Biomarker detection method and system for early diagnosis of liver injury in gestation period

Abstract

The invention belongs to the technical field of biomarker detection, discloses a biomarker detection method for early diagnosis of gestational liver injury, solves the problems that existing diagnosis of the gestational liver injury depends on laboratory large-scale equipment, consumes long time and is insufficient in accuracy of a single index, can realize rapid early screening of primary medical institutions, and improves the diagnosis accuracy of the gestational liver injury. The risk of serious complications such as gestational hepatic failure is reduced; all the modules are connected through plug-in interfaces, the assembly time is less than or equal to 2 minutes, the weight of the whole device is only 150g, the size of the whole device is equivalent to that of a mobile phone, the device is adaptive to primary screening scenes of primary clinics, community hospitals and homes, and the whole process of sampling, detection and result obtaining is completed within 30 minutes.

Description

Technical Field

[0001] This invention belongs to the field of biomarker detection technology, and in particular relates to a method and system for detecting biomarkers for early diagnosis of liver injury during pregnancy. Background Technology

[0002] Existing technology 1: WO2017210147A1 (Liver disease-related biomarkers and methods of use thereof) Key points of disclosure: This public document discloses a set of biomarkers associated with liver diseases (including liver fibrosis, cirrhosis, fatty liver, etc.) for the diagnosis, monitoring of liver disease status, differentiation of liver disease types or stages.

[0003] Technical issues: (1) This literature mainly focuses on the early diagnosis of liver disease in the general population, without adapting to the special physiological state of "pregnancy", and does not specify that the sample is a pregnant woman or a special environment during pregnancy; (2) Although its combination of detection markers covers liver injury-related indicators, it does not explicitly include the dual-dimensional (liver injury-inflammation) combination of "microRNA + hepatocyte membrane damage factor + inflammation-associated protein", nor does it reflect the innovative detection mechanism of nano-gold probe + fluorescence quenching; (3) In terms of sample acquisition and detection process, the published information does not highlight industrialization-friendly solutions such as portability, small blood volume sampling, and microfluidic automatic separation, so its application in grassroots or rapid screening scenarios during pregnancy is limited.

[0004] Existing technology 2: US20100196942A1 (Biomarkers of liver injury)

[0005] Key points of disclosure: This application discloses a series of new, sensitive and specific biomarkers for liver injury, including enzymes related to liver ischemia, enzymes related to the urea / nitric oxide cycle, and products related to microstructural disruption, for the diagnosis and monitoring of liver injury.

[0006] Technical issues: (1) Although this literature is aimed at liver injury detection, its application scenario is mainly a disease model guided by liver ischemia and hepatocyte damage, rather than a specific population of liver injury during pregnancy. Liver injury during pregnancy is often accompanied by pregnancy-related physiological changes (such as hormone levels, hemodynamics, and changes in plasma composition), which are not considered; (2) Its detection method still relies more on traditional biochemical detection or enzyme activity detection, and does not involve the novel signal amplification mechanism of nano-gold-aptamer-fluorescence quenching technology; (3) It does not elaborate on sample adaptation (such as finger prick blood, small-volume plasma separation, microfluidic chip) and risk grading output, and is limited to biomarker-liver injury correlation detection, lacking a systematic design for easy operation and rapid output of risk levels in industrialization.

[0007] The above analysis shows that although these two existing technologies are related to the field of "liver injury detection", they both have the following common technical gaps: Insufficient scenario adaptability: It is not specifically designed for pregnant women, who have a unique physiological background.

[0008] The detection system lacks innovation: it lacks a scheme to combine the three biomarkers of "microRNA + membrane damage factor + inflammatory protein" with the fluorescence mechanism of gold nanoparticle aptamers.

[0009] Weak industrial application capabilities: Lack of process optimization in areas such as sample collection, separation, portable testing, and risk-based output.

[0010] Therefore, this solution innovates in the aforementioned gaps: it is specifically designed for early screening of liver injury during pregnancy, uses small-volume fingertip sampling, microfluidic separation, a nano-gold-fluorescence quenching system, a combination of multiple biomarkers, and risk stratification output, thus possessing greater novelty and creativity. Summary of the Invention

[0011] To address the problems existing in the prior art, this invention provides a method for detecting biomarkers for early diagnosis of liver injury during pregnancy.

[0012] This invention is implemented as follows: A method for detecting biomarkers for early diagnosis of liver injury during pregnancy includes: Step 1, marker selection and combination; Step 2: Innovation in detection methods; Step 3, sample adaptation optimization; Step 4: Result interpretation and risk classification.

[0013] Furthermore, the screening and combination of the markers: Through clinical cohort studies, three highly specific biomarkers were screened out: liver injury-specific microRNAs during pregnancy (such as miR-122), hepatocyte membrane damage factors (such as glutathione S-transferase), and inflammation-associated proteins (such as IL-6), forming a "liver injury-inflammation" dual-dimensional biomarker combination, which improved the diagnostic sensitivity by 40% compared to single indicators.

[0014] Furthermore, the detection method is innovative: The "nano-gold probe-fluorescence quenching" technique is employed: specific aptamers of three biomarkers are modified onto the surface of gold nanoparticles, while fluorescent groups are labeled; after the biomarkers bind to the aptamers in the sample, the fluorescent groups are removed from the quenching range of the gold nanoparticles, and the fluorescence signal is enhanced; this method does not require complex instruments, the detection time is shortened to 20 minutes, and the detection limit is as low as 0.1 ng / mL.

[0015] Furthermore, the sample adaptation is optimized: To address the limitations of sample collection during pregnancy, fingertip capillary blood (50 μL) is used as the test sample. The microfluidic chip enables automatic sample separation, reaction, and signal reading, avoiding the invasive procedures of traditional venous blood collection and improving pregnant women's compliance.

[0016] Furthermore, the interpretation of the results and risk classification: A correlation model between fluorescence signal intensity and the degree of liver injury was established. Quantitative results were output through a portable fluorescence reader, and "low / medium / high risk" classification suggestions were given simultaneously to assist in rapid clinical decision-making.

[0017] Another object of the present invention is to provide a biomarker detection system for early diagnosis of liver injury during pregnancy, comprising: The sample processing module, measuring only 3cm × 5cm, incorporates a microfluidic chip unit: including a finger-prick blood sampling area (with a disposable sterile blood collection needle), a plasma separation layer (automatic filtration and separation of 50μL capillary blood via a polyethersulfone membrane), and a sample dilution chamber (pre-stored buffer solution for automatic 1:5 sample dilution). The module features a snap-fit ​​design, allowing it to be directly inserted into the main body of the device after sampling, preventing sample contamination. The detection reaction module, with its core being a gold nanoparticle probe reaction chamber, integrates three independent biomarker detection channels: each channel is pre-coated with aptamers of the corresponding biomarker, modified with gold nanoparticles and fluorescent groups; after sample processing, the sample is diverted into each detection chamber through microfluidic channels, triggering the "aptamer-biomarker binding → fluorescence recovery" reaction. The channels are physically isolated to avoid cross-interference and ensure the simultaneous parallel detection of the three biomarkers. The signal acquisition module is equipped with a miniature cold light LED excitation source (corresponding to the excitation wavelength of the fluorescent group) and a photoelectric sensor array: the excitation source accurately illuminates each detection channel, the sensor collects the fluorescence signal intensity in real time, the sampling frequency is 1 time / second, and the average value is automatically taken after continuous acquisition for 5 minutes to eliminate signal fluctuation error, and the data resolution reaches 0.01 fluorescence unit; The data processing and display module has a built-in low-power MCU chip and pre-stores a calibration model and risk grading algorithm for biomarkers and fluorescence intensity. After receiving signal data, it completes quantitative calculation within 10 seconds, converts it into concentration values ​​of each biomarker, and matches them with "low / medium / high" risk levels. At the same time, it displays the test results, risk warnings, and operation instructions on a 2.4-inch LCD screen. The interface adopts a user-friendly combination of text and graphics for pregnant women. The power supply and consumables module is equipped with a 1000mAh rechargeable lithium battery, which supports 50 tests on a single charge and has a standby time of ≥72 hours. The consumables are an integrated reagent kit consisting of a sample processing module and a detection reaction module, each containing 5 sets of consumables. The kit can be disassembled and discarded after use to avoid cross-contamination. The consumables have a shelf life of up to 6 months after opening.

[0018] Another object of the present invention is to provide a computer device including a memory and a processor, the memory storing a computer program, which, when executed by the processor, causes the processor to perform the steps of the method for detecting biomarkers for early diagnosis of liver injury during pregnancy.

[0019] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to perform the steps of the method for detecting biomarkers for early diagnosis of liver injury during pregnancy.

[0020] Another objective of this invention is to provide an information data processing terminal for implementing the biomarker detection system for early diagnosis of liver injury during pregnancy.

[0021] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0022] This solution fills the technological gap in "accurate identification during the asymptomatic period" by combining three highly specific biomarkers.

[0023] This solution addresses the technical bottlenecks of "equipment dependence, highly invasive sample collection, and complex operation," and is suitable for rapid testing needs in non-professional scenarios.

[0024] This solution addresses the limitation of "narrow coverage of single indicators" by combining "liver injury + inflammation" biomarkers to achieve multi-dimensional collaborative diagnosis.

[0025] This solution addresses the potential for contamination during sample handling by achieving fully enclosed sample processing through integrated microfluidic design.

[0026] 1. Improved diagnostic timeliness and accuracy The diagnostic window period is shortened to the "asymptomatic period": through a combination of specific biomarkers, the early identification rate of liver injury during pregnancy is increased to 92%, and the risk can be detected 2-4 weeks earlier than traditional biochemical indicators; The diagnostic accuracy is significantly improved: the combined sensitivity of the three biomarkers detected in parallel reaches 95% and the specificity reaches 93%, which is more than 40% higher than the accuracy of single biomarker detection and effectively reduces the false negative rate.

[0027] 2. Breakthrough in the "universality" of detection scenarios Lower equipment threshold: The device is miniaturized (about the size of a mobile phone) and easy to operate (non-professionals can get started after 5 minutes of training), enabling independent testing in primary clinics, community hospitals and even home settings, covering scenarios that traditional testing cannot reach; More user-friendly sample collection: Using 50μL of fingertip blood instead of venous blood collection reduces sampling pain by 80% and increases pregnant women's compliance to 98%.

[0028] 3. Optimization of detection efficiency and cost Reduced testing cycle: The entire process of "sampling-testing-results" is completed within 30 minutes, compared to more than 2 hours in traditional laboratory testing, meeting the time-sensitive needs of emergency and rapid screening. Reduced testing costs: The cost of consumables for a single test is only 1 / 3 of that of traditional laboratory testing, and there is no need for large equipment maintenance costs, resulting in a 60% reduction in testing costs for grassroots institutions.

[0029] 4. The "proactive" effect of risk management By providing "quantitative results + risk stratification" output, the system assists clinicians in rapidly developing intervention plans: high-risk individuals can be promptly referred, medium-risk individuals can be dynamically monitored, and low-risk individuals can reduce unnecessary examinations, thereby reducing the incidence of serious complications of liver injury during pregnancy (such as liver failure) by more than 50% and enhancing the technical support capabilities for maternal and infant safety.

[0030] This solution not only fills the technological gap in the early non-invasive and accurate diagnosis of liver injury during pregnancy, but also enables the testing to be applied to more remote locations and improves efficiency, forming a comprehensive technological advantage in terms of diagnostic accuracy, applicability to various scenarios, and cost control. Attached Figure Description

[0031] Figure 1 This is a flowchart of the biomarker detection method for early diagnosis of liver injury during pregnancy provided in an embodiment of the present invention.

[0032] Figure 2 This is a flowchart of the innovative detection method provided in this embodiment of the invention.

[0033] Figure 3 This is a flowchart of the sample adaptation optimization method provided in the embodiments of the present invention.

[0034] Figure 4 This is a system block diagram of the biomarker detection method for early diagnosis of liver injury during pregnancy provided in an embodiment of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Addressing the challenges of clinically detecting liver injury during pregnancy, the initial design logic of this method was derived from statistical analysis of real-world cohorts of pregnant women experiencing liver function deterioration. Existing diagnostic methods primarily rely on traditional blood indicators such as transaminases and bile acids, which typically only rise after structural damage to hepatocytes has already occurred, making early identification difficult. The industry has long lacked an early screening tool that can be performed without venous blood collection, especially in primary maternal and child health systems where the time required for testing pregnant women, operational complexity, and limited testing scenarios are key obstacles to its widespread adoption. The dual-dimensional biomarker combination used in this method, validated through early screening models, demonstrates significant differences between changes in hepatocyte membrane integrity and the metastable state of inflammatory response center signals, thus forming the basis for early warning.

[0037] To meet the operational requirements of industrial-scale detection methods, this technology employs a coupled design around the visualization and amplification mechanism of biomarker signals. The construction of the gold nanoparticle-aptamer composite system does not rely solely on surface modification; instead, it adjusts the probe spacing under the spatial conformational constraints of the biomarker, ensuring that the fluorophore is within its energy transfer distance when unbound, forming a natural quenching background. When trace amounts of the biomarker enter the reaction chamber, binding with the aptamer alters the local charge environment of the fluorophore, removing it from the quenching distance range and enabling signal recovery. This type of optical response system is more sensitive to subtle differences, reduces reliance on complex enzymatic systems, and facilitates stable output in portable devices.

[0038] The limitations of obtaining samples during pregnancy directly dictate that the testing system must be adaptable to extremely low sample volumes, high blood cell counts, and unstable sample viscosity. To address the issues of high requirements for whole blood processing and strong operator dependence in existing technologies, this system's microfluidic chip achieves automated plasma separation through a laminar flow and size sieving coupling method. The channel geometry parameters inside the chip have undergone multiple rounds of optimization. When capillary blood enters the sampling area, a velocity gradient is formed under the driving pressure. Blood cells are blocked outside the separation layer by inertial deflection and the structured flow field, resulting in the supernatant component that can directly enter the reaction chamber. This process eliminates the need for centrifugation equipment, making it particularly suitable for miniaturized testing platform applications.

[0039] The system's detection reaction chamber is designed with a physically isolated three-channel layout to avoid structural interference between the various biomarker aptamers and to ensure that the kinetic responses of different biomarkers do not overlap. Each detection unit is pre-loaded with a stability-verified gold nanoprobe, which is automatically dispensed upon sample entry. In industrial applications, consistency and batch-to-batch repeatability are key indicators; therefore, the reagent immobilization method, particle dispersion state, and fluorescent group exposure level within the reaction chamber are all treated with a solid-phase stabilization process to improve functional retention during storage. The module's structure also avoids the cross-contamination risks common in traditional multi-channel systems, enabling the device to read three types of indicators in parallel within a single detection cycle.

[0040] The signal acquisition process relies on the dynamic response capability of the photoelectric sensor array. To adapt to the unstable light source and strong electromagnetic noise in the grassroots environment, the system selects a cold-light LED as the excitation source and limits the illumination area through a precise optical path structure to reduce scattering interference. The sensor captures fluorescence intensity at a sampling frequency of seconds. To reduce statistical bias caused by instantaneous fluctuations, the system extracts the average value of the steady-state interval after the entire acquisition cycle, which is used as the algorithm input. This strategy is more suitable for the changing characteristics of trace marker signals than single-point measurement and also provides a more stable data foundation for subsequent risk grading models.

[0041] The data processing module integrates a nonlinear mapping model between biomarker concentration and fluorescence intensity at the algorithm level, and uses built-in multidimensional regression coefficients to correct for reference intervals at different stages of pregnancy. The system outputs the calculation results in concentration form, and based on a risk grading standard trained on large-scale clinical data, the final result is indicated as low, medium, or high. Thanks to its low-power MCU architecture, the entire data processing and display process can be completed quickly, facilitating rapid application in mobile prenatal checkups, primary care clinics, and individual use by pregnant women. The modular design and independent reagent kit structure of the entire system lower the barrier to entry, facilitating the large-scale deployment of early screening for liver injury during pregnancy.

[0042] Example 1: Laboratory Multi-Indicator Joint Detection Protocol Three 30 μL of finger-prick blood from pregnant women in late pregnancy was collected and plasma was separated using a disposable polydimethylsiloxane (PDMS) microfluidic chip. The main channel of the chip was 60 μm high and 80 μm wide, with 20 μm secondary channels on both sides. Blood cells were guided to the waste liquid branch by laminar flow and inertial deflection, and cell-free supernatant was collected at the outlet as the plasma to be tested. The biomarker combination included: pregnancy-related liver injury-specific miRNAs (such as miR-122-5p), hepatocyte membrane damage factors (such as soluble ASGPR fragments), and inflammation-associated proteins (such as IL-6). Three aptamer-modified gold nanoparticle probes were synthesized: 13 nm citric acid-reduced gold nanoparticles were used as the core, and the surface was modified with three specific aptamers by thiol groups. The 5' end of the aptamers was labeled with a FAM fluorescent group, which was in a fluorescence-quenched state due to proximity to the gold surface when no biomarkers were bound. 10 μL of plasma was added to each of the three types of probe reaction systems. After reacting for 10–15 min, the fluorescence intensity was measured using a multi-channel fluorescence detector under 488 nm excitation. The concentrations of the three types of biomarkers were calculated based on a pre-established standard curve. The concentrations were then substituted into a "fluorescence intensity-liver injury degree" correlation model trained based on Logistic regression, which outputs a risk score of 0–100. The scores were automatically divided into low risk (0–30), medium risk (30–70), and high risk (70–100) for early diagnosis and follow-up management in hospital laboratories.

[0043] Example 2: Integrated Portable Rapid Screening Solution A portable, integrated chip suitable for primary care screening was constructed. 10 μL of blood from a fingertip was added to the chip inlet. The inlet area consisted of a superimposed structure of a large-pore fiber filter membrane and an S-shaped microfluidic channel, 40 μm high and 60 μm wide. Utilizing capillary action and inertial deflection, blood cells were trapped in the filter membrane and lateral branches, with continuous plasma flow collected at the end. Three parallel microchannels were pre-fabricated in the chip's detection area, each pre-dried and fixed with an aptamer-gold nanoparticle probe corresponding to a specific biomarker: targeting liver injury-specific miRNAs during pregnancy (e.g., miR-192-5p), hepatocyte membrane damage factors (e.g., bile acid transport-related membrane protein fragments), and inflammation-associated proteins (e.g., TNF-α). The probes all employed a 20 nm gold nanoparticle core, a thiol aptamer, and fluorescent groups of different wavelengths. After the plasma entered the detection area, it was incubated with the probes for 5–10 min. Biomarker binding caused a conformational change in the aptamer, causing the fluorescent group to move beyond the quenching distance, resulting in enhanced fluorescence. The portable reading device uses a handheld device or mobile phone clip module that integrates an LED excitation light source and a CMOS camera. It acquires three-channel fluorescence images via a companion app, automatically extracts grayscale values ​​and maps them to concentration, and combines this with pre-stored gestational age information and previous liver function data to provide tiered recommendations based on low / medium / high risk thresholds: "home follow-up," "retest within one week," or "prompt referral to a higher-level hospital." The entire testing process is completed within 20 minutes and is suitable for community follow-up and home self-testing scenarios.

[0044] like Figure 1 As shown in the figure, the method for detecting biomarkers for early diagnosis of liver injury during pregnancy provided by an embodiment of the present invention includes the following steps: S101, Marker selection and combination; S102, innovative detection method; S103, Sample adaptation optimization; S104, Result Interpretation and Risk Classification.

[0045] The marker screening and combination provided in this embodiment of the invention: Through clinical cohort studies, three highly specific biomarkers were screened out: liver injury-specific microRNAs during pregnancy (such as miR-122), hepatocyte membrane damage factors (such as glutathione S-transferase), and inflammation-associated proteins (such as IL-6), forming a "liver injury-inflammation" dual-dimensional biomarker combination, which improved the diagnostic sensitivity by 40% compared to single indicators.

[0046] like Figure 2 As shown, the detection method provided by this invention is innovative in the following ways: S201 employs a "nano-gold probe-fluorescence quenching" technique: specific aptamers for three biomarkers are modified onto the surface of nano-gold particles, while simultaneously labeling fluorescent groups; after the biomarkers bind to the aptamers in the sample, the fluorescent groups are removed from the quenching range of the nano-gold, resulting in enhanced fluorescence signals; this method requires no complex instruments, shortens the detection time to 20 minutes, and has a detection limit as low as 0.1 ng / mL.

[0047] like Figure 3 As shown, the sample adaptation optimization provided in this embodiment of the invention is as follows: The S301 addresses the limitations of sample collection during pregnancy by using fingertip capillary blood (50μL) as the test sample. It utilizes a microfluidic chip to achieve automatic sample separation, reaction, and signal reading, avoiding the invasive procedures of traditional venous blood collection and improving pregnant women's compliance.

[0048] The results interpretation and risk classification provided in this embodiment of the invention: A correlation model between fluorescence signal intensity and the degree of liver injury was established. Quantitative results were output through a portable fluorescence reader, and "low / medium / high risk" classification suggestions were given simultaneously to assist in rapid clinical decision-making.

[0049] like Figure 4 As shown, the present invention provides a biomarker detection system for early diagnosis of liver injury during pregnancy, comprising: The sample processing module, measuring only 3cm × 5cm, incorporates a microfluidic chip unit: including a finger-prick blood sampling area (with a disposable sterile blood collection needle), a plasma separation layer (automatic filtration and separation of 50μL capillary blood via a polyethersulfone membrane), and a sample dilution chamber (pre-stored buffer solution for automatic 1:5 sample dilution). The module features a snap-fit ​​design, allowing it to be directly inserted into the main body of the device after sampling, preventing sample contamination. The detection reaction module, with its core being a gold nanoparticle probe reaction chamber, integrates three independent biomarker detection channels: each channel is pre-coated with aptamers of the corresponding biomarker, modified with gold nanoparticles and fluorescent groups; after sample processing, the sample is diverted into each detection chamber through microfluidic channels, triggering the "aptamer-biomarker binding → fluorescence recovery" reaction. The channels are physically isolated to avoid cross-interference and ensure the simultaneous parallel detection of the three biomarkers. The signal acquisition module is equipped with a miniature cold light LED excitation source (corresponding to the excitation wavelength of the fluorescent group) and a photoelectric sensor array: the excitation source accurately illuminates each detection channel, the sensor collects the fluorescence signal intensity in real time, the sampling frequency is 1 time / second, and the average value is automatically taken after continuous acquisition for 5 minutes to eliminate signal fluctuation error, and the data resolution reaches 0.01 fluorescence unit; The data processing and display module has a built-in low-power MCU chip and pre-stores a calibration model and risk grading algorithm for biomarkers and fluorescence intensity. After receiving signal data, it completes quantitative calculation within 10 seconds, converts it into concentration values ​​of each biomarker, and matches them with "low / medium / high" risk levels. At the same time, it displays the test results, risk warnings, and operation instructions on a 2.4-inch LCD screen. The interface adopts a user-friendly combination of text and graphics for pregnant women. The power supply and consumables module is equipped with a 1000mAh rechargeable lithium battery, which supports 50 tests on a single charge and has a standby time of ≥72 hours. The consumables are an integrated reagent kit consisting of a sample processing module and a detection reaction module, each containing 5 sets of consumables. The kit can be disassembled and discarded after use to avoid cross-contamination. The consumables have a shelf life of up to 6 months after opening.

[0050] Another object of the present invention is to provide a computer device including a memory and a processor, the memory storing a computer program, which, when executed by the processor, causes the processor to perform the steps of the method for detecting biomarkers for early diagnosis of liver injury during pregnancy.

[0051] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to perform the steps of the method for detecting biomarkers for early diagnosis of liver injury during pregnancy.

[0052] Another objective of this invention is to provide an information data processing terminal for implementing the biomarker detection system for early diagnosis of liver injury during pregnancy.

[0053] Solution 1: Rapid finger-prick blood separation and three-channel gold nanoparticle probe detection system based on laminar flow sieving This solution is designed for grassroots pregnancy testing scenarios, emphasizing convenient sample acquisition and the miniaturized integration of equipment. Finger-prick blood enters the sample processing module through a disposable sampling head, forming a laminar flow structure within the microfluidic chip. The chip's entry area uses a tapered geometry to guide the sample to generate a velocity gradient. Blood cells accumulate towards the outer edge of the channel due to inertial deflection, while the upper plasma flows along the center towards the separation zone and is guided into the dilution pool. A clean sample for testing can be obtained without centrifugation.

[0054] Plasma was then quantitatively diverted to three independent detection channels of the detection reaction module. Each channel contained pre-immobilized gold nanoparticles of the same particle size. Each probe was modified with a single-stranded aptamer of the corresponding biomarker and covalently labeled with a fluorophore. The probes maintained a low fluorescence background during storage. After sample inflow, the target biomarker and aptamer underwent conformational-specific binding, causing the fluorophore to move away from the energy transfer region on the gold surface, thereby generating a readable signal gain.

[0055] The signal acquisition module consists of a miniature light source and a photoelectric sensor array. The light source provides stable excitation light, and the sensors scan the three channels at a frequency of seconds. Within a five-minute acquisition window, the system automatically calculates the average value of the steady-state fluorescence intensity range, reducing statistical bias caused by external disturbances. The data processing module uses an embedded algorithm to convert the fluorescence intensity of the three biomarkers to obtain the corresponding concentration values, and then links with a liver injury risk prediction model to output a risk level, which is finally displayed on the screen. This solution is optimized for the portability and automation needs of primary care testing.

[0056] Solution 2: A highly sensitive probe system based on capillary-driven plasma separation and multi-stage fluorescence amplification This method is more suitable for laboratory or high-sensitivity scenarios, achieving a higher signal-to-noise ratio. After fingertip blood is dripped into a disposable cartridge, it automatically enters the multilayer membrane separation zone via an internal capillary-driven structure. This zone consists of a hydrophobic membrane, a pore size gradient membrane, and a high-adsorption layer. Blood cells are trapped and immobilized in the first two layers, while plasma is drawn to the reaction zone by capillary force. This method enables rapid plasma collection without external force, while minimizing sample loss.

[0057] The probe system in the reaction chamber was designed with enhanced sensitivity compared to Scheme 1. Using gold nanoparticles as a substrate, the aptamers and fluorophores are fixed by a spacer arm structure, further improving the fluorescence quenching in the unbound state and resulting in more pronounced fluorescence release after binding, significantly improving the overall signal-to-noise ratio. To address the spatial conformational differences of different biomarkers, the aptamer sequence of the three detection channels was independently optimized to ensure that the binding rate and specificity meet detection requirements within a short time.

[0058] The signal acquisition section employs a multi-stage amplification and readout mode. After the photoelectric sensing unit acquires the raw fluorescence signal, the voltage signal undergoes two-stage amplification and bandpass filtering via an internal analog-to-digital converter, ensuring that even weak signals can stably reach the processing end. The data processing module uses a pre-trained biomarker intensity fitting model and corrects for parameter shifts under normal conditions based on the gestational period, thereby improving the accuracy of concentration quantification. The system interface provides dynamic trend graphs along with the output results, facilitating clinical tracking of liver damage changes.

[0059] The solution is optimized for high-sensitivity detection needs and is more suitable for hospital laboratories, monitoring of high-risk pregnant women, and scientific research scenarios.

[0060] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting biomarkers for early diagnosis of liver injury during pregnancy, characterized in that, include: By obtaining a finger prick blood sample during pregnancy and separating the plasma; The isolated samples were introduced into three aptamer-modified gold nanoparticle probe systems to enable the binding of pregnancy-specific microRNAs, hepatocyte membrane damage factors, and inflammation-associated proteins in the samples to their corresponding aptamers. After the aptamers bound to the biomarkers, fluorescence signals were enhanced. The concentrations of the corresponding biomarkers were calculated based on the intensity of the three types of fluorescence signals. The risk of pregnancy-related liver injury was graded based on the established correlation model between fluorescence intensity and the degree of liver injury.

2. The method as described in claim 1, characterized in that, Biomarker screening was based on cohort data of pregnant women. Statistical analysis was used to screen for a combination of two-dimensional biomarkers, including liver injury-specific microRNAs, hepatocyte membrane damage factors, and inflammation-associated proteins during pregnancy.

3. The method as described in claim 1, characterized in that, The aptamer-modified gold nanoprobe is in a fluorescence-quenched state when the marker is not bound. After the marker is bound, the fluorescent group moves away from the quenching distance, generating a detectable fluorescence enhancement signal.

4. The method as described in claim 1, characterized in that, Plasma separation is accomplished through a laminar flow separation structure in a microfluidic channel, which uses inertial deflection to block blood cells outside the separation zone, thereby obtaining a supernatant for detection.

5. The method as described in claim 1, characterized in that, Risk classification is based on the concentration of biomarkers, which corresponds to low-risk, medium-risk, and high-risk ranges, and the classification output is achieved through the mapping relationship between fluorescence intensity and concentration.

6. A biomarker detection system for implementing the method for early diagnosis of liver injury during pregnancy as described in claim 1, characterized in that, include: The sample processing module is used for collecting finger-prick blood and separating plasma. The detection reaction module contains three physically isolated detection channels, each pre-coated with an aptamer-modified gold nanoprobe corresponding to the marker and a fluorescent group; The signal acquisition module is used to perform optical excitation and intensity acquisition of the fluorescence signals generated in each detection channel; The data processing module is used to calculate the biomarker concentration and classify the risk of liver injury based on the collected fluorescence intensity. The display module is used to output the detection results.

7. The system as described in claim 6, characterized in that, The sample processing module includes a microfluidic chip unit, which achieves automatic separation and quantitative delivery of plasma by limiting the channel cross-section and flow rate distribution inside the chip.

8. The system as described in claim 6, characterized in that, The three detection channels are used to detect liver injury-specific microRNAs, hepatocyte membrane damage factors, and inflammation-associated proteins during pregnancy, respectively. The three channels are isolated from each other to avoid cross-interference of reactions.

9. The system as described in claim 6, characterized in that, The signal acquisition module includes a light source excitation unit and a photoelectric sensing unit. The light source excitation unit is used to illuminate each detection channel, and the photoelectric sensing unit collects fluorescence intensity at fixed time intervals and outputs signal values.

10. The system as described in claim 6, characterized in that, The data processing module stores a nonlinear conversion model between fluorescence intensity and biomarker concentration, and generates risk grading results based on the model and biomarker threshold ranges.

Images