7, characterized in that: The "optimized reaction buffer" comprises 50 mM PB, 1% PVP, 1% mannitol, and 1% BSA.

10. An analytical method for a microfluidic immunoassay chip integrating lateral and vertical flow, characterized in that: Includes the following steps: (1) Sample acquisition: The sample acquisition steps include: Venous blood samples were collected from the subjects, centrifuged, and the supernatant was extracted as the test sample; the test sample was used to detect the concentration of the target analyte in vitro. The sample acquisition method includes the following steps: a) Blood collection: Collect 3-5 mL of venous blood from the subject using an anticoagulant vacuum blood collection tube; b) Centrifugation: Place the blood collection tube in a centrifuge and centrifuge at 1500-3000×g for 8-15 minutes; c) Layered extraction: Separating the supernatant layer of the sample after centrifugation, which contains serum or plasma; d) Sample storage: Transfer the extracted supernatant to sterile EP tubes and store at -20°C to -80°C; (2) Add sample: Add the test sample containing the target antigen to the chip inlet; The sample flows through the binding pad 105 under the drive of capillary force, releasing antibody-modified SERS nanotags. The two mix thoroughly in the serpentine channel to form an immune complex. The immune complex enters the AAO membrane region with the fluid and is specifically captured by the capture antibody immobilized on the membrane surface. (3) Signal acquisition: After the reaction, the color change of the detection area is observed by the naked eye for qualitative analysis, and the SERS signal is acquired by confocal Raman microscopy for quantitative analysis. (4) Data analysis: The data analysis methods include: The SERS signal spectrum of the sample is input into the quantitative detection model, which outputs the concentration value of the target analyte; wherein, the quantitative detection model establishes a mapping relationship between the intensity of SERS characteristic peaks and concentration through machine learning algorithms.

Images