A tear detection device and method for early screening of alzheimer's disease
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING TECH & BUSINESS UNIV
- Filing Date
- 2026-04-09
- Publication Date
- 2026-06-26
Smart Images
Figure CN122283142A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of in vitro diagnostic medical device technology, and in particular to a tear film detection device and method for early screening of Alzheimer's disease. Background Technology
[0002] Alzheimer's disease is an age-related, progressive neurodegenerative brain disorder and the most common form of dementia. It causes severe health damage and mortality worldwide, and treatment options are limited. Diagnosis of Alzheimer's disease presents significant challenges, necessitating timely identification and intervention. Currently, while some medications may slow its progression, there is no effective cure.
[0003] At the molecular level, the pathophysiological mechanisms of Alzheimer's disease primarily include extracellular amyloid-β peptide deposition (forming amyloid plaques) and intracellular hyperphosphorylated tau protein aggregation (forming neurofibrillary tangles). These processes, in turn, trigger oxidative stress, chronic neuroinflammation, neuronal dysfunction, and neurodegeneration. Currently, the discovery of biomarkers for Alzheimer's disease, such as magnetic resonance imaging (MRI), positron emission tomography (PET-CT), and cerebrospinal fluid (CSF) molecules, has greatly enhanced our understanding of the disease and is crucial for identifying early neuropathological changes before clinical changes and cognitive decline.
[0004] However, these current standard diagnostic methods have significant limitations. Brain imaging techniques are expensive and the equipment is not widely available, so they are usually not used for initial screening but only for definitive diagnosis. CSF collection is an invasive procedure that requires hospitalization and is performed by trained professionals, posing risks such as infection and headaches, and has low public acceptance. These factors make it difficult to widely apply the above methods to early screening and population surveillance of Alzheimer's disease. Summary of the Invention
[0005] The purpose of this invention is to provide a tear film detection device and method for early screening of Alzheimer's disease. By designing a tear film sampler that can be directly coupled to a microfluidic chip, the sample transfer step is eliminated, ensuring the stability of biomarkers. By integrating a multi-channel detection unit on the microfluidic chip, parallel analysis of protein biomarkers and microRNA biomarkers in tears is achieved, thereby obtaining more comprehensive disease characteristic information with extremely low sample consumption. By combining electrochemical immunosensing and fluorescence hybridization signal amplification technology, and equipping it with a portable reader, the detection process does not require a professional laboratory environment, significantly reducing the cost and operation time of a single test.
[0006] To achieve the above objectives, the present invention provides a tear film detection device for early screening of Alzheimer's disease, comprising a tear film sampler, a microfluidic chip, and a portable reader. The tear film sampler is used to deliver collected and pre-processed tear film samples to the microfluidic chip. The microfluidic chip is provided with a sample input port, and the tear film sampler is connected to the sample input port. The microfluidic chip is provided with a microchannel structure for detecting biomarkers, and the microfluidic chip can be placed in the slot of the portable reader. The portable reader is used to perform signal detection and analyze the monitoring data to output diagnostic results.
[0007] Preferably, the tear sampler includes a microsponge head, a polymer rod connected to the microsponge head, and a handle body connected to the other end of the polymer rod.
[0008] Preferably, the handle body has a buffer reservoir inside, a sealing membrane on the buffer reservoir, a pressable sealing plug at the end of the handle body, and an interface end at the end of the handle body, which is connected to the sample input port.
[0009] Preferably, the microfluidic chip includes a top cover layer and a microchannel layer, with the sample inlet located on the top cover layer. The top cover layer is provided with a negative pressure port for driving the liquid to flow within the microfluidic chip, and the microchannel structure is located on the microchannel layer.
[0010] Preferably, the microchannel structure includes a serpentine hybrid channel, one end of which is connected to a sample inlet, and the other end of which is connected to a flow divider. The other end of the flow divider is connected to a first detection microchannel, a second detection microchannel, a third detection microchannel for protein biomarker detection, and a fourth detection microchannel for microRNA biomarker detection. The first, second, and third detection microchannels are each provided with an immunosensor region, and the fourth detection microchannel is provided with a nucleic acid hybridization region.
[0011] Preferably, protein marker antibodies are provided on the first, second, and third detection microchannels, and a DNA capture probe is provided on the fourth detection microchannel.
[0012] Preferably, the microfluidic chip further includes a substrate layer on which a microelectrode assembly for protein biomarker detection is disposed, and the corresponding portion of the substrate layer and the nucleic acid hybridization region is transparent.
[0013] A tear film detection method for early screening of Alzheimer's disease includes the following steps: S1: During sampling, place the micro-sponge head of the tear sampler into the conjunctival sac of the patient's lower eyelid to absorb tears for 1-2 minutes; S2: After sampling, insert the interface of the tear sampler into the sample input port of the microfluidic chip and tighten it. This tightening action will simultaneously press down the sealing plug in the tear sampler, puncture the sealing membrane of the buffer reservoir, and allow the buffer to flow through the polymer rod under gravity and capillary action, flushing the microsponge head and eluting the adsorbed biomarkers into the microfluidic chip. S3: Place the microfluidic chip connected to the tear sampler into the slot of the portable reader. The portable reader automatically applies negative pressure through the negative pressure port, driving 100 μL of sample-buffer mixture to flow through four detection microchannels within 5 minutes. During this process, Aβ42 protein in the sample is specifically captured by the anti-Aβ42 monoclonal antibody in the first detection microchannel, forming an antigen-antibody complex. T-tau protein in the sample is specifically captured by the anti-T-tau monoclonal antibody in the second detection microchannel, forming an antigen-antibody complex. Lactoferrin in the sample is specifically captured by the anti-lactoferrin monoclonal antibody in the third detection microchannel, forming an antigen-antibody complex. microRNA-200b-5p in the sample hybridizes with the captured DNA probe in the fourth detection microchannel. S4: The portable reader automatically draws cleaning fluid from a cleaning fluid reservoir on the microfluidic chip to flush each detection microchannel and remove unbound impurities; S5: For the first, second, and third detection microchannels, an electrochemical immunoassay was used. A portable reader injected a secondary antibody solution containing horseradish peroxidase into each of the three detection microchannels. After incubation and washing, an electrochemical substrate was injected. Subsequently, a potentiostat inside the portable reader applied a specific voltage to the microelectrode assembly and measured the resulting reduction current. The current intensity was proportional to the concentration of the captured target protein. S6: For the fourth detection microchannel, a hybridization chain reaction is used to amplify the fluorescence signal. The portable reader sequentially injects two partially complementary DNA hairpin structures, H1 and H2, labeled with fluorescent groups, into the fourth detection microchannel. If the target microRNA is present, it will trigger the alternating hybridization of H1 and H2 to form a long double-stranded DNA polymer, thereby fixing a large number of fluorescent groups in the nucleic acid hybridization region. Subsequently, the LED light source built into the portable reader excites fluorescence, and the emitted fluorescence intensity is detected by a photodiode. The fluorescence intensity is proportional to the concentration of the target microRNA. S7: The portable reader has a built-in microprocessor that takes the collected current and fluorescence signal values, substitutes them into a pre-stored calibration curve, calculates the concentration of various biomarkers, and then integrates these concentration values into a comprehensive Alzheimer's disease risk index through a diagnostic model based on machine learning algorithms. The result of "low risk", "medium risk" or "high risk" is displayed on the screen. At the same time, the data can be transferred to a smartphone or cloud via Bluetooth or USB.
[0014] Preferably, the electrochemical immunoassay in S5 is replaced with the chemiluminescent immunoassay. The portable reader injects the chemiluminescent substrate into the three detection microchannels respectively, and the luminescence intensity is detected by the photomultiplier tube in the portable reader. The remaining steps remain unchanged.
[0015] Preferably, the hybridization chain reaction amplification of the fluorescence signal in S6 is replaced with electrochemiluminescence. The captured DNA probe can be labeled with an electrochemiluminescence tag, which generates a light signal after voltage is applied and is detected by a photodiode inside the portable reader. The remaining steps remain unchanged.
[0016] Therefore, this invention employs the aforementioned tear film detection device and method for early screening of Alzheimer's disease. By designing a tear film sampler that can be directly coupled to a microfluidic chip, the sample transfer step is eliminated, ensuring the stability of biomarkers. By integrating a multi-channel detection unit on the microfluidic chip, parallel analysis of protein biomarkers and microRNA biomarkers in tears is achieved, thereby obtaining more comprehensive disease characteristic information with extremely low sample consumption. By combining electrochemical immunosensing and fluorescence hybridization signal amplification technology, and equipping it with a portable reader, the detection process does not require a professional laboratory environment, significantly reducing the cost and operation time of a single test.
[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0018] Figure 1 This is a schematic diagram of the overall structure of the tear film detection device for early screening of Alzheimer's disease in this invention. Figure 2 This is a schematic diagram of the specific structure of the tear sampler in this invention; Figure 3 This is a schematic diagram of the specific structure of the microchannel layer of the microfluidic chip in this invention; Figure 4 This is a schematic diagram of the specific structure of the substrate layer of the microfluidic chip in this invention.
[0019] Figure Labels 100. Tear sampler; 101. Microsponge tip; 102. Polymer rod; 103. Handle body; 104. Buffer reservoir; 105. Sealing plug; 106. Interface end; 200. Microfluidic chip; 201. Sample inlet; 202. Negative pressure port; 203. Serpentine mixing channel; 204. Flow splitter; 205a. First detection microchannel; 205b. Second detection microchannel; 205c. Third detection microchannel; 205d. Fourth detection microchannel; 206. Immunosensor region; 207. Nucleic acid hybridization region; 208. Microelectrode assembly; 300. Portable reader. Detailed Implementation
[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The terms "first," "second," and similar terms used in this invention do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.
[0022] Example 1 like Figure 1 As shown, a tear film detection device for early screening of Alzheimer's disease includes a tear film sampler 100, a microfluidic chip 200, and a portable reader 300. The tear film sampler 100 is used to collect and pre-process tear film samples and deliver the processed samples to the microfluidic chip 200. The microfluidic chip 200 is provided with a sample input port 201, which has a built-in threaded structure. The tear film sampler 100 is threadedly connected to the sample input port 201. The microfluidic chip 200 is etched with a microchannel structure, which is used to detect biomarkers. The microfluidic chip 200 can be placed inside the slot of the portable reader 300. The portable reader 300 is used to perform signal detection and analyze the monitoring data to output diagnostic results.
[0023] like Figure 2As shown, the tear sampler 100 adopts an improved microsponge structure, the core of which is to integrate sampling with preliminary sample elution and pretreatment functions. Specifically, the tear sampler 100 includes a microsponge head 101, a polymer rod 102 and a handle body 103. The microsponge head 101 is connected to the handle body 103 through the polymer rod 102. The microsponge head 101 is made of medical-grade hydrophilic cellulose material and is shaped like a sphere with a diameter of 2 mm. The polymer rod 102 is a flexible hollow structure.
[0024] The handle body 103 has a sealed buffer reservoir 104 inside, which is covered with a sealing membrane. The buffer reservoir 104 is pre-stored with 150 μL of phosphate buffer containing RNase inhibitors for subsequent microRNA analysis. The handle body 103 has a sealing plug 105 at its end. The sealing plug 105 is pressable and has a channel for sample passage. The handle body 103 also has an interface end 106 at its end. The interface end 106 has a thread structure that matches the thread structure on the sample input port 201, and the interface end 106 is threadedly connected to the sample input port 201.
[0025] The microfluidic chip 200 includes a top cover layer and a microchannel layer. The sample inlet 201 is located on the top cover layer, and a negative pressure port 202 is provided on the top cover layer. The negative pressure port 202 is used to connect an external micro-injection pump (not shown in the figure) during the initial sample addition to drive the liquid to flow inside the microfluidic chip 200. The microchannel structure is located in the microchannel layer. Preferably, both the top cover layer and the microchannel layer are made of PDMS material.
[0026] like Figure 3 As shown, the microchannel structure includes a serpentine mixing channel 203. One end of the serpentine mixing channel 203 is connected to the sample inlet 201. After the sample enters from the sample inlet 201, it flows through the serpentine mixing channel 203 to ensure that the sample and buffer are fully mixed. The other end of the serpentine mixing channel 203 is connected to a splitter 204. The other end of the splitter 204 is connected to a first detection microchannel 205a, a second detection microchannel 205b, a third detection microchannel 205c, and a fourth detection microchannel 205d, respectively. The first detection microchannel 205a, the second detection microchannel 205b, and the third detection microchannel 205c are used to detect protein biomarkers, and the fourth detection microchannel 205d is used to detect microRNA biomarkers. The mixed sample solution is precisely distributed to the first detection microchannel 205a, the second detection microchannel 205b, the third detection microchannel 205c, and the fourth detection microchannel 205d.
[0027] The first detection microchannel 205a, the second detection microchannel 205b, and the third detection microchannel 205c are each provided with an immunosensor region 206, and the fourth detection microchannel 205d is provided with a nucleic acid hybridization region 207.
[0028] The electrode surface of the first detection microchannel 205a is immobilized with anti-Aβ42 monoclonal antibody, the electrode surface of the second detection microchannel 205b is immobilized with T-tau monoclonal antibody, the electrode surface of the third detection microchannel 205c is immobilized with anti-lactoferrin monoclonal antibody, and the fourth detection microchannel 205d is covalently immobilized with a capture DNA probe with the sequence 5'-NH2-(C6 Spacer)-GCAGCACGUUACCAUACUAC-3', which is completely complementary to the target microRNA-200b-5p.
[0029] like Figure 4 As shown, the microfluidic chip 200 also includes a substrate layer on which a microelectrode assembly 208 is disposed. The microelectrode assembly 208 is used for protein marker detection. The microelectrode assembly 208 includes a gold working electrode, a gold counter electrode, and an Ag / AgCl reference electrode. Meanwhile, the portion of the substrate layer corresponding to the nucleic acid hybridization region is transparent to facilitate optical detection. Preferably, the substrate layer is made of glass.
[0030] A tear film detection method for early screening of Alzheimer's disease, the specific steps of which are as follows: S1: During sampling, place the micro-sponge head 101 of the tear sampler 100 into the conjunctival sac of the patient's lower eyelid to absorb tears for 1.5 minutes; S2: After sampling is completed, insert the interface end 106 of the tear sampler 100 into the sample input port 201 of the microfluidic chip 200 and tighten it. This tightening action will simultaneously press down the sealing plug 105 in the tear sampler 100, puncture the sealing membrane of the buffer reservoir 104, and allow the buffer to flow through the polymer rod 102 under gravity and capillary action, flushing the microsponge head 101 and eluting the adsorbed biomarkers into the microfluidic chip 200. S3: Place the microfluidic chip 200 connected to the tear sampler 100 into the slot of the portable reader 300. The portable reader 300 automatically applies negative pressure through the negative pressure port 202, causing 100 μL of sample-buffer mixture to flow through four detection microchannels within 5 minutes. During this process, Aβ42 protein in the sample is specifically captured by the anti-Aβ42 monoclonal antibody in the first detection microchannel 205a, forming an antigen-antibody complex. T-tau protein in the sample is specifically captured by the anti-T-tau monoclonal antibody in the second detection microchannel 205b, forming an antigen-antibody complex. Lactoferrin in the sample is specifically captured by the anti-lactoferrin monoclonal antibody in the third detection microchannel 205c, forming an antigen-antibody complex. microRNA-200b-5p in the sample hybridizes with the captured DNA probe in the fourth detection microchannel 205d. S4: The portable reader 300 automatically draws in cleaning fluid from a cleaning fluid reservoir (not shown in the figure) on the microfluidic chip 200, rinses each detection microchannel, and removes unbound impurities; S5: For the first detection microchannel 205a, the second detection microchannel 205b, and the third detection microchannel 205c, an electrochemical immunoassay was used. The portable reader 300 injected a secondary antibody solution containing horseradish peroxidase into the three detection microchannels, respectively. After incubation and washing, TMB was injected. Subsequently, the potentiostat inside the portable reader 300 applied a defined voltage to the microelectrode assembly 208 and measured the generated reduction current. The current intensity was proportional to the concentration of the captured target protein. S6: For the fourth detection microchannel 205d, a hybridization chain reaction is used to amplify the fluorescence signal. The portable reader 300 sequentially injects two partially complementary DNA hairpin structures H1 and H2, labeled with FAM, into the fourth detection microchannel 205d. If the target microRNA is present, it will trigger the alternating hybridization of H1 and H2 to form a long double-stranded DNA polymer, thereby fixing a large number of fluorescent groups in the nucleic acid hybridization region 207. Subsequently, the LED light source with a center wavelength of 485nm built into the portable reader 300 excites the fluorescence, and the fluorescence intensity with a center wavelength of 520nm is detected by a photodiode. The fluorescence intensity is proportional to the concentration of the target microRNA. S7: The Portable Reader 300 has a built-in microprocessor that takes the collected current and fluorescence signal values, substitutes them into a pre-stored calibration curve, calculates the concentration of various biomarkers, and then integrates these concentration values into a comprehensive Alzheimer's disease risk index through a diagnostic model based on logistic regression. The result is displayed as "low risk," "medium risk," or "high risk" on the screen. Data can be transferred to a smartphone or the cloud via Bluetooth or USB.
[0031] Example 2 Based on Example 1, the electrochemical immunoassay in S5 is replaced with the chemiluminescent immunoassay.
[0032] A tear film detection method for early screening of Alzheimer's disease, the specific steps of which are as follows: S1: During sampling, place the micro-sponge head 101 of the tear sampler 100 into the conjunctival sac of the patient's lower eyelid to absorb tears for 1.5 minutes; S2: After sampling is completed, insert the interface end 106 of the tear sampler 100 into the sample input port 201 of the microfluidic chip 200 and tighten it. This tightening action will simultaneously press down the sealing plug 105 in the tear sampler 100, puncture the sealing membrane of the buffer reservoir 104, and allow the buffer to flow through the polymer rod 102 under gravity and capillary action, flushing the microsponge head 101 and eluting the adsorbed biomarkers into the microfluidic chip 200. S3: Place the microfluidic chip 200 connected to the tear sampler 100 into the slot of the portable reader 300. The portable reader 300 automatically applies negative pressure through the negative pressure port 202, causing 100 μL of sample-buffer mixture to flow through four detection microchannels within 5 minutes. During this process, Aβ42 protein in the sample is specifically captured by the anti-Aβ42 monoclonal antibody in the first detection microchannel 205a, forming an antigen-antibody complex. T-tau protein in the sample is specifically captured by the anti-T-tau monoclonal antibody in the second detection microchannel 205b, forming an antigen-antibody complex. Lactoferrin in the sample is specifically captured by the anti-lactoferrin monoclonal antibody in the third detection microchannel 205c, forming an antigen-antibody complex. microRNA-200b-5p in the sample hybridizes with the captured DNA probe in the fourth detection microchannel 205d. S4: The portable reader 300 automatically draws cleaning fluid from a cleaning fluid reservoir on the microfluidic chip 200 to flush each detection microchannel and remove unbound impurities; S5: For the first detection microchannel 205a, the second detection microchannel 205b and the third detection microchannel 205c, chemiluminescence immunoassay is used. The portable reader 300 injects luminol into the three detection microchannels respectively, and the luminescence intensity is detected by the photomultiplier tube in the portable reader 300. S6: For the fourth detection microchannel 205d, a hybridization chain reaction is used to amplify the fluorescence signal. The portable reader 300 sequentially injects two partially complementary DNA hairpin structures H1 and H2, labeled with FAM, into the fourth detection microchannel 205d. If the target microRNA is present, it will trigger the alternating hybridization of H1 and H2 to form a long double-stranded DNA polymer, thereby fixing a large number of fluorescent groups in the nucleic acid hybridization region 207. Subsequently, the LED light source with a center wavelength of 485nm built into the portable reader 300 excites the fluorescence, and the fluorescence intensity with a center wavelength of 520nm is detected by a photodiode. The fluorescence intensity is proportional to the concentration of the target microRNA. S7: The Portable Reader 300 has a built-in microprocessor that takes the collected current and fluorescence signal values, substitutes them into a pre-stored calibration curve, calculates the concentration of various biomarkers, and then integrates these concentration values into a comprehensive Alzheimer's disease risk index through a diagnostic model based on logistic regression. The result is displayed as "low risk," "medium risk," or "high risk" on the screen. Data can be transferred to a smartphone or the cloud via Bluetooth or USB.
[0033] Example 3 Based on Example 1, the hybridization chain reaction amplification of fluorescence signal in S6 was replaced with electrochemiluminescence.
[0034] A tear film detection method for early screening of Alzheimer's disease, the specific steps of which are as follows: S1: During sampling, place the micro-sponge head 101 of the tear sampler 100 into the conjunctival sac of the patient's lower eyelid to absorb tears for 1.5 minutes; S2: After sampling is completed, insert the interface end 106 of the tear sampler 100 into the sample input port 201 of the microfluidic chip 200 and tighten it. This tightening action will simultaneously press down the sealing plug 105 in the tear sampler 100, puncture the sealing membrane of the buffer reservoir 104, and allow the buffer to flow through the polymer rod 102 under gravity and capillary action, flushing the microsponge head 101 and eluting the adsorbed biomarkers into the microfluidic chip 200. S3: Place the microfluidic chip 200 connected to the tear sampler 100 into the slot of the portable reader 300. The portable reader 300 automatically applies negative pressure through the negative pressure port 202, causing 100 μL of sample-buffer mixture to flow through four detection microchannels within 5 minutes. During this process, Aβ42 protein in the sample is specifically captured by the anti-Aβ42 monoclonal antibody in the first detection microchannel 205a, forming an antigen-antibody complex. T-tau protein in the sample is specifically captured by the anti-T-tau monoclonal antibody in the second detection microchannel 205b, forming an antigen-antibody complex. Lactoferrin in the sample is specifically captured by the anti-lactoferrin monoclonal antibody in the third detection microchannel 205c, forming an antigen-antibody complex. microRNA-200b-5p in the sample hybridizes with the captured DNA probe in the fourth detection microchannel 205d. S4: The portable reader 300 automatically draws cleaning fluid from a cleaning fluid reservoir on the microfluidic chip 200 to flush each detection microchannel and remove unbound impurities; S5: For the first detection microchannel 205a, the second detection microchannel 205b, and the third detection microchannel 205c, an electrochemical immunoassay was used. The portable reader 300 injected a secondary antibody solution containing horseradish peroxidase into the three detection microchannels, respectively. After incubation and washing, TMB was injected. Subsequently, the potentiostat inside the portable reader 300 applied a defined voltage to the microelectrode assembly 208 and measured the generated reduction current. The current intensity was proportional to the concentration of the captured target protein. S6: For the fourth detection microchannel 205d, an electrochemiluminescence method is used to capture DNA probes labeled with ruthenium bipyridine, which generates a light signal after voltage is applied, and is detected by a photodiode inside the portable reader 300; S7: The Portable Reader 300 has a built-in microprocessor that takes the collected current and fluorescence signal values, substitutes them into a pre-stored calibration curve, calculates the concentration of various biomarkers, and then integrates these concentration values into a comprehensive Alzheimer's disease risk index through a diagnostic model based on logistic regression. The result is displayed as "low risk," "medium risk," or "high risk" on the screen. Data can be transferred to a smartphone or the cloud via Bluetooth or USB.
[0035] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A tear film detection device for early screening of Alzheimer's disease, characterized in that: The device includes a tear sampler, a microfluidic chip, and a portable reader. The tear sampler is used to deliver collected and pre-processed tear samples to the microfluidic chip. The microfluidic chip has a sample input port, and the tear sampler is connected to the sample input port. The microfluidic chip has microchannel structures for detecting biomarkers, and the microfluidic chip can be placed in the slot of the portable reader. The portable reader is used to perform signal detection, analyze the monitoring data, and output diagnostic results.
2. The tear film detection device for early screening of Alzheimer's disease according to claim 1, characterized in that: The tear sampler includes a microsponge head, a polymer rod connected to the microsponge head, and a handle body connected to the other end of the polymer rod.
3. The tear film detection device for early screening of Alzheimer's disease according to claim 2, characterized in that: The handle body has a buffer reservoir inside, and a sealing membrane is provided on the buffer reservoir. The handle body has a pressable sealing plug at its end, and an interface end at its end, which is connected to the sample input port.
4. A tear film detection device for early screening of Alzheimer's disease according to claim 1, characterized in that: The microfluidic chip includes a top cover layer and a microchannel layer. The sample input port is disposed on the top cover layer. The top cover layer is provided with a negative pressure port for driving liquid to flow within the microfluidic chip. The microchannel structure is disposed on the microchannel layer.
5. A tear film detection device for early screening of Alzheimer's disease according to claim 4, characterized in that: The microchannel structure includes a serpentine hybrid channel. One end of the serpentine hybrid channel is connected to the sample inlet, and the other end of the serpentine hybrid channel is connected to a flow divider. The other end of the flow divider is connected to a first detection microchannel, a second detection microchannel, a third detection microchannel for protein biomarker detection, and a fourth detection microchannel for microRNA biomarker detection. The first, second, and third detection microchannels are each provided with an immunosensor region, and the fourth detection microchannel is provided with a nucleic acid hybridization region.
6. A tear film detection device for early screening of Alzheimer's disease according to claim 5, characterized in that: The first, second, and third detection microchannels are all equipped with protein marker antibodies, and the fourth detection microchannel is equipped with a DNA capture probe.
7. A tear film detection device for early screening of Alzheimer's disease according to claim 5, characterized in that: The microfluidic chip also includes a substrate layer on which a microelectrode assembly for protein biomarker detection is disposed. The portion of the substrate layer corresponding to the nucleic acid hybridization region is transparent.
8. A tear film detection method for early screening of Alzheimer's disease, based on the device according to any one of claims 1-7, characterized in that, Includes the following steps: S1: During sampling, place the micro-sponge head of the tear sampler into the conjunctival sac of the patient's lower eyelid to absorb tears for 1-2 minutes; S2: After sampling, insert the interface of the tear sampler into the sample input port of the microfluidic chip and tighten it. This tightening action will simultaneously press down the sealing plug in the tear sampler, puncture the sealing membrane of the buffer reservoir, and allow the buffer to flow through the polymer rod under gravity and capillary action, flushing the microsponge head and eluting the adsorbed biomarkers into the microfluidic chip. S3: Place the microfluidic chip connected to the tear sampler into the slot of the portable reader. The portable reader automatically applies negative pressure through the negative pressure port, driving 100 μL of sample-buffer mixture to flow through four detection microchannels within 5 minutes. During this process, Aβ42 protein in the sample is specifically captured by the anti-Aβ42 monoclonal antibody in the first detection microchannel, forming an antigen-antibody complex. T-tau protein in the sample is specifically captured by the anti-T-tau monoclonal antibody in the second detection microchannel, forming an antigen-antibody complex. Lactoferrin in the sample is specifically captured by the anti-lactoferrin monoclonal antibody in the third detection microchannel, forming an antigen-antibody complex. microRNA-200b-5p in the sample hybridizes with the captured DNA probe in the fourth detection microchannel. S4: The portable reader automatically draws cleaning fluid from a cleaning fluid reservoir on the microfluidic chip to flush each detection microchannel and remove unbound impurities; S5: For the first, second, and third detection microchannels, an electrochemical immunoassay was used. A portable reader injected a secondary antibody solution containing horseradish peroxidase into each of the three detection microchannels. After incubation and washing, an electrochemical substrate was injected. Subsequently, a potentiostat inside the portable reader applied a specific voltage to the microelectrode assembly and measured the resulting reduction current. The current intensity was proportional to the concentration of the captured target protein. S6: For the fourth detection microchannel, a hybridization chain reaction is used to amplify the fluorescence signal. The portable reader sequentially injects two partially complementary DNA hairpin structures, H1 and H2, labeled with fluorescent groups, into the fourth detection microchannel. If the target microRNA is present, it will trigger the alternating hybridization of H1 and H2 to form a long double-stranded DNA polymer, thereby fixing a large number of fluorescent groups in the nucleic acid hybridization region. Subsequently, the LED light source built into the portable reader excites fluorescence, and the emitted fluorescence intensity is detected by a photodiode. The fluorescence intensity is proportional to the concentration of the target microRNA. S7: The portable reader has a built-in microprocessor that takes the collected current and fluorescence signal values, substitutes them into a pre-stored calibration curve, calculates the concentration of various biomarkers, and then integrates these concentration values into a comprehensive Alzheimer's disease risk index through a diagnostic model based on machine learning algorithms. The result is displayed as "low risk," "medium risk," or "high risk." Data can be transferred to a smartphone or the cloud via Bluetooth or USB.
9. A tear film detection method for early screening of Alzheimer's disease according to claim 8, characterized in that: Replace the electrochemical immunoassay in S5 with the chemiluminescent immunoassay. Inject the chemiluminescent substrate into the three detection microchannels using a portable reader, and detect the luminescence intensity using a photomultiplier tube inside the portable reader. The remaining steps remain unchanged.
10. A tear film detection method for early screening of Alzheimer's disease according to claim 8, characterized in that: The hybridization chain reaction in S6 that amplifies the fluorescence signal is replaced with electrochemiluminescence. The DNA capture probe can be labeled with an electrochemiluminescence tag, which generates a light signal after voltage is applied. This signal is then detected by a photodiode inside a portable reader. The remaining steps remain unchanged.