A dry-type bipolar electrochemiluminescence microfluidic chip and application thereof in protein and nucleic acid detection

Abstract

The application discloses a dry-type bipolar electrochemiluminescence microfluidic chip and application thereof in protein and nucleic acid detection, wherein the chip comprises a bottom plate, an electrode sheet, a detection sheet, a combination sheet and a sample adding sheet which are stacked in sequence; the integrated bipolar electrode of the electrode sheet is in a "one" shape and has more than one bipolar electrode anode and one bipolar electrode cathode; the multiple bipolar electrode anodes are in a series connection relationship; the number of report channels is consistent with the number of bipolar electrode anodes; the buffer channel and the sample channel on the sample adding sheet correspond to the channel positions on the combination sheet; and the sample channel on the sample adding sheet is one or more. The bipolar electrochemiluminescence microfluidic chip can be used for simultaneous detection of multiple biomarkers in the same sample, and can also be used for simultaneous detection of single or multiple biomarkers in different samples, so as to meet different application requirements in different scenes.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chips, specifically relating to a dry bipolar electrochemiluminescence microfluidic chip and its application in protein and nucleic acid detection. Background Technology

[0002] Microfluidic technology aims to integrate sample introduction, sample processing, and detection onto a single microchip. Compared to traditional detection methods and instruments, microfluidic chips offer advantages such as fluid controllability, low sample and reagent consumption, and rapid analysis. In recent years, bipolar electrodes combined with electrochemiluminescence (ECL) technology have achieved diverse developments, ranging from single-pass detection to high-throughput and multivariate detection. On microfluidic chips, bipolar electrodes are often designed in different shapes and sizes to meet various detection needs. ECL technology is used for immunoassay or nucleic acid detection, typically by labeling electrochemiluminescent probes onto antibodies (or antigens) or nucleic acids. Under electrical triggering, a specific electrochemiluminescence reaction is induced, enabling rapid, sensitive, and specific detection of protein and nucleic acid biomarkers. Bipolar ECL microfluidic chips have the potential to provide rapid analytical tools for in vitro diagnostics, point-of-care testing, and other fields.

[0003] Simultaneous identification of two analytes in the same sample using a single detection platform not only offers economic benefits but also greatly aids in disease diagnosis, postoperative evaluation, drug discovery, precision medicine, and early diagnosis. Early methods for simultaneous detection of nucleic acids and proteins include multi-parameter cell analysis systems, DNA arrays combined with polymerase chain reaction (PCR), and protein arrays combined with enzyme-linked immunosorbent assay (ELISA). However, these analytical methods typically employ wet chemistry (wet chromatography) techniques, requiring multiple liquid additions and washing steps, resulting in complex operations, long analysis times, and the need for large analytical instruments and specialized operators. These factors limit their application in on-site, point-of-care testing. Summary of the Invention

[0004] The purpose of this invention is to provide a dry bipolar electrochemiluminescence microfluidic chip for protein and nucleic acid detection, solving the problem of detecting different biomarkers in the same sample or biomarkers in different samples. By combining dry chemical analysis technology with a microfluidic chip, the reagents required for the reaction are immobilized on the microfluidic chip. During detection, only the sample to be tested and a small amount of buffer solution need to be added. The operation is simple and the detection is rapid, providing a new approach for point-of-care testing.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A dry bipolar electrochemiluminescence microfluidic chip includes a substrate, electrode sheets, detection sheets, binding sheets, and sample loading sheets.

[0007] The electrode sheet includes an integrated bipolar electrode, driving electrodes positive and negative, a reporting channel and a support channel; the integrated bipolar electrode is in the shape of an "I" and has one or more (also referred to as "multiple" in this application) bipolar electrode anodes and one bipolar electrode cathode; the multiple bipolar electrode anodes are connected in series; the number of reporting channels is the same as the number of bipolar electrode anodes;

[0008] Each bipolar electrode anode is connected to the negative electrode of the driving electrode through its corresponding reporting channel, and the bipolar electrode cathode is connected to the positive electrode of the driving electrode through the support channel.

[0009] The detection strip is stacked on top of the bipolar electrode of the electrode sheet; the detection strip contains hydrophilic channels corresponding to the position and number of the reporting channel and support channel on the electrode sheet, wherein the hydrophilic channel corresponding to the support channel on the electrode sheet is a buffer channel, and the rest are channels for the sample to be tested.

[0010] The binding plate is stacked on top of the detection plate and contains hydrophilic channels corresponding to the position and number of hydrophilic channels on the detection plate. It is also divided into buffer channels and test sample channels.

[0011] The sample loading strip is stacked on top of the conjugate strip; the buffer solution channel and the sample channel on the sample loading strip correspond to the channel positions on the conjugate strip;

[0012] The sample loading plate can have one test sample channel (connected to multiple test sample channels on the binding plate) for detecting different biomarkers in the same sample;

[0013] The number of test sample channels on the sample application strip is the same as that on the binding strip (multiple channels), used for the detection of biomarkers in different samples;

[0014] The electrode sheet and the bonding sheet are preferably made of cotton cloth as a substrate; the detection sheet is preferably made of absorbent paper as a substrate, which has good light transmittance; the sample application sheet is preferably made of non-woven fabric as a substrate, which has good liquid flowability.

[0015] A microfluidic chip detection box includes the aforementioned dry bipolar electrochemiluminescence microfluidic chip, a transparent cover, and a shell;

[0016] The outer casing includes an upper cover and a lower cover;

[0017] The top cover is provided with a sample addition hole, a buffer addition hole, an observation window, and an electrode contact area;

[0018] The sample application well corresponds to the sample channel on the sample application strip, and the buffer solution addition well corresponds to the buffer solution channel on the sample application strip. The observation window corresponds to the sample channel on the detection strip.

[0019] The lower cover has a chip fixing area for placing microfluidic chips;

[0020] The transparent cover sheet covers the microfluidic chip (including the overlapping part of the sample application sheet and the bonding sheet, the bonding sheet, the detection sheet and the electrode sheet; it does not affect sample application) to prevent the fiber microfluidic chip from directly contacting the outside world.

[0021] The dry bipolar electrochemiluminescence microfluidic chip and microfluidic chip detection kit described herein can be used for the detection of different biomarkers in the same sample, or for the detection of biomarkers in different samples.

[0022] The biomarkers mentioned are proteins and / or nucleic acids.

[0023] The aforementioned dry bipolar electrochemiluminescence microfluidic chip is used for the detection of biomarkers, and includes the following steps:

[0024] (1) Add the treatment solution to the test sample channel of the detection strip and the conjugate strip, then fix the capture antibody (or capture probe) that can specifically bind to the biomarker in the test sample channel of the detection strip, and dry and fix the labeled antibody (or signal probe) that can specifically bind to the biomarker in the test sample channel of the conjugate strip.

[0025] When detecting different biomarkers simultaneously, capture antibodies or capture probes that specifically bind to the biomarkers are immobilized in different test sample channels on the detection strip, and labeled antibodies or signal probes that specifically bind to the biomarkers are dried and immobilized in different test sample channels on the binding strip.

[0026] The test sample channels on the detection chip and the binding chip are pretreated with a processing solution to increase the hydrophilicity and storage stability of the chip.

[0027] Furthermore, the treatment solution contains 0.1% Tween-20, 50 mg / mL sucrose, 10 mg / mL bovine serum albumin, and 10 mg / mL polyvinylpyrrolidone.

[0028] Furthermore, the labeled antibody or signal probe contains an electrochemiluminescent group and an antibody or nucleic acid chain that specifically binds to the biomarker to be tested;

[0029] Furthermore, the electrochemiluminescent group is a coupling complex of an electrochemiluminescent substance (such as ruthenium bipyridine) and an intramolecular co-reaction reagent (such as polylysine).

[0030] (2) Add the test sample solution to the sample well. The solution flows from the sample application plate to the conjugation plate. The biomarker specifically binds to the labeled antibody (or signal probe) on the conjugation plate to form a "labeled antibody-biomarker" (or "signal probe-biomarker") complex. This complex further flows with the liquid to the detection plate and specifically binds to the capture antibody (or capture probe) on the detection plate to form a "labeled antibody-biomarker-capture antibody" (or "signal probe-biomarker-capture probe") complex. After the test sample solution is added, add buffer solution to the buffer addition well. The buffer solution flows to the electrode plate and fills the support channels on the electrode plate.

[0031] (3) Add a certain volume of rinsing buffer to the sample well to promote the further generation of the “labeled antibody-biomarker-capture antibody” (or “signal probe-biomarker-capture probe”) complex, while rinsing the unbound substances on the detection sheet and filling the reporter channel on the electrode sheet.

[0032] Preferably, after adding the sample solution to be tested for 4 minutes in step (2), the rinsing buffer is added in step (3);

[0033] (4) Push the dry bipolar electrochemiluminescence microfluidic chip into the electrochemiluminescence analyzer, apply a driving voltage to the driving electrode to trigger the electrochemiluminescence reaction on the sample channel on the detection chip, and the generated light signal is collected by the camera and transmitted to the data analysis and storage unit to realize the detection of biomarkers.

[0034] The preferred driving voltage is 8V;

[0035] The rinsing buffer is a phosphate buffer (10×PBS, pH 7.2-7.4) containing 0.1% Tween-20.

[0036] The present invention has the following advantages and effects compared with the prior art:

[0037] 1. This invention proposes a dry bipolar electrochemiluminescence microfluidic chip, which overcomes the shortcomings of traditional electrochemiluminescence biosensor electrodes, such as difficulty in modification, multiple liquid additions, and repeated rinsing, and simplifies the chip modification and detection process.

[0038] 2. Currently, most diagnostic methods detect proteins or nucleic acids separately. The fiber microfluidic chip in this invention has multiple liquid pathways for the sample to be tested, which do not interfere with each other, allowing for simultaneous protein immunoreaction and nucleic acid hybridization reactions, enabling the simultaneous detection of proteins and nucleic acids.

[0039] 3. Most currently developed biosensors for the simultaneous detection of proteins and nucleic acids are based on colorimetric, fluorescence, and electrochemical methods, which have drawbacks such as low sensitivity, susceptibility to background light sources, and difficulty in signal differentiation. The dry bipolar electrochemiluminescence microfluidic chip proposed in this invention overcomes these shortcomings, offering high sensitivity, low background, and easy control. In particular, the multiple luminescent signals are physically separated, eliminating signal cross-interference and increasing the accuracy of the detection results.

[0040] 4. Most current detection methods have a single detection mode. The bipolar electrochemiluminescence microfluidic chip proposed in this invention can not only be used for the simultaneous detection of multiple biomarkers in the same sample, but also for the simultaneous detection of single or multiple biomarkers in different samples, meeting different application needs in different scenarios.

[0041] 5. The dry bipolar electrochemiluminescence microfluidic chip proposed in this invention only requires the addition of the sample and buffer solution to obtain detection results within 6 minutes. Compared with traditional electrochemiluminescence analyzers, it is more convenient and faster, making it suitable for rapid on-site detection. Attached Figure Description

[0042] Figure 1 This is a structural decomposition diagram of a dry bipolar electrochemiluminescence microfluidic chip (with structure A plus sample).

[0043] Figure 2 This is a schematic diagram of the overall structure of a dry bipolar electrochemiluminescence microfluidic chip (with structure A plus sample).

[0044] Figure 3 This is an exploded view of the microfluidic chip detection box (with structure A plus sample).

[0045] Figure 4 This is a schematic diagram of the overall structure of a microfluidic chip detection box (with structure A plus a sample).

[0046] Figure 5 This is a structural decomposition diagram of a microfluidic chip (with structure B plus a sample).

[0047] Figure 6 This is a schematic diagram of the overall structure of a microfluidic chip (with structure B plus a sample).

[0048] Figure 7 The graph shows the relationship between the electrochemiluminescence intensity and the AD7c-NTP concentration on a microfluidic chip (with structure A plus sample).

[0049] Figure 8 The graph shows the relationship between the electrochemiluminescence intensity on a microfluidic chip (with structure A sample) and the concentrations of AD7c-NTP and ApoEε4 in the same sample.

[0050] Figure 9 For comparison, see the exploded view of the chip structure;

[0051] Figure 10 For comparison, see the overall structural diagram of the chip;

[0052] Figure 11 To compare the relationship between the electrochemiluminescence intensity values ​​on the chip and the concentrations of AD7c-NTP and ApoEε4 in the same sample;

[0053] 1-Sample loading plate; 1-1, 1-2-Sample loading plate sample channel; 1-3-Sample loading plate buffer channel; 2-Binding plate; 2-1, 2-2-Binding plate sample channel; 2-3-Binding plate buffer channel; 3-Detection plate; 3-1, 3-2-Detection plate sample channel; 3-3-Detection plate buffer channel; 4-Electrode plate; 4-1, 4-2-Closed bipolar electrode anode; 4-3-Closed bipolar electrode cathode; 4-4, 4-5-Report channel; 4-6-Support channel; 4-7-Drive electrode negative electrode; 4-8-Drive electrode positive electrode; 5-Base plate; 6-Top cover; 6-1-Sample loading hole; 6-2-Buffer adding hole; 6-3-Observation window; 6-4-Electrode contact area; 7-Transparent cover plate; 8-Fiber microfluidic chip; 9-Lower cover; 9-1-Chip fixing area. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0055] Example 1

[0056] The fabrication process of the dry bipolar electrochemiluminescence microfluidic chip is as follows:

[0057] 1. The microfluidic chip configuration (including sample loading plate 1, binding plate 2, detection plate 3, and electrode plate 4) was designed using the drawing software Adobe Illustrator CS6. The carbon electrodes (including driving electrodes and closed bipolar electrodes) were made using carbon ink screen printing technology, and the hydrophilic channels (including support channels, reporter channels, sample channels, and buffer channels) were made using nylon ink screen printing technology.

[0058] 2. Design the shell structure (upper cover 6 and lower cover 9) using the drawing software Solidworks 2020. The shell is printed by a 3D printer.

[0059] The resulting microfluidic chip, such as Figure 1 , Figure 2 , Figure 5 , Figure 6As shown, it includes a sample plate 1, a bonding plate 2, a detection plate 3, an electrode plate 4, and a base plate 5; the sample plate 1, the bonding plate 2, the detection plate 3, and the electrode plate 4 are respectively covered with non-woven fabric, cotton cloth, absorbent paper, and cotton cloth as substrates;

[0060] Electrode 4 includes an integrated bipolar electrode, a driving electrode negative electrode 4-7 and a driving electrode positive electrode 4-8, reporting channels 4-4 and 4-5, and a support channel 4-6. The integrated bipolar electrode is in the shape of an "I" and has two bipolar electrode anodes 4-1 and 4-2 and one bipolar electrode cathode 4-3. The two bipolar electrode anodes 4-1 and 4-2 are connected in series. The bipolar electrode anodes 4-1 and 4-2 are connected to the driving electrode negative electrode 4-7 through the corresponding reporting channels 4-4 and 4-5, and the bipolar electrode cathode 4-3 is connected to the driving electrode positive electrode 4-8 through the support channel 4-6.

[0061] The detection strip 3 is stacked on top of the bipolar electrode 4. The detection strip 3 is designed with three hydrophilic channels 3-1, 3-2 and 3-3, one of which is a buffer channel 3-3, and the other two are test sample channels 3-1 and 3-2. The buffer channel 3-3 on the detection strip 3 corresponds to and overlaps with the bipolar cathode 4-3 on the electrode 4. The test sample channels 3-1 and 3-2 on the detection strip 3 correspond to and overlap with the bipolar anodes 4-1 and 4-2 on the electrode 4, respectively.

[0062] The binding plate 2 is stacked on top of the detection plate 3. The binding plate 2 is designed with three hydrophilic channels 2-1, 2-2 and 2-3, one of which is a buffer channel 2-3, and the other two are test sample channels 2-1 and 2-2. The buffer channel 2-3 on the binding plate 2 corresponds to the buffer channel 3-3 on the detection plate 3 and overlaps by 1.5 mm. The test sample channels 2-1 and 2-2 on the binding plate 2 correspond to the test sample channels 3-1 and 3-2 on the detection plate 3 and overlap by 1.5 mm, respectively.

[0063] Sample patch 1 is stacked on top of conjugate patch 2; sample patch 1 has two hydrophilic channels 1-1 and 1-3 (structure A), one of which is a buffer channel 1-3, and the other is a "T"-shaped sample channel 1-1; the buffer channel 1-3 on sample patch 1 corresponds to the buffer channel 2-3 on conjugate patch 2 and overlaps by 1.5 mm; the wider portion of the sample channel 1-1 on sample patch 1 corresponds to the sample channels 2-1 and 2-2 on conjugate patch 2 and overlaps by 1.5 mm. Figure 1 , Figure 2 );

[0064] The sample application piece 1 can be further designed with three hydrophilic channels 1-1, 1-2, and 1-3 (structure B), one of which is a buffer channel 1-3, and the other two are test sample channels 1-1 and 1-2; the buffer channel 1-3 on the sample application piece 1 corresponds to the buffer channel 2-3 on the binding piece 2 and overlaps by 1.5 mm; the test sample channels 1-1 and 1-2 on the sample application piece 1 correspond to the test sample channels 2-1 and 2-2 on the binding piece 2 and overlap by 1.5 mm, respectively. Figure 5 , Figure 6 );

[0065] The resulting outer shell ( Figure 3 , Figure 4 The device includes an upper cover 6 and a lower cover 9. The upper cover 6 has a sample application hole 6-1, a buffer addition hole 6-2, an observation window 6-3, and an electrode contact area 6-4. The sample application hole 6-1 corresponds to the narrower part of the sample channel 1-1 on the sample application sheet 1 (structure A) or the sample channels 1-1 and 1-2 on the sample application sheet 1 (structure B). The buffer addition hole 6-2 corresponds to the buffer channel 1-3 on the sample application sheet 1. The observation window 6-3 corresponds to the sample channels 3-1 and 3-2 on the detection sheet 3. The electrode contact area 6-4 corresponds to the positive electrode 4-8 and the negative electrode 4-7 of the driving electrode on the electrode sheet 4. The lower cover 9 has a chip fixing area 9-1 for placing the microfluidic chip 8. A transparent cover 7 covers the fiber microfluidic chip 8 to prevent the fiber microfluidic chip 8 from directly contacting the outside world.

[0066] Example 2

[0067] The dry bipolar electrochemiluminescence microfluidic chip (with structure A sample loading plate) of Example 1 is used for the accurate detection of proteins, including the following steps:

[0068] (1) Microfluidic chip modification

[0069] Add 3 μL of treatment solution (a mixture of 0.1% Tween-20, 50 mg / mL sucrose, 10 mg / mL bovine serum albumin, and 10 mg / mL polyvinylpyrrolidone) to the two test sample channels 2-1 and 2-2 of the conjugate tablet 2, and dry under vacuum at 37 °C for 30 min; then add 3 μL of labeled antibody (70 μg / mL) against Alzheimer's disease-associated neurofilament protein (AD7c-NTP), and dry under vacuum at 37 °C; the labeled antibody contains a polylysine-bipyridine ruthenium complex.

[0070] Add 3 μL of treatment solution to each of the two test sample channels 3-1 and 3-2 of the test strip 3, and vacuum dry at 37°C for 30 min; then add 3 μL of capture antibody against AD7c-NTP (120 μg / mL), and incubate at 37°C for 30 min. The capture antibody is then immobilized in the two test sample channels 3-1 and 3-2 by covalent bonding of chitosan and glutaraldehyde.

[0071] (2) Chip Assembly

[0072] like Figure 2 As shown, the base plate 5 is single-sided adhesive. The electrode sheet 4, the detection sheet 3, the bonding sheet 2 and the sample application sheet 1 are stacked and fixed on the base plate 5 in sequence to assemble the microfluidic chip 8.

[0073] like Figure 3 , Figure 4 As shown, the assembled microfluidic chip 8 is placed in the chip fixing area 9-1 of the lower cover 9; the transparent cover 7 covers the microfluidic chip 8 to prevent the microfluidic chip 8 from directly contacting the outside world; the upper cover 6 is pressed tightly to complete the assembly of the dry bipolar electrochemiluminescence microfluidic chip.

[0074] (3) Protein immune response

[0075] Add 20 μL of the test sample solution (containing AD7c-NTP) to sample well 6-1. The solution flows from the test sample channel 1-1 of sample plate 1 to the two test sample channels 2-1 and 2-2 of binding plate 2. The target protein AD7c-NTP specifically binds to the labeled antibody on binding plate 2 to form a "labeled antibody-AD7c-NTP" complex. This complex further flows to detection plate 3 and specifically binds to the capture antibody on detection plate 3 to form a "labeled antibody-AD7c-NTP-capture antibody" complex. After the test sample solution is added, add 20 μL of phosphate buffer to buffer addition well 6-2. The buffer flows to electrode plate 4 and fills the support channels 4-6 of electrode plate 4.

[0076] Subsequently, after waiting for the protein immunoreaction to proceed for 4 minutes, 20 μL of rinsing buffer (containing 0.1% Tween-20 phosphate buffer) was added to sample well 6-1 to promote the further formation of the "labeled antibody-AD7c-NTP-capture antibody" complex. At the same time, the unbound material on the detection strip 3 was rinsed and the two reporter channels 4-4 and 4-5 of the electrode strip 4 were filled.

[0077] (4) Electrochemiluminescence detection

[0078] The dry bipolar electrochemiluminescence microfluidic chip is pushed into the electrochemiluminescence analyzer (patent application number 202210518683.4). The detection start button is pressed, and an 8V driving voltage is applied to the negative electrode 4-7 and the positive electrode 4-8 of the driving electrode to simultaneously trigger the electrochemiluminescence reaction on the two sample channels 3-1 and 3-2 on the detection chip 3. The generated light signal is collected by the camera and transmitted to the data analysis and storage unit to achieve accurate detection of AD7c-NTP in the sample solution.

[0079] The relationship between electrochemiluminescence intensity and AD7c-NTP concentration is as follows: Figure 7 As shown, the dry bipolar electrochemiluminescence microfluidic chip has the ability to accurately detect proteins.

[0080] Example 3

[0081] The dry bipolar electrochemiluminescence microfluidic chip (with structure A sample loading plate) of Example 1 is used for the simultaneous detection of proteins and nucleic acids in the same sample, including the following steps:

[0082] (1) Modification of fiber microfluidic chip

[0083] Add 3 μL of processing solution to each of the two test sample channels 2-1 and 2-2 of the conjugate patch 2, and dry under vacuum at 37 °C for 30 min. Then add 3 μL of labeled antibody against AD7c-NTP (70 μg / mL) and 3 μL of signal probe complementary to the apolipoprotein E gene (ApoEε4) (0.7 μM), and dry under vacuum at 37 °C for 30 min. Both the labeled antibody and the signal probe contain a polylysine-bipyridine ruthenium complex.

[0084] Add 3 μL of treatment solution to each of the two test sample channels 3-1 and 3-2 of the test strip 3, and dry under vacuum at 37°C for 30 min. Then add 3 μL of capture antibody against AD7c-NTP (120 μg / mL) and 3 μL of capture probe complementary to ApoEε4 (1 μM), and incubate at 37°C for 30 min. The capture antibody and capture probe are then immobilized in the test sample channels 3-1 and 3-2 by covalent bonding of chitosan and glutaraldehyde.

[0085] (2) Chip assembly is the same as in Example 2

[0086] (3) Protein immune response and nucleic acid hybridization reaction

[0087] Add 20 μL of the test sample solution (containing AD7c-NTP and ApoEε4) to sample well 6-1. The solution flows from the test sample channel 1-1 of sample patch 1 to the two test sample channels 2-1 and 2-2 of binding patch 2. The target protein AD7c-NTP and the target nucleic acid ApoEε4 specifically bind to the labeled antibody and signal probe on binding patch 2, respectively, forming "labeled antibody-AD7c-NTP" and "signal probe-ApoEε4" complexes. The complexes further flow to detection patch 3 and specifically bind to the capture antibody and capture probe on detection patch 3, forming "labeled antibody-AD7c-NTP-capture antibody" and "signal probe-ApoEε4-capture probe" complexes. After the test sample solution is added, add 20 μL of phosphate buffer to buffer addition well 6-2. The buffer flows to electrode patch 4 and fills the support channel 4-6 of electrode patch 4.

[0088] Subsequently, after waiting for the protein immunoassay and nucleic acid hybridization reaction to proceed for 4 minutes, 20 μL of washing buffer was added to sample well 6-1 to promote the further formation of the "labeled antibody-AD7c-NTP-capture antibody" and "signal probe-ApoEε4-capture probe" complexes. At the same time, unbound material on the detection strip 3 was washed and the reporting channels 4-4 and 4-5 of the electrode strip 4 were filled.

[0089] (4) Electrochemiluminescence detection

[0090] The dry bipolar electrochemiluminescence microfluidic chip is pushed into the electrochemiluminescence analyzer. The detection start button is pressed, and an 8V driving voltage is applied to the negative electrode 4-7 and the positive electrode 4-8 of the driving electrode to simultaneously trigger the electrochemiluminescence reaction on the two sample channels 3-1 and 3-2 on the detection chip 3. The generated light signal is collected by the camera and transmitted to the data analysis and storage unit to realize the simultaneous detection of AD7c-NTP and ApoEε4 in the same sample.

[0091] The relationship between electrochemiluminescence intensity and AD7c-NTP concentration and ApoEε4 concentration is as follows: Figure 8 As shown, the dry bipolar electrochemiluminescence microfluidic chip has the ability to simultaneously detect proteins and nucleic acids in the same sample.

[0092] Comparative Example

[0093] A dry bipolar electrochemiluminescence microfluidic chip (patent application number 202210518635.5, hereinafter referred to as the comparison chip) is used for the simultaneous detection of proteins and nucleic acids in the same sample, including the following steps:

[0094] (1) Fabrication of the comparison chip

[0095] The chip configuration was designed using Adobe Illustrator CS6 drawing software. The carbon electrodes (including the driving electrode and the closed bipolar electrode) were fabricated using carbon ink screen printing technology, while the hydrophilic channels (including the support channel, the reporter channel, the sample channel, and the buffer channel) were fabricated using nylon ink screen printing technology.

[0096] The resulting comparison chip is as follows Figure 9 , Figure 10 As shown, it includes a sample plate 1, a bonding plate 2, a detection plate 3, an electrode plate 4, and a base plate 5. The sample plate 1, the bonding plate 2, the detection plate 3, and the electrode plate 4 are respectively based on non-woven fabric, cotton cloth, absorbent paper, and cotton cloth.

[0097] The electrode sheet 4 includes an integrated bipolar electrode, a positive driving electrode 4-8, a negative driving electrode 4-7, a reporting channel 4-4, and a support channel 4-6. The integrated bipolar electrode is "E"-shaped and has bipolar anodes 4-1 and 4-2 and a bipolar cathode 4-3. The two bipolar anodes 4-1 and 4-2 are connected in parallel. The bipolar anodes 4-1 and 4-2 are connected to the negative driving electrode 4-7 through the reporting channel 4-4, and the bipolar cathode 4-3 is connected to the positive driving electrode 4-8 through the support channel 4-6.

[0098] The detection strip 3 is stacked on top of the electrode sheet 4. The detection strip 3 is designed with three hydrophilic channels 3-1, 3-2, and 3-3, one of which is a buffer solution channel 3-3, and the other two are test sample channels 3-1 and 3-2. The buffer solution channel 3-3 on the detection strip 3 corresponds to and overlaps with the anode of the bipolar electrode 4-3 on the electrode sheet 4. The test sample channels 3-1 and 3-2 on the detection strip 3 correspond to and overlap with the anode of the bipolar electrode 4-1 and 4-2 on the electrode sheet 4, respectively.

[0099] The binding plate 2 is stacked on top of the detection plate 3; the binding plate 2 is designed with two hydrophilic channels 2-1 and 2-3, one of which is a buffer channel 2-3 and the other is a sample channel 2-1; the buffer channel 2-3 on the binding plate 2 corresponds to the buffer channel 3-3 on the detection plate 3 and overlaps by 1.5 mm; the sample channel 2-1 on the binding plate 2 also corresponds to the two sample channels 3-1 and 3-2 on the detection plate 3 and overlaps by 1.5 mm.

[0100] Sample patch 1 is stacked on top of conjugate patch 2; sample patch 1 is designed with two hydrophilic channels 1-1 and 1-3, one of which is a buffer channel 1-3 and the other is a sample channel 1-1; the buffer channel 1-3 on sample patch 1 corresponds to and partially overlaps with the buffer channel 2-3 on conjugate patch 2; the sample channel 1-1 on sample patch 1 corresponds to and overlaps with the sample channel 2-1 on conjugate patch 2 by 1.5 mm.

[0101] (2) Comparison of chip modification

[0102] Add 5 μL of processing solution to the test sample channel 2-1 of the conjugate 2 and vacuum dry at 37 °C for 30 min; then add 6 μL of a mixture of labeled antibody and signal probe (containing a labeled antibody against AD7c-NTP (70 μg / mL) and a signal probe complementary to ApoEε4 (0.7 μM)) and vacuum dry at 37 °C for 30 min. Both the labeled antibody and the signal probe contain a polylysine-bipyridine ruthenium complex.

[0103] Add 3 μL of treatment solution to each of the two test sample channels 3-1 and 3-2 of the test strip 3, and dry under vacuum at 37°C for 30 min. Then add 3 μL of capture antibody against AD7c-NTP (120 μg / mL) and 3 μL of capture probe complementary to ApoEε4 (1 μM), and incubate at 37°C for 30 min. The capture antibody and capture probe are then immobilized in the test sample channels 3-1 and 3-2 by covalent bonding of chitosan and glutaraldehyde.

[0104] (3) Comparison of chip assembly

[0105] like Figure 10 As shown, the base plate 5 is single-sided adhesive. The electrode sheet 4, the detection sheet 3, the bonding sheet 2 and the sample application sheet 1 are stacked and fixed on the base plate 5 in sequence to assemble the microfluidic chip 8.

[0106] The assembled microfluidic chip 8 is placed in the chip fixing area 9-1 of the lower cover 9; the transparent cover 7 covers the fiber microfluidic chip 8 to prevent the fiber microfluidic chip 8 from directly contacting the outside world; the upper cover 6 is pressed tightly to complete the assembly of the dry bipolar electrochemiluminescence microfluidic chip.

[0107] (4) Protein immune response and nucleic acid hybridization reaction

[0108] Add 20 μL of the test sample solution (containing AD7c-NTP and ApoEε4) to sample well 6-1. The solution flows from the test sample channel 1-1 of sample patch 1 to the test sample channel 2-1 of binding patch 2. The target protein AD7c-NTP and the target nucleic acid ApoEε4 specifically bind to the labeled antibody and signal probe on binding patch 2, respectively, forming "labeled antibody-AD7c-NTP" and "signal probe-ApoEε4" complexes. The complexes further flow to the two test sample channels 3-1 and 3-2 of detection patch 3, and specifically bind to the capture antibody and capture probe on test sample channels 3-1 and 3-2, respectively, forming "labeled antibody-AD7c-NTP-capture antibody" and "signal probe-ApoEε4-capture probe" complexes, respectively. After the test sample solution is added, add 20 μL of phosphate buffer to buffer addition well 6-2. The buffer flows to electrode patch 4 and fills the support channel 4-6 of electrode patch 4.

[0109] Subsequently, after waiting 4 minutes for the protein immunoassay and nucleic acid hybridization reaction to proceed, 20 μL of washing buffer was added to sample well 6-1 to promote the further formation of the "labeled antibody-AD7c-NTP-capture antibody" and "signal probe-ApoEε4-capture probe" complexes. At the same time, unbound material on the detection strip 3 was washed and the reporter channel 4-4 of the electrode strip 4 was filled.

[0110] (5) The electrochemiluminescence detection process is the same as in Example 3.

[0111] Figure 11 This represents the relationship between electrochemiluminescence intensity and the concentrations of AD7c-NTP and ApoEε4. As can be seen from the figure, the corresponding luminescence intensity at the same concentration is approximately... Figure 8 The luminescence intensity is reduced to half of that in the control sample. One possible reason for this phenomenon is that, in the control sample 2, the labeled antibody and signal probe on the test sample channel 2-1 can flow into the test sample channels 3-1 and 3-2 of the detection sample 3 simultaneously, which means that the labeled antibody and signal probe cannot all flow into the corresponding test sample channels 3-1 and 3-2 on the detection sample 3, resulting in a significant reduction in luminescence intensity.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A dry bipolar electrochemiluminescence microfluidic chip, comprising a substrate, an electrode sheet, a detection sheet, a binding sheet, and a sample loading sheet; the electrode sheet includes an integrated bipolar electrode, driving electrodes (positive and negative), a reporting channel, and a support channel; the detection sheet is stacked on top of the bipolar electrode sheet; the binding sheet is stacked on top of the detection sheet; and the sample loading sheet is stacked on top of the binding sheet. Its features are: The integrated bipolar electrode of the electrode sheet is in the shape of an "I" and has one or more bipolar anodes and one bipolar cathode. Multiple bipolar electrode anodes are connected in series; the number of reporting channels is the same as the number of bipolar electrode anodes. The detection strip contains hydrophilic channels corresponding to the position and number of reporting channels and support channels on the electrode strip, wherein the hydrophilic channels corresponding to the support channels on the electrode strip are buffer channels, and the rest are channels for the test sample. The binding sheet contains hydrophilic channels corresponding to the position and number of hydrophilic channels on the detection sheet; The buffer solution channel and the test sample channel on the sample application strip correspond to the channel positions on the binding strip; the test sample channel on the sample application strip can be one or more.

2. The microfluidic chip according to claim 1, characterized in that: On the electrode sheet, each bipolar electrode anode is connected to the negative electrode of the driving electrode through its corresponding reporting channel, and the bipolar electrode cathode is connected to the positive electrode of the driving electrode through the support channel.

3. The microfluidic chip according to claim 1, characterized in that: The test strip uses absorbent paper as a substrate.

4. The microfluidic chip according to claim 1, characterized in that: The sample sheet is backed by a non-woven fabric.

5. The microfluidic chip according to claim 1, characterized in that: The electrode sheet and the bonding sheet are backed with cotton cloth.

6. The application of the microfluidic chip according to any one of claims 1-5 in the detection of different biomarkers in the same sample, and in the detection of biomarkers in different samples.

7. The application according to claim 6, characterized in that: The biomarkers mentioned are proteins and / or nucleic acids.

8. A method for detecting biomarkers using a microfluidic chip according to any one of claims 1-5, characterized in that... Includes the following steps: (1) Add the treatment solution to the test sample channel of the detection strip and the conjugate strip, then fix the capture antibody or capture probe that can specifically bind to the biomarker in the test sample channel of the detection strip, and dry and fix the labeled antibody or signal probe that can specifically bind to the biomarker in the test sample channel of the conjugate strip. (2) Add the sample solution to be tested. The solution flows from the sample application strip to the conjugation strip. The biomarker specifically binds to the labeled antibody or signal probe on the conjugation strip to form a "labeled antibody-biomarker" or "signal probe-biomarker" complex. The complex further flows with the liquid to the detection strip and specifically binds to the capture antibody or capture probe on the detection strip to form a "labeled antibody-biomarker-capture antibody" or "signal probe-biomarker-capture probe" complex. After the sample solution to be tested is added, buffer solution is added dropwise. The buffer solution flows to the electrode plate and fills the support channels on the electrode plate. (3) Add a certain volume of rinsing buffer to promote the further generation of the "labeled antibody-biomarker-capture antibody" or "signal probe-biomarker-capture probe" complex, while rinsing the unbound substances on the detection sheet and filling the reporter channel on the electrode sheet. (4) The dry bipolar electrochemiluminescence microfluidic chip is pushed into the electrochemiluminescence analyzer, and a driving voltage is applied to the driving electrode to trigger the electrochemiluminescence reaction on the sample channel on the detection chip. The generated light signal is collected by the camera and transmitted to the data analysis and storage unit to realize the detection of biomarkers.

9. The detection method according to claim 8, characterized in that: In step (1), when different biomarkers are detected simultaneously, capture antibodies or capture probes that specifically bind to the biomarkers are fixed in different test sample channels on the detection sheet, and labeled antibodies or signal probes that specifically bind to the biomarkers are dried and fixed in different test sample channels on the binding sheet.

10. The detection method according to claim 8, characterized in that: The labeled antibody or signal probe described in step (1) contains an electrochemiluminescent group and an antibody or nucleic acid chain that specifically binds to the biomarker to be tested.

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