PCOS early screening micro-fluidic dual-enhanced electrochemical chip based on saliva and detection method thereof

Abstract

The invention provides a saliva-based PCOS early-screening microfluidic dual-enhanced electrochemical chip and a detection method thereof, the electrochemical chip comprises: a microfluidic pretreatment module, in which a filter area is used for intercepting insoluble impurities in saliva injected through a sample introduction area, an enrichment area is used for enriching target hormones, and a detection area is used for bearing an enriched sample; the dual-enhancement electrochemical detection module is used for detecting a sample located in a detection area, a signal acquisition unit is used for acquiring an electrochemical signal of the sample, a signal amplification unit is used for performing physical and chemical dual enhancement on the electrochemical signal, and a signal processing unit is used for calculating hormone concentration; the collaborative integration module is arranged between the detection area of the microfluidic pretreatment module and the dual-enhancement electrochemical detection module, the connection unit is used for realizing electric connection, and the trigger unit is used for automatically starting a detection process based on an electrode contact signal of the detection area. The performance of the electrochemical chip in the aspects of detection efficiency, sensitivity, portability and the like is remarkably improved.

Description

A saliva-based microfluidic dual-enhanced electrochemical chip for early PCOS screening and its detection method Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a saliva-based microfluidic dual-enhanced electrochemical chip for early PCOS screening and its detection method. Background Technology

[0002] Early screening for PCOS (Polycystic Ovary Syndrome) currently relies primarily on laboratory testing of plasma samples. The current gold standard for clinical diagnosis of PCOS is entirely based on plasma sample testing. Medical institutions commonly obtain plasma samples through venous blood collection and diagnose by detecting the concentrations of biomarkers such as AMH (Anti-Müllerian Hormone) and testosterone. While this method has high accuracy, it has significant limitations. Obtaining plasma samples requires specialized medical personnel and must be performed in qualified medical facilities, which greatly restricts the accessibility and widespread availability of screening.

[0003] The testing process for plasma samples involves multiple complex steps, from sample collection, transportation, preprocessing to final analysis, all of which require strict quality control standards and professional equipment support. This highly specialized requirement makes it difficult to promote and implement early PCOS screening in primary healthcare institutions or communities, let alone realize the application scenario of home self-testing.

[0004] Currently, while traditional ELISA (enzyme-linked immunosorbent assay) technology is mature, its sensitivity is limited when detecting low concentrations of biomarkers. This is particularly true for early PCOS screening, where the accuracy of traditional methods often falls short when hormone concentrations are at critical levels. While electrochemical detection techniques have improved sensitivity to some extent, they are still limited by sample matrix effects and interfering substances. Although plasma samples contain relatively high concentrations of target hormones, they also contain a large number of interfering proteins and lipids, which can easily form non-specific bindings during the detection process, affecting the accuracy of the results. Furthermore, existing detection technologies typically require long incubation times, making the entire detection process time-consuming and difficult to meet the needs of rapid screening.

[0005] In summary, existing PCOS early screening technologies have significant shortcomings in terms of sample processing, detection sensitivity, and device portability. Summary of the Invention

[0006] The purpose of this invention is to provide a saliva-based microfluidic dual-enhanced electrochemical chip for early PCOS screening and its detection method, so as to solve the technical problems of existing PCOS early screening technologies in terms of sample processing, detection sensitivity, and device portability.

[0007] The technical solution of this invention to solve the above-mentioned technical problems is as follows: Firstly, this invention provides a saliva-based microfluidic dual-enhanced electrochemical chip for early PCOS screening, comprising: a microfluidic pretreatment module integrating a sample injection area, a filtration area, an enrichment area, and a detection area; the filtration area is used to retain insoluble impurities in the saliva injected through the sample injection area; the enrichment area is used to enrich the target hormone; and the detection area is used to hold the enriched sample; a dual-enhanced electrochemical detection module for detecting the sample located in the detection area, including a signal acquisition unit, a signal amplification unit, and a signal processing unit; the signal acquisition unit is used to acquire the electrochemical signal of the sample; the signal amplification unit is used to perform physical and chemical dual enhancement of the electrochemical signal; and the signal processing unit is used to calculate the hormone concentration based on a log concentration-current calibration curve algorithm; and a collaborative integration module located between the detection area of ​​the microfluidic pretreatment module and the dual-enhanced electrochemical detection module, including a connection unit and a trigger unit; the connection unit is used to achieve electrical connection; and the trigger unit is used to automatically start the detection process based on the electrode contact signal of the detection area.

[0008] Based on the above technical solution, the microfluidic pretreatment module further adopts a multi-channel branch structure design, and the inner wall of the channel is hydrophilic, so that the sample can be automatically flowed by capillary force or air pressure.

[0009] Furthermore, the filtration zone has a built-in hydrophilic filter membrane; the inner wall of the enrichment zone channel is coated with a 3D gold nanoflower coating.

[0010] Furthermore, the signal amplification unit integrates a gold nanosphere substrate, a sandwich immunorecognition structure, and a tyramine signal amplification reagent to enhance the electrochemical signal of the sample through both physical and chemical means.

[0011] Furthermore, the gold nanosphere substrate of the signal amplification unit is modified on the surface of the SPCE working electrode to increase the specific capture surface area; the sandwich immunorecognition structure is used to sequentially capture antibodies, recognize target hormones, and detect antibodies; the tyramine signal amplification reagent generates a precipitate through HRP enzyme catalysis for secondary signal amplification.

[0012] Furthermore, the signal acquisition unit consists of a miniature potentiostat chip and an SPCE three-electrode system, used to acquire the electrochemical signals of the sample.

[0013] Furthermore, the electrode surface of the SPCE three-electrode system is pre-modified with capture antibody, BSA blocking layer and SA-HRP, and is preserved by lyophilization.

[0014] Furthermore, it also includes a communication unit for synchronizing detection data to the user terminal.

[0015] Secondly, the present invention provides a PCOS early screening detection method using the above-mentioned microfluidic electrochemical chip, comprising the following steps: S1, sample injection: injecting an untreated saliva sample into the sample injection area of ​​the microfluidic pretreatment module; S2, automatic pretreatment: the sample is driven by capillary force to flow sequentially through the filtration area to remove impurities and the enrichment area to enrich the target hormone, and then reaches the detection area; S3, signal acquisition and amplification: automatic triggering of dual-enhanced electrochemical detection through electrode contact, acquiring electrochemical signals and performing physical-chemical dual amplification; S4, concentration calculation: calculating the target hormone concentration based on the log concentration-current calibration curve algorithm; S5, data synchronization: synchronizing the detection results to the user end through the communication unit.

[0016] Furthermore, it also includes information processing steps: generating a hormone level change trend chart based on historical detection data; comparing the received user life cycle information with the hormone level change trend chart, and outputting display data.

[0017] Compared with existing technologies, this invention successfully solves the technical problems faced by traditional PCOS detection technology, such as cumbersome sample processing, insufficient detection sensitivity, and strong equipment dependence, by organically integrating three major modules: a microfluidic pretreatment module, a dual-enhanced electrochemical detection module, and a collaborative integration module.

[0018] Specifically, the beneficial effects of this invention are reflected in the following aspects: 1. Significantly improved sample processing efficiency: Traditional saliva sample pretreatment requires multiple centrifugation and filtration operations, taking more than 30 minutes. This invention, through the integrated design of a microfluidic chip, shortens the pretreatment time to less than 1 minute. 2. Order-of-magnitude improvement in detection sensitivity: The dual-enhanced electrochemical detection module achieves high-sensitivity detection through a dual physical and chemical enhancement mechanism, enabling stable detection of low concentrations of hormones at the pg / mL level, with sensitivity improved by 5 orders of magnitude compared to traditional ELISA methods. 3. Significantly improved detection stability: The collaborative integration module automatically triggers the detection process, avoiding manual operation errors. This intelligent process control effectively eliminates human error and improves the repeatability of detection results. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 is a schematic diagram of the microfluidic pretreatment module in the saliva-based PCOS early screening microfluidic dual-enhanced electrochemical chip provided in an embodiment of the present invention; Figure 2 is a schematic diagram of sample processing in the saliva-based PCOS early screening microfluidic dual-enhanced electrochemical chip provided in an embodiment of the present invention; Figure 3 is a schematic diagram of the detection principle of the dual-enhanced electrochemical detection module in the saliva-based PCOS early screening microfluidic dual-enhanced electrochemical chip provided in an embodiment of the present invention. Detailed Implementation

[0021] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the reagents and raw materials used are all commercially available.

[0024] Example 1 The microfluidic dual-enhanced electrochemical chip for early PCOS screening based on saliva provided in this example is characterized by comprising: a fluidic pretreatment module, a dual-enhanced electrochemical detection module, and a collaborative integration module.

[0025] As shown in Figure 1, the microfluidic pretreatment module integrates a sample injection zone, a filtration zone, an enrichment zone, and a detection zone. The filtration zone is used to retain insoluble impurities in the saliva injected through the sample injection zone, the enrichment zone is used to enrich the target hormone, and the detection zone is used to hold the enriched sample.

[0026] Specifically, as shown in Figure 1, the microfluidic pretreatment module adopts a multi-channel branch structure design, preferably Y-shaped, and the inner wall of the channel is hydrophilic. The sample is automatically circulated by capillary force or air pressure.

[0027] During sample processing, "in-situ filtration + antigen enrichment" is used to ensure automatic pretreatment of saliva samples, which improves diagnostic accuracy by 15%.

[0028] Preferably, the filtration zone has a built-in 0.22μm hydrophilic filter membrane, which can be selected from polyethersulfone filter membrane, polyethersulfone, nylon, etc., to only intercept insoluble impurities in saliva (such as mucin, epithelial cells, bacteria, etc.), because these impurities will block subsequent microchannels, contaminate electrodes, or interfere with electrochemical signals. The filtered saliva still contains "target protein AMH + other soluble impurities (such as albumin, lactoferrin, etc.)", and these soluble proteins will enter the next step of the 3D AuNS channel together.

[0029] The inner wall of the enrichment zone channel is coated with a 3D gold nanoflower coating. The "non-specific adsorption" of 3D AuNS is non-selective—it adsorbs all soluble proteins (including AMH and other proteins) flowing through the channel, rather than "adsorbing only other proteins and releasing AMH." In practical scenarios, for children with extremely low AMH concentrations in their saliva (e.g., 0.1-1 pg / mL), AuNS adsorption "captures" trace amounts of AMH in saliva on the channel surface and enriches them (for example, if there is only 0.1 pg AMH in 100 μL of saliva, after enrichment, more than 90% of the AMH will be attached to the AuNS surface, which is equivalent to "concentrating" the amount of AMH).

[0030] Furthermore, instead of simply "letting AMH continue to flow away" after enrichment, AMH is involved in detection through the following method: after in-situ elution, it flows to the detection area: the AuNS channel is rinsed with a mild elution buffer (such as 0.05% Tween-20) to elute the enriched protein (containing AMH) and flow together to the detection area—at this point, the AMH concentration is 10-100 times higher than that of the original saliva, making it easier for the electrode to capture.

[0031] The enrichment zone described above serves to "concentrate" (increase the total amount per unit volume), while the detection zone aims to utilize antibodies for "specific capture." By increasing the local concentration of the sample through non-specific enrichment, combined with specific antibody recognition in the detection zone, the problem of low salivary hormone abundance can be effectively solved.

[0032] The dual-enhanced electrochemical detection module is used to detect samples located in the detection area. It includes a signal acquisition unit, a signal amplification unit, and a signal processing unit. The signal acquisition unit is used to acquire the electrochemical signal of the sample, the signal amplification unit is used to perform physical and chemical dual enhancement of the electrochemical signal, and the signal processing unit is used to calculate the hormone concentration based on the log concentration-current calibration curve algorithm.

[0033] As shown in Figure 3, the signal amplification unit integrates a gold nanosphere substrate, a sandwich immunorecognition structure, and a tyramine signal amplification reagent to enhance the electrochemical signal of the sample through both physical and chemical means. This integration of three technologies achieves high sensitivity amplification of extremely low levels of hormone signals in saliva.

[0034] Specifically, the gold nanosphere substrate of the signal amplification unit is modified on the surface of the SPCE working electrode to increase the specific capture surface area.

[0035] The sandwich-shaped immune recognition structure is used to sequentially capture antibodies, recognize target hormones, and detect antibodies.

[0036] Tyramine signal amplification reagent produces a precipitate through HRP enzyme catalysis, which is used for secondary signal amplification.

[0037] Preferably, the gold nanosphere substrate is modified onto the SPCE working electrode by electrochemical deposition, increasing the capture surface area by 50 times; the sandwich immunorecognition sequentially immobilizes anti-AMH monoclonal antibody, AMH hormone in the sample, and HRP-labeled polyclonal antibody; the tyramine signal amplification reagent contains 0.05% TMB substrate, which produces a precipitate under HRP catalysis, thereby achieving secondary signal amplification.

[0038] The aforementioned signal acquisition unit consists of a miniature potentiostat chip and an SPCE three-electrode system, used to acquire the electrochemical signals of the sample. Preferably, the electrode surfaces of the SPCE three-electrode system are pre-modified with a capture antibody (1 μg / mL), a BSA blocking layer (1%), and SA-HRP (0.5 μg / mL), and are lyophilized. This setup simplifies user operation to just two steps: adding the sample and the TMB substrate, improving stability. The SPCE three-electrode system can optionally include a working electrode (modified with gold nanospheres), a reference electrode (Ag / AgCl), and a counter electrode (platinum wire). The potentiostat chip output potential is -0.2V for the it method and -0.3~+0.7V for the DPV method, its function being to accurately capture electrochemical signals and adapt to low-concentration detection.

[0039] Specifically, the SPCE electrode surface is modified with an AMH-specific capture antibody. Whether AMH is "washed over" or "directly enriched on AuNS", it will ultimately only bind to the AMH antibody on the electrode, while other proteins, which cannot bind to the antibody, will be removed by subsequent rinsing steps.

[0040] The specific detection pathway is as follows: low concentration of AMH → first enrichment with AuNS (to increase the total amount of AMH) → then specific binding of electrode antibody (to distinguish AMH from other proteins) → finally achieving high-sensitivity detection.

[0041] The collaborative integration module is located between the detection area of ​​the microfluidic pretreatment module and the dual-enhanced electrochemical detection module. It includes a connection unit and a trigger unit. The connection unit is used to realize electrical connection, and the trigger unit is used to automatically start the detection process based on the electrode contact signal of the detection area.

[0042] Furthermore, this electrochemical chip also includes a communication unit for synchronizing detection data to the user terminal. For example, a Bluetooth 5.0 module is integrated into the detection terminal, with a transmission distance of 10m, synchronizing data to a mobile app. This enables real-time transmission and long-term dynamic management of detection results.

[0043] The following is a specific example: In a concrete implementation scenario, the microfluidic pretreatment module is injection molded from PDMS material, with dimensions of 25mm × 15mm × 5mm. The sample injection area is designed in a funnel shape for easy injection of saliva samples; the filtration area incorporates a 0.22μm hydrophilic polyethersulfone filter membrane with a thickness of 100μm to retain impurities such as mucin; the inner wall of the enrichment area channel is coated with a 3D gold nanoflower coating to enrich AMH / T hormones through non-specific adsorption; the detection area has an electrode interface. The signal acquisition unit of the dual-enhanced electrochemical detection module uses an AD5941 miniature potentiostat chip (4mm × 4mm, current detection range covering 10) -12 ~10 - 6 A) The SPCE three-electrode system; the signal amplification unit integrates a gold nanosphere substrate, a sandwich immunorecognition structure, and a tyramine signal amplification reagent; the signal processing unit uses an STM32L4 microcontroller with a built-in log concentration-current calibration curve algorithm. The connection unit of the collaborative integration module is a 16-channel spring probe slot, and the trigger unit automatically starts detection based on the electrode contact signal with a 30-second delay. Specifically, the detection is automatically started upon electrode contact, and signal acquisition is performed after a 30-second wait to ensure sample preprocessing is complete.

[0044] The channel adopts a Y-shaped structure, with separate waste liquid separation and inlet liquid separation units. The inner wall is treated with oxygen plasma hydrophilic treatment, with a contact angle of <10°. The sample is automatically circulated by capillary force at a flow rate of 1-5 μL / s. The Y-shaped design can avoid waste liquid backflow and improve filtration efficiency.

[0045] The polyethersulfone filter membrane in the filtration zone is integrated via hot pressing, with a pore size of 0.22 μm. The 3D gold nanoflower coating in the enrichment zone is prepared by PEI / PAA template electrodeposition, with a thickness of 5-10 μm, a specific surface area of ​​500 m² / g, and an enrichment efficiency of 2-3 times. Its function is to retain impurities in the filter membrane and enrich target hormones in the coating, thereby improving detection sensitivity.

[0046] Example 2 This example provides a PCOS early screening detection method using the microfluidic electrochemical chip of Example 1, characterized by the following steps: S1, Sample injection: Injecting an untreated saliva sample into the sample injection area of ​​the microfluidic pretreatment module; S2, Automatic pretreatment: The sample is driven by capillary force to flow sequentially through the filtration area to remove impurities and the enrichment area to enrich the target hormone, before reaching the detection area; S3, Signal acquisition and amplification: Automatically triggering dual-enhanced electrochemical detection through electrode contact, acquiring electrochemical signals and performing physical-chemical dual amplification; S4, Concentration calculation: Calculating the target hormone concentration based on the log concentration-current calibration curve algorithm; S5, Data synchronization: Synchronizing the detection results to the user terminal through the communication unit.

[0047] Specifically, in one implementation scenario, sample injection: 10 μL of untreated saliva is collected and injected into the injection area under pressure; automatic pretreatment: the sample is filtered and enriched within 30 seconds by capillary force; signal acquisition and amplification: it / DPV detection is triggered by electrode contact; concentration calculation: the microcontroller executes the calibration curve algorithm; data synchronization: data is transmitted to the APP via Bluetooth.

[0048] Furthermore, optionally, the method also includes an information processing step: generating a hormone level change trend chart based on historical detection data; comparing the received user life cycle information with the hormone level change trend chart, and outputting display data.

[0049] Specifically, a hormone level trend chart is generated based on historical data from the APP, and the data is displayed in combination with the user's input menstrual cycle information. Preferably, when three consecutive tests show that hormone levels exceed the preset range (e.g., AMH>8.5pg / mL), health management suggestions are generated.

[0050] Overall, PCOS patients experienced improved quality of life, with their MPCOSQ scores increasing by 60 points.

[0051] In summary, the technical solution proposed in this invention has achieved a breakthrough in the field of early PCOS screening through systematic modular innovative design. The overall technical effect is reflected in a significant improvement in four dimensions: detection efficiency, sensitivity, portability, and user experience.

[0052] In this specification, the specific features, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, those skilled in the art can combine and integrate the different embodiments or examples described herein, as well as the features of those different embodiments or examples, without contradiction.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A saliva-based microfluidic dual-enhanced electrochemical chip for early PCOS screening, characterized in that, include: The microfluidic pretreatment module integrates a sample injection zone, a filtration zone, an enrichment zone, and a detection zone. The filtration zone is used to retain insoluble impurities in the saliva injected through the sample injection zone, the enrichment zone is used to enrich target hormones, and the detection zone is used to hold the enriched sample. A dual-enhanced electrochemical detection module is used to detect samples located in the detection area. It includes a signal acquisition unit, a signal amplification unit, and a signal processing unit. The signal acquisition unit is used to acquire the electrochemical signal of the sample. The signal amplification unit is used to perform physical and chemical dual enhancement of the electrochemical signal. The signal processing unit is used to calculate the hormone concentration based on the log concentration-current calibration curve algorithm. A collaborative integration module is located between the detection area of ​​the microfluidic pretreatment module and the dual-enhanced electrochemical detection module. It includes a connection unit and a triggering unit. The connection unit is used to realize electrical connection. The triggering unit is used to automatically start the detection process based on the electrode contact signal of the detection area.

2. The microfluidic electrochemical chip according to claim 1, characterized in that, The microfluidic pretreatment module adopts a multi-channel branch structure design, and the inner wall of the channel is hydrophilic. The sample is automatically circulated by capillary force or air pressure.

3. The microfluidic electrochemical chip according to claim 1, characterized in that, The filtration zone has a built-in hydrophilic filter membrane; the inner wall of the enrichment zone channel is coated with a 3D gold nanoflower coating.

4. The microfluidic electrochemical chip according to claim 1, characterized in that, The signal amplification unit integrates a gold nanosphere substrate, a sandwich immunorecognition structure, and a tyramine signal amplification reagent to enhance the electrochemical signal of the sample through both physical and chemical means.

5. The microfluidic electrochemical chip according to claim 4, characterized in that, The gold nanosphere substrate of the signal amplification unit is modified on the surface of the SPCE working electrode to increase the specific capture surface area; the sandwich immunorecognition structure is used to sequentially capture antibodies, recognize target hormones, and detect antibodies; the tyramine signal amplification reagent generates a precipitate through HRP enzyme catalysis for secondary signal amplification.

6. The microfluidic electrochemical chip according to claim 1, characterized in that, The signal acquisition unit consists of a miniature potentiostat chip and an SPCE three-electrode system, and is used to acquire the electrochemical signals of the sample.

7. The microfluidic electrochemical chip according to claim 1, characterized in that, The electrode surfaces of the SPCE three-electrode system are pre-modified with capture antibodies, a BSA blocking layer, and SA-HRP, and are preserved by lyophilization.

8. The microfluidic electrochemical chip according to claim 1, characterized in that, It also includes a communication unit for synchronizing detection data to the user terminal.

9. A method for early screening and detection of PCOS using the microfluidic electrochemical chip according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Sample Injection: Inject the untreated saliva sample into the injection area of ​​the microfluidic pretreatment module; S2. Automatic Pretreatment: Driven by capillary force, the sample flows sequentially through the filtration area to remove impurities and the enrichment area to enrich the target hormone, before reaching the detection area; S3. Signal Acquisition and Amplification: Automatically triggered dual-enhanced electrochemical detection is achieved through electrode contact, and the electrochemical signal is acquired and amplified by both physical and chemical methods. S4. Concentration Calculation: Calculate the target hormone concentration based on the log concentration-current calibration curve algorithm; S5. Data Synchronization: Synchronize the detection results to the user terminal through the communication unit.

10. The detection method according to claim 9, characterized in that, It also includes information processing steps: generating a hormone level change trend chart based on historical detection data; comparing the received user life cycle information with the hormone level change trend chart, and outputting display data.

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