Device and Method for Detecting Five Biomarkers of Secondary Hypertension Based on Microfluidic Chips

Abstract

This invention belongs to the field of detection devices, specifically a device and method for detecting five biomarkers of secondary hypertension based on a microfluidic chip. It includes a reading device body, a chip slot, and a chip body. The reading device body is equipped with a chip slot, a touchscreen, and function buttons. The chip body is housed within the chip slot. This invention shortens the turnaround time for clinical reports of the five biomarkers through an integrated automated process. By reducing the output time of the five biomarker detection results, it improves the continuity and automation of the in vitro sample detection process, solving the problem of excessively long detection times in traditional methods. This provides a reliable basis for timely clinical diagnosis and treatment planning, reducing the risk of disease progression. Furthermore, by integrating an AEC enzyme catalytic signal amplification system with a chip-integrated automated temperature control system, it transforms PRA enzyme activity detection into a one-step rapid detection method, while avoiding human temperature control errors and ensuring accurate detection results.

Description

Technical Field

[0001] This invention belongs to the field of detection devices, specifically a device and method for detecting five biomarkers of secondary hypertension based on a microfluidic chip. Background Technology

[0002] Secondary hypertension is a type of hypertension with a clear cause that can be cured or significantly improved through targeted treatment. Currently, hypertension worldwide is showing a serious trend of high incidence and younger onset. In my country, the prevalence of hypertension among people aged 18 and above has reached 23.2%, affecting over 300 million people, with the proportion of young hypertension patients under 35 years old increasing year by year. Among young hypertension patients, the prevalence of secondary hypertension is as high as 29.6%, and even higher in those with refractory hypertension, reaching 20%-30%.

[0003] The core biomarkers for clinical screening of secondary hypertension include plasma renin activity (PRA), aldosterone, methoxyadrenaline (MN), nomethoxyadrenaline (NMN), and vanillylmandelic acid (VMA). Accurate detection of these biomarkers is crucial for diagnosing diseases such as pheochromocytoma, primary aldosteronism, and renovascular hypertension.

[0004] Currently, the mainstream clinical detection methods are liquid chromatography-tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay. Although LC-MS / MS is the recognized reference method, the equipment is expensive; ELISA and chemiluminescence have lower equipment requirements, but are cumbersome to operate. These methods generally suffer from problems such as long detection cycles, high equipment requirements, and complex operation.

[0005] The excessively long testing cycle severely restricts the efficiency of diagnosis and treatment: LC-MS / MS methods involve complex pretreatment and have a total cycle of up to 4 working days; ELISA methods take a cumulative 2-3 hours; and traditional PRA testing takes 4-6 hours. Patients typically have to wait 3-5 days from blood collection to obtaining complete results for all five items, making it impossible to formulate a timely treatment plan.

[0006] High equipment and technical barriers hinder widespread adoption at the grassroots level: LC-MS / MS equipment requires an investment of 3-8 million yuan, has stringent environmental requirements, and necessitates operation by professional personnel, making it suitable only for large tertiary hospitals. While methods such as ELISA are lower in cost, their procedures are cumbersome, and result interpretation is easily influenced by human factors, making them difficult to promote at the grassroots level.

[0007] Plasma renin activity (PRA) detection faces a core technical bottleneck: PRA is an enzyme activity indicator and cannot be directly measured using immunoassays. Traditional two-step incubation-detection methods are complex and time-consuming; furthermore, the clinical diagnostic significance of direct renin concentration immunoassays is not entirely equivalent to that of PRA, becoming the biggest technical obstacle to rapid detection.

[0008] Sample processing is cumbersome, resulting in low convenience and compliance: Urine biomarkers (such as VMA) require complex pretreatment processes such as acidification and extraction, and the 24-hour urine sample collection process is cumbersome. Whole blood samples require manual centrifugation, which involves multiple steps and is prone to introducing errors. In addition, the matrix differences between blood and urine samples are significant, and if they are simply combined in the same chip, cross-contamination, incompatible reaction systems, and optical signal crosstalk can easily occur.

[0009] The detection of multiple biomarkers cannot be integrated, resulting in high detection costs: each biomarker needs to be detected separately using different technical platforms, leading to multiple samplings, high costs, and difficulty in unifying and integrating the results for analysis.

[0010] Delayed testing leads to increased clinical risks and medical costs: Excessive testing cycles directly result in longer hospital stays, increased medical expenses, and may delay the diagnosis of diseases such as pheochromocytoma, leading to serious risks such as hypertensive crisis.

[0011] Therefore, the present invention provides a device and method for detecting five biomarkers of secondary hypertension based on a microfluidic chip. Summary of the Invention

[0012] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0013] The technical solution adopted by this invention to solve its technical problem is as follows: A microfluidic chip-based device for detecting five biomarkers of secondary hypertension, comprising a reading device body, a chip slot, and a chip body. The reading device body is provided with a chip slot, a touch screen, and function buttons. The chip body is disposed within the chip slot. The chip body is provided with a sample loading area, a plasma separation area, a urine concentration and enrichment area, a PRA temperature-controlled reaction area, an aldosterone / MN / NMN homogeneous reaction area, a VMA chromatography detection area, and a waste liquid area. The sample loading area is used for separately loading isolated blood samples and isolated urine samples, with separate, isolated blood loading areas. The sample inlet and urine sample inlet, and the blood sample inlet are connected to the PRA temperature-controlled reaction zone and the aldosterone / MN / NMN homogeneous reaction zone via the plasma separation sub-zone, respectively. The urine sample inlet is connected to the VMA chromatography detection zone via the urine concentration and enrichment sub-zone. The plasma separation sub-zone is used for plasma separation of whole blood samples. The urine concentration and enrichment sub-zone is used for 10-20 times enrichment of VMA in urine, matrix removal, and buffer condition adjustment. The PRA temperature-controlled reaction zone is used to contain the pre-placed angiotensinogen fragment-enzyme conjugate (AEC) and reacts specifically with renin in isolated plasma samples at a controlled temperature to generate an on-chip optically readable signal related to renin activity. The main body of the reading device is internally equipped with a communication module, a multi-channel optical detection module, and a temperature control module. The communication module is used for data transmission. The multi-channel optical detection module is equipped with a data processing unit, which is used to analyze the detection signals corresponding to PRA, aldosterone, MN, NMN, and VMA, and output the in vitro quantitative intermediate results and quality control status of the in vitro sample. The temperature control module is equipped with a temperature control unit and an optical signal reading module. A microfluidic channel is set above the optical signal reading module. The microfluidic channel is directly opposite the chip slot, and the chip body can be inserted into the microfluidic channel through the chip slot.

[0014] Furthermore, the plasma separation sub-region integrates an asymmetric PES / PVDF gradient filter membrane, and the urine concentration and enrichment sub-region integrates a C18 solid-phase extraction microcolumn.

[0015] Furthermore, the PRA temperature-controlled reaction zone has a built-in thin-film heater, temperature sensor, and PID temperature control module. The constant temperature range of the PRA temperature-controlled reaction zone is 35℃-39℃. The aldosterone / MN / NMN homogeneous reaction zone has three independent parallel reaction chambers. The aldosterone / MN / NMN homogeneous reaction zone is used to pre-place lyophilized specific antibodies and enzyme markers.

[0016] Furthermore, the VMA chromatography detection zone integrates a quantum dot immunochromatographic test strip, and the VMA chromatography detection zone is used for chromatographic reaction and fluorescent signal labeling.

[0017] Furthermore, the waste liquid area is designed as a sealed collection chamber, which is used to seal and collect the waste liquid after testing.

[0018] Furthermore, the chip body contains lyophilized reagents, and the chip body is connected to various functional areas through microchannels.

[0019] Furthermore, the multi-channel optical inspection module is equipped with a multi-wavelength LED excitation source and a PMT / CMOS detector. The data processing unit has a built-in microprocessor for baseline subtraction, temperature compensation, chip batch number calibration, calibration curve conversion, quality control judgment, and abnormal signal identification. The temperature control module is equipped with semiconductor cooling and heating components, and a power module is provided at the end of the main body of the reading device.

[0020] A detection method for a microfluidic chip-based device for detecting five biomarkers of secondary hypertension, comprising the following steps: S1. Add the isolated blood sample to the blood sampling inlet in the sample application area, and add the isolated urine sample to the urine sampling inlet in the sample application area, so that the two enter the blood branch and urine branch that are isolated from each other respectively. S2, the sample loading area relies on capillary force to drive sample flow, and performs plasma separation, urine VMA enrichment, matrix removal and buffer adjustment respectively; S3. After pretreatment, the sample is automatically distributed to each reaction zone of the aldosterone / MN / NMN homogeneous reaction zone through a microchannel. Under a temperature-controlled environment of 37℃, the PRA temperature-controlled reaction zone, the aldosterone / MN / NMN homogeneous reaction zone and the VMA chromatography detection zone are started simultaneously to perform PRA enzymatic reaction, aldosterone / MN / NMN homogeneous immunoreaction and VMA fluorescence immunochromatographic reaction. S4. Insert the completed reaction chip body into the microfluidic channel through the chip slot. The device automatically collects the detection signals of each reaction zone for signal analysis, concentration calculation and quality control judgment, and generates a structured test report containing the in vitro quantitative results of five markers, quality control status, ARR ratio and optional VMA correction results. S5. The test results are displayed on the touch screen and simultaneously uploaded to the hospital's LIS / HIS system via the communication module for clinicians to view.

[0021] The beneficial effects of this invention are as follows: 1. The present invention discloses a microfluidic chip-based device and method for detecting five biomarkers of secondary hypertension. Through an integrated automated process, the turnaround time for clinical reports of the five biomarkers is shortened. By reducing the output time of the five biomarker detection results, the continuity and automation of the in vitro sample detection process are improved, solving the problem of excessively long detection times in traditional methods. This provides a reliable basis for timely clinical diagnosis and treatment planning and reduces the risk of disease progression. The device integrates an AEC enzyme catalytic signal amplification system with a chip-integrated automated temperature control system, transforming PRA enzyme activity detection into a one-step rapid detection method, while avoiding human temperature control errors and ensuring accurate detection results.

[0022] 2. The present invention discloses a microfluidic chip-based detection device and method for five biomarkers of secondary hypertension, integrating homogeneous enzyme immunoassay, quantum dot fluorescence immunochromatography, and microfluidic chip to achieve full automation of sample pretreatment, detection reaction, and signal acquisition with extremely low human intervention. It possesses excellent repeatability and precision, meeting clinical precision requirements and significantly reducing the risk of human error and sample cross-contamination. The detection chip uses medical-grade polymer materials, and its compact size, achieved through structural optimization, allows for large-scale, standardized production via injection molding, reducing chip costs. The accompanying portable reading device is significantly cheaper than LC-MS / MS equipment; operation is simple, requiring only basic training for ordinary laboratory personnel, and it is suitable for various scenarios including hospitals at all levels, community health service centers, and bedside visits, facilitating the implementation of the hierarchical diagnosis and treatment policy for hypertension. It supports the detection of trace amounts of whole blood (≤50μL) and random urine (≤100μL), eliminating the need for large-volume blood collection; relying on a creatinine-corrected random urine VMA conversion algorithm, it eliminates the tedious and painful 24-hour urine collection for patients, greatly improving patient cooperation. The price of the testing terminal is much lower than the cost of LC-MS / MS testing; after the completion of clinical application evaluation, it is expected to shorten the testing waiting time and reduce the cost of repeated sampling and manual pretreatment, thereby improving the efficiency of medical resource utilization; through the independent setting of blood and urine branches, cross-contamination between different sample matrices can be reduced, and the stability of multi-reaction systems running in parallel within the same chip can be improved.

[0023] 3. The microfluidic chip-based device and method for detecting five biomarkers of secondary hypertension described in this invention demonstrate good consistency potential between the five biomarker detection results and clinical reference methods. In subsequent methodological validation, the cross-reactivity with structural analogs and the resistance to common interfering factors such as hemolysis and lipemia can be further evaluated, ensuring the accuracy and reliability of the detection results. The device incorporates an intelligent algorithm that automatically performs concentration calculation, ARR ratio calculation, quality control judgment, and clinical diagnostic prompts, eliminating the need for manual interpretation and calculation, thus reducing the workload of clinicians. It supports integration with hospital LIS / HIS systems to achieve information management of detection data and assist in real-time clinical diagnosis and treatment decisions.

[0024] 4. The microfluidic chip-based device and method for detecting five biomarkers of secondary hypertension described in this invention utilizes lyophilized core reagents within the chip, eliminating the need for cold chain transportation and storage, significantly reducing reagent transportation costs and warehousing requirements, and adapting to the storage conditions of primary healthcare institutions. The microfluidic chip platform features an open design, allowing for rapid expansion to the detection of other hypertension-related biomarkers; the accompanying homogeneous enzyme immunoassay reagents are compatible with existing fully automated biochemical analyzers in hospitals, eliminating the need for additional equipment purchases and further reducing the cost of technology adoption for hospitals. Attached Figure Description

[0025] The invention will now be further described with reference to the accompanying drawings.

[0026] Figure 1 This is a schematic diagram of the device structure in this invention; Figure 2 This is a schematic diagram of the microfluidic detection chip structure in this invention; Figure 3 This is a schematic diagram of the internal structure of the device in this invention; Figure 4 This is a schematic diagram of the detection process in this invention; Figure 5 This is a schematic diagram of the PRA temperature-controlled reaction zone and AEC enzyme-catalyzed signal conversion in this invention.

[0027] In the diagram: 1. Reading device main body; 11. Touch screen; 12. Function buttons; 13. Power module; 2. Chip slot; 21. Chip main body; 22. Sample loading area; 23. Plasma separation sub-area; 24. Urine concentration and enrichment sub-area; 25. PRA temperature-controlled reaction area; 26. Aldosterone / MN / NMN homogeneous reaction area; 27. VMA chromatography detection area; 28. Waste liquid area; 3. Communication module; 4. Multi-channel optical detection module; 5. Data processing unit; 6. Temperature control module; 7. Temperature control unit; 8. Optical signal reading module; 9. Microfluidic channel. Detailed Implementation

[0028] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0029] like Figures 1 to 5As shown in the figure, an embodiment of the present invention provides a microfluidic chip-based device for detecting five biomarkers of secondary hypertension, including a reading device body 1, a chip slot 2, and a chip body 21. The reading device body 1 is provided with a chip slot 2, a touch screen 11, and function buttons 12. The chip body 21 is disposed in the chip slot 2. The chip body 21 is provided with a sample loading area 22, a plasma separation sub-area 23, a urine concentration and enrichment sub-area 24, a PRA temperature-controlled reaction area 25, an aldosterone / MN / NMN homogeneous reaction area 26, a VMA chromatography detection area 27, and a waste liquid area 28. The sample loading area 22 is used for loading isolated blood samples and isolated urine samples separately, and the blood loading area is isolated from each other. The sample inlet and urine sample inlet, and the blood sample inlet are connected to the PRA temperature-controlled reaction zone 25 and the aldosterone / MN / NMN homogeneous reaction zone 26 via the plasma separation sub-region 23, respectively. The urine sample inlet is connected to the VMA chromatography detection zone 27 via the urine concentration and enrichment sub-region 24. The plasma separation sub-region 23 is used for plasma separation of whole blood samples. The urine concentration and enrichment sub-region 24 is used for 10-20 times enrichment of VMA in urine, matrix removal and buffer condition adjustment. The PRA temperature-controlled reaction zone 25 is used to contain the pre-placed angiotensinogen fragment-enzyme conjugate AEC, and reacts specifically with renin in the isolated plasma sample at a controlled temperature to generate an on-chip optically readable signal related to renin activity. The main body 1 of the reading device is internally equipped with a communication module 3, a multi-channel optical detection module 4, and a temperature control module 6. The communication module 3 is used for data transmission. The multi-channel optical detection module 4 is equipped with a data processing unit 5, which is used to analyze the detection signals corresponding to PRA, aldosterone, MN, NMN, and VMA, and output the in vitro quantitative intermediate results and quality control status of the in vitro sample. The temperature control module 6 is equipped with a temperature control unit 7 and an optical signal reading module 8. A microfluidic channel 9 is provided above the optical signal reading module 8. The microfluidic channel 9 is directly opposite the chip slot port 2, and the chip body 21 can be inserted into the microfluidic channel 9 through the chip slot port 2.

[0030] Specifically, the main body of the reading device 1 is a handheld dry chemical analyzer with an overall size of 15cm×8cm×4cm and a weight of <500g. It is highly portable and can be adapted to various testing scenarios such as bedside and grassroots communities. As the core terminal of the integrated testing system, it can achieve seamless docking with the microfluidic chip.

[0031] Chip slot 2 adopts a foolproof design, which can realize quick chip insertion and removal. The equipment can automatically identify the detection items and avoid operational errors.

[0032] The chip body 21 is a medical-grade microfluidic integrated compact structure, with overall dimensions of 5cm × 3cm × 0.5cm. It is made of medical-grade PMMA / COC polymer material, suitable for large-scale injection molding production, reducing manufacturing costs. The chip integrates lyophilized core reagents, connecting various functional areas through microchannels to achieve fully automated closed-loop processing from sample pretreatment to detection reaction, requiring no manual intervention. The microchannels are 50-100μm wide and 30-50μm deep, employing a shallow channel design driven by capillary forces to achieve autonomous sample flow. The chip's optical detection window uses a high-transmittance material with a transmittance >95%, ensuring lossless signal acquisition.

[0033] The sample loading area 22 supports micro-volume loading of whole blood / urine (whole blood ≤50μL, urine ≤100μL), and has separate blood loading inlet and urine loading inlet; hydrophobic barriers, capillary valves or check valves can be installed downstream of each inlet to achieve autonomous sample loading by relying on capillary force or negative pressure, while preventing cross-flow of different sample matrices.

[0034] The temperature control module 6 is equipped with a semiconductor cooling / heating component, with a temperature control range of 4-50℃ and a temperature control accuracy of ±0.5℃; the reading device provides external environmental temperature control, and the chip's built-in module provides precise temperature control of the reaction chamber. Dual temperature control ensures the stability of the detection reaction temperature.

[0035] The multi-channel optical detection module 4 is equipped with a multi-wavelength LED excitation source and a PMT / CMOS high-sensitivity detector, which can simultaneously detect multiple signals such as ultraviolet-visible absorption, fluorescence, and time-resolved fluorescence. The whole chip signal scanning time is less than 30 seconds, resulting in high detection efficiency.

[0036] Data processing unit 5 incorporates a high-performance microprocessor and storage module, and is equipped with core pre-built algorithms for fully automated processing, specifically including: (1) Signal preprocessing: baseline subtraction (to eliminate background interference), temperature compensation (to correct the effects of temperature fluctuations), reagent batch calibration (to eliminate reagent batch differences), and abnormal signal identification (to filter out noise). (2) Quality control algorithm: Westgard multi-rule quality control, using 1-3s and 2-2s core rules, automatically determines whether the quality control is qualified; (3) Concentration quantification: The main algorithm uses four-parameter Logistic fitting (using the core equation y=A+(BA) / (1+(x / C)^D) to adapt to the immunoquantitative detection of PRA, aldosterone, MN and NMN), and the auxiliary algorithm uses linear regression, piecewise fitting (adapted to VMA wide concentration detection) and table lookup interpolation (for backup when fitting is abnormal). (4) Result Output: Automatically calculates the ARR ratio (aldosterone concentration ÷ PRA concentration) and outputs VMA correction results, quality control status, and sample validity prompts; the above results are intermediate results of in vitro testing and do not directly form disease diagnosis conclusions. Execution Flow: Initialization → Signal Acquisition → Preprocessing → Quality Control Judgment → Concentration Conversion → Result Output.

[0037] The touchscreen 11 can directly display structured test reports; the communication module 3 supports Bluetooth 5.0 / Wi-Fi and can seamlessly connect with the hospital's LIS / HIS system to realize the informatization and systematic management of test data, making it easy for clinicians to quickly retrieve the data.

[0038] like Figure 2 As shown, the plasma separation sub-region 23 integrates an asymmetric PES / PVDF gradient filter membrane, and the urine concentration and enrichment sub-region 24 integrates a C18 solid-phase extraction microcolumn. The sample loading region 22 and / or its downstream microchannels may be equipped with hydrophobic barriers, capillary valves, check valves, or perforated membrane valves to prevent cross-flow between blood and urine samples.

[0039] Specifically, the pore size of the plasma separation sub-region 23 is 5μm→0.22μm, the membrane porosity is >80%, and the plasma separation of whole blood samples can be automatically completed within 2-3 minutes, with a plasma recovery rate of >80% and a cell removal rate of >99%.

[0040] The packing material in the urine concentration enrichment sub-region 24 has a particle size of 40-60μm, which can achieve 10-20 times enrichment of VMA in urine, significantly improving the detection sensitivity of VMA and meeting the needs of low concentration sample detection.

[0041] like Figure 3 As shown, the PRA temperature-controlled reaction zone 25 incorporates a thin-film heater, a temperature sensor, and a PID temperature control module. The constant temperature range of the PRA temperature-controlled reaction zone 25 is 35℃-39℃. The aldosterone / MN / NMN homogeneous reaction zone 26 has three independent parallel reaction chambers. The aldosterone / MN / NMN homogeneous reaction zone 26 is used for pre-positioning lyophilized specific antibodies and enzyme markers. The constant temperature range of the PRA temperature-controlled reaction zone 25 is preferably maintained at 37℃±0.5℃.

[0042] Specifically, the PRA temperature-controlled reaction zone 25 is the on-chip reaction chamber of the AEC system, pre-placed with lyophilized AEC microspheres or lyophilized reagent layers; after the ex vivo plasma sample enters this zone, the AEC is reconstituted under the action of the sample liquid, and undergoes a specific enzymatic reaction with renin in the sample under the constant temperature conditions of 37℃±0.5℃ provided by the temperature control module 6, causing a measurable change in reporter enzyme activity or reporter signal, and then the absorbance, fluorescence or time-resolved fluorescence signal generated by the reporter enzyme is collected through the corresponding optical detection window.

[0043] The homogeneous reaction zone 26 for aldosterone / MN / NMN is pre-loaded with lyophilized specific antibodies and enzyme markers, respectively. After the sample enters, a liquid-phase competitive immune reaction can occur simultaneously, enabling the parallel detection of the three.

[0044] like Figure 2 As shown, the VMA chromatography detection zone 27 integrates a quantum dot immunochromatographic test strip, and is used for chromatographic reaction and fluorescent signal labeling. An elution buffer chamber or delayed release structure can be provided between the urine concentration enrichment zone 24 and the VMA chromatography detection zone 27, allowing the enriched VMA to complete elution and buffer condition adjustment before entering the chromatography detection zone.

[0045] Specifically, VMA chromatography detection zone 27: After enrichment, the sample automatically enters this zone to complete the chromatography reaction and fluorescence signal labeling.

[0046] like Figure 2 As shown, the waste liquid area 28 is a sealed collection chamber design, which is used for the sealed collection of waste liquid after testing.

[0047] Specifically, the waste liquid area 28 can be sealed and collected after testing to prevent biological contamination and ensure the biosafety of the testing operation.

[0048] like Figure 2 As shown, the chip body 21 contains lyophilized reagents, and the chip body 21 is connected to various functional areas through microchannels.

[0049] Specifically, the chip body 21 has a shelf life of >18 months when stored at room temperature and sealed, and >24 months when stored at 4℃. After sample addition, the signal stability of the reaction system at room temperature is >4 hours with no significant signal attenuation, which is suitable for the operational needs of different clinical testing scenarios.

[0050] like Figure 3 As shown, the multi-channel optical detection module 4 is equipped with a multi-wavelength LED excitation source and a PMT / CMOS detector. The data processing unit 5 has a built-in microprocessor for baseline subtraction, temperature compensation, chip batch number calibration, calibration curve conversion, quality control judgment and abnormal signal identification. The temperature control module 6 is equipped with semiconductor cooling and heating components. The power module 13 is provided at the end of the main body 1 of the reading device.

[0051] Specifically, 1. Core Detection Principle: (1) Plasma renin activity (PRA): An angiotensinogen fragment-enzyme conjugate (AEC) was designed as an artificial specific substrate. This AEC consists of a polypeptide fragment containing a human angiotensinogen-derived renin recognition / cleavage site (the cleavage site or a conserved variant site between leucine at position 10 and valine at position 11), a flexible linker, and a reporter enzyme (glucose-6-phosphate dehydrogenase, G6PDH). Renin in the sample can specifically cleave the AEC, releasing biologically active G6PDH, which catalyzes the substrate reaction and generates a detectable signal. PRA quantification can be achieved by detecting the signal intensity. This method transforms renin activity detection into reporter enzyme activity detection, achieving rapid one-step detection and reducing the PRA detection time from the traditional 4-6 hours to 15-20 minutes.

[0052] (2) Homogeneous reaction zone 26 for aldosterone / MN / NMN: Homogeneous enzyme immunoassay is used, with specific labeling enzymes (G6PDH, malate dehydrogenase MDH, and G6PDH coupled with a time-resolved fluorescent probe) paired with the three biomarkers respectively. In the liquid phase system, the labeling enzyme-coupled antigen competes with the free antigen in the sample for a limited number of specific antibodies. The antigen-antibody binding reaction inhibits the biological activity of the labeling enzyme through steric hindrance. By detecting the enzyme catalytic signal at different wavelengths, the three biomarkers are simultaneously quantified using non-interfering optical signal channels, enabling parallel and interference-free quantitative detection of the three biomarkers within 10 minutes.

[0053] (3) VMA chromatography detection zone 27: Quantum dot fluorescence immunochromatography is used to detect the VMA using Eu. 3+ Chelated quantum dots, as high-brightness and high-photostability fluorescent markers, enable quantitative detection of VMA using a competitive assay. Simultaneously, to address the challenges of traditional 24-hour urine sample collection, creatinine is used as an endogenous marker of urine volume for correction. An algorithm for calculating 24-hour excretion from random urine samples is employed. By simultaneously detecting urinary creatinine concentration and combining it with clinical information such as patient gender, age, and weight, the total 24-hour VMA excretion is estimated, reducing the VMA detection time to 10-15 minutes.

[0054] 2. Reagent system design: (1) Angiotensinogen fragment-enzyme conjugate (AEC): composed of 1-14 amino acids from the N-terminus of angiotensinogen (containing Leu 10 -Leu 11 Cutting site), flexible connecting arm [(GGGGS)] 2-3 [or PEG] and reporter enzyme (G6PDH) are directionally coupled via a heterobifunctional cross-linking agent SMCC. This design ensures efficient recovery of enzyme activity and effective amplification of the detection signal.

[0055] (2) Homogeneous enzyme immunoassay reagents: Customized aldosterone-G6PDH (detects NADH ultraviolet absorbance signal at 340nm), MN-MDH (detects NADH fluorescence signal excitation at 340nm / emission at 460nm), and NMN-G6PDH (detects time-resolved fluorescence at 615nm). The detection signal wavelengths of the three reagents do not overlap, achieving synchronous quantification of the three signals without interference.

[0056] (3) Quantum dot immunochromatographic reagent: Eu 3+ Chelate fluorescent materials (excitation wavelength 100–400 nm) are used to label anti-VMA antibodies. These fluorescent materials have good photostability, high fluorescence intensity, and detection sensitivity can reach the pg / mL level.

[0057] (4) Calibrators and quality control products: The product is equipped with gradient concentration calibrators for five biomarkers (covering the linear range of clinical testing), as well as quality control products at three concentration levels (low, medium and high) to achieve quality control of the testing process and ensure the accuracy and repeatability of the test results.

[0058] A detection method for a microfluidic chip-based device for detecting five biomarkers of secondary hypertension, comprising the following steps: S1. Add the isolated blood sample to the blood sampling inlet in the sample application area 22, and add the isolated urine sample to the urine sampling inlet in the sample application area 22, so that the two enter the mutually isolated blood branch and urine branch respectively. S2, the sample loading area 22 relies on capillary force to drive sample flow, and performs plasma separation, urine VMA enrichment, matrix removal and buffer adjustment respectively; S3. The pre-treated sample is automatically distributed to each reaction zone of the aldosterone / MN / NMN homogeneous reaction zone 26 through a microchannel. Under a temperature-controlled environment of 37℃, the PRA temperature-controlled reaction zone 25, the aldosterone / MN / NMN homogeneous reaction zone 26 and the VMA chromatography detection zone 27 are simultaneously activated to carry out PRA enzymatic reaction, aldosterone / MN / NMN homogeneous immunochromatographic reaction and VMA fluorescence immunochromatographic reaction. S4. Insert the completed reaction chip body 21 into the microfluidic channel 9 through the chip slot 2. The device automatically collects the detection signals of each reaction zone for signal analysis, concentration calculation and quality control judgment, and generates a structured detection report containing the in vitro quantitative results of five markers, quality control status, ARR ratio and optional VMA correction results. The above results serve as intermediate results of in vitro detection for subsequent clinical judgment or laboratory evaluation reference, and do not directly form a disease diagnosis conclusion. S5. The test results are displayed on the touch screen 11 and simultaneously uploaded to the hospital's LIS / HIS system via the communication module 3 for clinical doctors to view.

[0059] Specifically, 1. A method for estimating 24-hour random urinary VMA excretion based on urinary creatinine correction: To address the challenge of 24-hour urine sample collection, creatinine was used as an endogenous marker of urine volume for correction. A VMA 24-hour excretion estimation algorithm based on urinary creatinine correction was employed, and the calculation formula is as follows: 24h VMA excretion (mg / 24h) = random urine VMA concentration (mg / L) × [expected 24h creatinine excretion (mg) / random urine creatinine concentration (mg / L)]; The expected 24-hour creatinine excretion is calculated based on the patient's gender and weight (20-25 mg / kg / 24h for males and 15-20 mg / kg / 24h for females). The accuracy of this estimation method can be evaluated in subsequent methodological validation by comparing it with the results of conventional 24-hour urine sample testing; the output is a corrected result for isolated urine testing and should not be used as an independent diagnosis.

[0060] 2. Detection chip characterization: (1) Physical characterization: The overall size of the chip is 5cm×3cm×0.5cm; the microchannel width is 50-100μm and the depth is 30-50μm. It adopts a shallow channel design driven by capillary force to realize the autonomous flow of samples; the plasma separation membrane is an asymmetric PES / PVDF membrane with a pore size gradient of 5μm→0.22μm and a membrane porosity of >80%; the urine enrichment area is equipped with C18 solid phase extraction packing material with a packing particle size of 40-60μm; the chip optical detection window is made of high transmittance material with a transmittance of >95% to ensure lossless signal acquisition.

[0061] (2) Reagent stability: The chip contains lyophilized reagents, which have a shelf life of more than 18 months when stored at room temperature and more than 24 months when stored at 4°C. After sample addition, the signal stability of the reaction system at room temperature is more than 4 hours with no obvious signal attenuation, which is suitable for the operation requirements of different clinical testing scenarios.

[0062] (3) Repeatability and precision: In subsequent method validation, the repeatability and stability of this device can be evaluated by intra-batch repeatability, inter-batch repeatability and test results under different operator conditions. The specific CV value shall be based on the actual experimental data.

[0063] (4) Sample compatibility: It is compatible with four sample types: whole blood, plasma, serum and urine; it is compatible with two commonly used clinical anticoagulants: EDTA-K2 and lithium heparin; it can further evaluate the influence of common clinical interfering substances such as free hemoglobin and triglycerides on the test results. The specific bias range is subject to the actual experimental data.

[0064] 3. Characteristics of this reading device: (1) Optical performance: The equipment is equipped with a multi-wavelength excitation light source and a high-sensitivity optical detection unit, which can realize the acquisition and recognition of various signals such as ultraviolet absorption, fluorescence, and time-resolved fluorescence, and can meet the detection sensitivity requirements of different markers.

[0065] (2) Temperature control performance: The equipment is equipped with high-precision temperature control components, which can achieve stable temperature control over a wide temperature range, and the temperature rise response is rapid, providing reliable temperature protection for rapid response.

[0066] (3) Data processing performance: The equipment has high-speed signal acquisition capability, supports a variety of commonly used fitting algorithms for concentration calculation, and can realize local storage and traceability management of detection data.

[0067] (4) Communication and display: The equipment is equipped with a touch display unit, which can intuitively display the detection process and results; it is equipped with a wireless communication module, which can connect with the hospital information system to realize the real-time transmission and management of detection information.

[0068] 4. Methodological characterization of the detection: (1) Linear range: The detection range design of the present invention can cover the concentration range required for routine clinical testing, and the detection signal and concentration have a good correspondence within the designed range.

[0069] (2) Detection sensitivity: Through system amplification and chip enrichment design, the present invention can achieve effective detection of low concentration samples, which can meet the clinical demand for high sensitivity detection.

[0070] (3) Specificity: The present invention adopts a specific recognition system, which can reduce the interference of structurally similar substances and has high detection specificity.

[0071] (4) Recovery rate: The sample pretreatment and reaction system of the present invention is reasonably designed, which can make the detection results meet the general recovery rate requirements in the field of clinical in vitro diagnostics.

[0072] (5) Methodological comparison: The detection principle of this invention is consistent with the commonly used clinical reference method. After reasonable verification, it can achieve good correlation and accuracy with the reference method and meet the requirements of clinical use.

[0073] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip-based device for detecting five biomarkers of secondary hypertension, comprising a reading device body (1), a chip slot (2), and a chip body (21), characterized in that: The main body (1) of the reading device is provided with a chip slot (2), a touch screen (11) and function buttons (12). The chip slot (2) is provided with a chip body (21). The chip body (21) is provided with a sample loading area (22), a plasma separation sub-area (23), a urine concentration and enrichment sub-area (24), a PRA temperature-controlled reaction area (25), an aldosterone / MN / NMN homogeneous reaction area (26), a VMA chromatography detection area (27) and a waste liquid area (28). The sample loading area (22) is used for loading isolated blood samples and isolated urine samples, and is provided with separate blood loading inlets and urine loading inlets. The blood loading inlet is connected to the plasma separation sub-area. Sub-region (23) is connected to the PRA temperature-controlled reaction region (25) and the aldosterone / MN / NMN homogeneous reaction region (26), respectively. The urine sample inlet is connected to the VMA chromatography detection region (27) via the urine concentration and enrichment sub-region (24). The plasma separation sub-region (23) is used for plasma separation of whole blood samples. The urine concentration and enrichment sub-region (24) is used for 10-20 times enrichment of VMA in urine, matrix removal, and buffer condition adjustment. The PRA temperature-controlled reaction region (25) is used to contain the pre-placed angiotensinogen fragment-enzyme conjugate AEC, and reacts specifically with renin in the isolated plasma sample at a controlled temperature to generate an on-chip optically readable signal related to renin activity. The main body (1) of the reading device is equipped with a communication module (3), a multi-channel optical detection module (4) and a temperature control module (6). The communication module (3) is used for data transmission. The multi-channel optical detection module (4) is equipped with a data processing unit (5). The data processing unit (5) is used to analyze the detection signals corresponding to PRA, aldosterone, MN, NMN and VMA, and output the in vitro quantitative intermediate results and quality control status of the in vitro sample. The temperature control module (6) is equipped with a temperature control unit (7) and a light signal reading module (8). A microfluidic channel (9) is provided above the light signal reading module (8). The microfluidic channel (9) is directly opposite the chip slot (2), and the chip body (21) can be inserted into the microfluidic channel (9) through the chip slot (2).

2. The device for detecting five biomarkers of secondary hypertension based on a microfluidic chip according to claim 1, characterized in that: The plasma separation sub-region (23) integrates an asymmetric PES / PVDF gradient filter membrane, and the urine concentration and enrichment sub-region (24) integrates a C18 solid-phase extraction microcolumn.

3. The device for detecting five biomarkers of secondary hypertension based on a microfluidic chip according to claim 1, characterized in that: The PRA temperature-controlled reaction zone (25) has a built-in thin film heater, temperature sensor and PID temperature control module. The constant temperature range of the PRA temperature-controlled reaction zone (25) is 35℃-39℃. The aldosterone / MN / NMN homogeneous reaction zone (26) is provided with three independent parallel reaction chambers. The aldosterone / MN / NMN homogeneous reaction zone (26) is used to pre-place freeze-dried specific antibodies and enzyme markers.

4. The device for detecting five biomarkers of secondary hypertension based on a microfluidic chip according to claim 1, characterized in that: The VMA chromatography detection area (27) integrates a quantum dot immunochromatographic test strip, and the VMA chromatography detection area (27) is used for chromatography reaction and fluorescent signal labeling.

5. The device for detecting five biomarkers of secondary hypertension based on a microfluidic chip according to claim 1, characterized in that: The waste liquid area (28) is a sealed collection chamber design, which is used to seal and collect the waste liquid after testing.

6. The device for detecting five biomarkers of secondary hypertension based on a microfluidic chip according to claim 1, characterized in that: The chip body (21) contains lyophilized reagents, and the chip body (21) is connected to each functional area through microchannels.

7. The device and method for detecting five biomarkers of secondary hypertension based on a microfluidic chip according to claim 1, characterized in that: The multi-channel optical detection module (4) is equipped with a multi-wavelength LED excitation source and a PMT / CMOS detector. The data processing unit (5) has a built-in microprocessor for baseline subtraction, temperature compensation, chip batch number calibration, calibration curve conversion, quality control judgment and abnormal signal identification. The temperature control module (6) is equipped with semiconductor cooling and heating components. The end of the main body (1) of the reading device is provided with a power module (13).

8. A detection method for a microfluidic chip-based detection device for five biomarkers of secondary hypertension, used in any one of claims 1-7, characterized in that, The method includes the following steps: S1. Add the isolated blood sample to the blood sampling inlet in the sample application area (22) and add the isolated urine sample to the urine sampling inlet in the sample application area (22) so that the two enter the mutually isolated blood branch and urine branch respectively. S2, the sample loading area (22) relies on capillary force to drive the sample flow, and performs plasma separation, urine VMA enrichment, matrix removal and buffer adjustment respectively; S3. After pretreatment, the sample is automatically distributed to each reaction zone of the aldosterone / MN / NMN homogeneous reaction zone (26) through a microchannel. Under a temperature-controlled environment of 37°C, the PRA temperature-controlled reaction zone (25), the aldosterone / MN / NMN homogeneous reaction zone (26) and the VMA chromatography detection zone (27) are started simultaneously to carry out PRA enzymatic reaction, aldosterone / MN / NMN homogeneous immunoassay and VMA fluorescence immunochromatographic reaction. S4. Insert the completed reaction chip body (21) into the microfluidic channel (9) through the chip slot (2). The device automatically collects the detection signals of each reaction zone to perform signal analysis, concentration calculation and quality control judgment, and generates a structured detection report containing the in vitro quantitative results of five markers, quality control status, ARR ratio and optional VMA correction results. S5. The test results are displayed on the touch screen (11) and simultaneously uploaded to the hospital's LIS / HIS system via the communication module (3) for clinical doctors to view.

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