Paper-based nanofluid concentrator and preparation method and use method thereof

Abstract

The present invention discloses a paper-based nanofluid concentrator and a preparation method and a use method thereof. The concentrator is provided with a positive electrode electrolytic cell and a negative electrode electrolytic cell. The positive electrode electrolytic cell is composed of a cation exchange membrane and a cell body, and the negative electrode electrolytic cell is composed of an anion exchange membrane and a cell body. In this way, the target protein can be concentrated in the main channel while maintaining the stability of the pH value in the main channel. In addition, by adjusting the relative positions of the positive electrode electrolytic cell and the negative electrode electrolytic cell to control the overlap of the enrichment area and the detection line, the concentration and detection steps are carried out simultaneously, greatly shortening the time required for the overall detection.

Description

Technical Field

[0001] The present invention relates to the technical field of protein detection, and in particular to a paper-based nanofluid concentrator and a preparation method and a use method thereof. Background Art

[0002] Microfluidic paper-based analytical devices (μPADs) are low-cost, easy-to-use analytical detection platforms that have been widely used in in vitro diagnostics (IVDs) and point-of-care testing (POCT). Paper-based microfluidic chips use ordinary filter paper as a substrate, leveraging the capillary action of the microporous structure within the paper substrate to achieve directional flow of liquid samples within the hydrophilic region. Compared to traditional microfluidic chips (such as those based on glass, silicon wafers, and polymers), paper-based microfluidic chips do not require expensive equipment, complex and rigorous manufacturing processes, or additional pumping devices to control liquid flow. They offer advantages such as low cost, simple structure, and ease of operation, and are now widely used in biology, chemistry, and medicine. Currently, paper-based microfluidic chips can achieve rapid and immediate detection of target biomolecules (such as nucleic acids and proteins).

[0003] However, in the diagnosis of certain diseases (such as early diabetes, coronary artery disease, and early ovarian cancer), the concentrations of target biomolecules (such as glycated hemoglobin, myeloperoxidase, and lactoferrin) are often very low (in the fM to pM range). Detection of these low-abundance biomolecules still suffers from deficiencies such as insufficient sensitivity and limited quantitation limits. Therefore, in early disease screening and diagnosis, it is necessary to simultaneously achieve pre-concentration and in situ detection of target biomolecules in liquid samples in paper-based microfluidic chips. Ion concentration polarization (ICP) technology, which utilizes the electrokinetic effect of nanofluidics, is considered an effective method for achieving high-fold pre-concentration of low-abundance analytes and can be applied to the pre-concentration of low-abundance target molecules, especially for the pre-concentration of protein molecules that cannot be amplified by PCR. For the currently widely used paper-based detection kits, the following factors mainly affect the concentration and detection of low-abundance target protein molecules: First, although the inherent micron-scale porous structure in the paper substrate can stabilize the eddy currents generated in the ICP, making the concentration of target molecules in the working area of ​​the device more stable, inducing ion concentration polarization requires an external electric field, and the redox reaction at the two end electrodes will cause a sharp change in the pH value in the paper-based device, which may change the net surface charge of the amphiphilic molecules (i.e., protein molecules), and ultimately lead to the inability to continuously concentrate specific protein molecules; Second, the current pre-concentration technology has a low throughput and cannot achieve pre-concentration of target biological molecules in the entire liquid sample (tens to hundreds of microliters) in a short time, resulting in the inability to accurately diagnose diseases, especially for the detection of low-abundance marker protein molecules in early disease screening; Third, the current pre-concentration technology is difficult to achieve concentration and in situ detection at the same time, which increases the time required for detection and reduces the detection efficiency of paper-based detection kits. Summary of the Invention

[0004] The embodiments of the present application provide a paper-based nanofluid concentrator and a preparation method and use method thereof. The concentrator is provided with a positive electrode electrolytic cell and a negative electrode electrolytic cell. The positive electrode electrolytic cell is composed of a cation exchange membrane and a cell body, and the negative electrode electrolytic cell is composed of an anion exchange membrane and a cell body. In this way, the target protein can be concentrated in the main channel while maintaining the stability of the pH value in the main channel. In addition, by adjusting the relative positions of the positive electrode electrolytic cell and the negative electrode electrolytic cell to control the overlap of the enrichment area and the detection line, the concentration and detection steps are carried out simultaneously, greatly shortening the time required for the overall detection.

[0005] The present invention provides a paper-based nanofluid concentrator, comprising a paper-based microfluidic chip and a concentrating module;

[0006] The paper-based microfluidic chip comprises a sample pad, a conjugation pad, a main channel, a water-absorbing pad and a bottom plate, wherein the sample pad, the conjugation pad, the main channel and the water-absorbing pad are all located on the bottom plate; one end of the sample pad covers one end of the conjugation pad; the other end of the conjugation pad covers one end of the main channel; the conjugation pad contains a non-specifically adsorbed detection reagent, wherein the detection reagent includes a fluorescently labeled specific antibody; after the sample solution to be tested passes through the conjugation pad, the detection reagent and the target protein in the sample solution to be tested undergo specific immune binding to form an immunoconjugate containing a fluorescent label; a detection line is provided on the main channel, wherein the detection line is used to capture the target protein; one end of the water-absorbing pad covers the other end of the main channel;

[0007] The concentration module includes a positive electrode electrolysis module and a negative electrode electrolysis module, the positive electrode electrolysis module includes a positive electrode electrolysis cell and a first inert metal electrode, one end of the first inert metal electrode is inserted into the positive electrode electrolysis cell, the other end of the first inert metal electrode is connected to a voltage source, and the material of the bottom of the positive electrode electrolysis cell is a cation exchange membrane; the negative electrode electrolysis module includes a negative electrode electrolysis cell and a second inert metal electrode, one end of the second inert metal electrode is inserted into the negative electrode electrolysis cell, the other end of the second inert metal electrode is connected to the voltage source, and the material of the bottom of the negative electrode electrolysis cell is an anion exchange membrane.

[0008] In some embodiments, a control line is set on the main channel at a position after the detection line and at a preset distance from the detection line according to the flow direction of the sample solution to be tested. The control line is used to capture fluorescently labeled specific antibodies that are not bound to the target protein.

[0009] In some embodiments, the sample pad is made of glass fiber paper.

[0010] In some embodiments, the material of the conjugate pad is glass fiber paper.

[0011] In some embodiments, the material of the main channel is nitrocellulose membrane.

[0012] In some embodiments, a hydrophobic film is disposed on the back side of the main channel.

[0013] In some embodiments, the absorbent pad is made of pure cotton linter pulp filter paper.

[0014] The present invention also provides a method for preparing a paper-based nanofluid concentrator, comprising:

[0015] Bond the main channel to the base plate;

[0016] Adhere one end of the bonding pad to one end of the main channel, and adhere the other end of the bonding pad to the base plate;

[0017] Adhere one end of the sample pad to the other end of the conjugate pad, and adhere the other end of the sample pad to the base plate;

[0018] Adhere one end of the water-absorbing pad to the other end of the main channel, and adhere the other end of the water-absorbing pad to the bottom plate;

[0019] Processing and preparing a cell body and a cell bottom of a positive electrode electrolytic cell, and bonding the cell body and the cell bottom of the positive electrode electrolytic cell to form a positive electrode electrolytic cell;

[0020] The cell body and cell bottom of the negative electrode electrolytic cell are processed and prepared, and the cell body and cell bottom of the negative electrode electrolytic cell are bonded together to form the negative electrode electrolytic cell.

[0021] The present invention also provides a method for using the paper-based nanofluid concentrator, comprising:

[0022] The sample solution to be tested is injected into the sample pad, so that the sample pad absorbs the sample solution to be tested to a supersaturated state, and then the sample solution to be tested flows from the sample pad into the conjugate pad. The target protein in the sample solution to be tested is specifically immunologically bound to the detection reagent in the conjugate pad to form an immunoconjugate containing a fluorescent marker. After the conjugate pad absorbs the sample solution to a supersaturated state, the immunoconjugate flows into the main channel along with the sample solution to be tested. The detection line on the main channel captures the target protein, and the immunoconjugate containing the target protein is retained on the detection line, causing the detection line to exhibit a certain level of fluorescence intensity. After flowing through the main channel, the sample solution to be tested is absorbed by the absorbent pad and is led out of the channel.

[0023] After the main channel is completely soaked by the test sample solution, the positive electrode electrolysis module and the negative electrode electrolysis module are pressed onto the main channel, wherein when the target protein in the test sample solution is concentrated into positively charged molecules, the negative electrode electrolysis module is pressed upstream of the main channel, and the positive electrode electrolysis module is pressed downstream of the main channel; when the target protein in the test sample solution is pre-concentrated into loaded molecules, the positive electrode electrolysis cell is located upstream of the main channel, and the negative electrode electrolysis cell is located downstream of the main channel;

[0024] applying power to the first inert metal electrode and the second inert metal electrode using a voltage source to introduce a potential difference between the positive electrolytic cell and the negative electrolytic cell in the main channel;

[0025] Adjusting the positions of the positive and negative electrolytic cells on the main channel and / or the voltage of the voltage source to form an immunoconjugate enrichment region on the main channel, and to make the enrichment region coincide with the detection line;

[0026] After the voltage source is powered on for a preset time, power is turned off, the fluorescence intensity of the detection line is detected, and the concentration of the target protein is determined based on the fluorescence intensity and a predetermined curve, wherein the predetermined curve includes different concentrations of the target protein and the fluorescence intensities corresponding to the concentrations.

[0027] In some embodiments, the method further comprises: detecting the fluorescence intensity of the control line.

[0028] The present invention provides a paper-based nanofluid concentrator and a preparation method and use method thereof. The concentrator is provided with a positive electrode electrolytic cell and a negative electrode electrolytic cell. The positive electrode electrolytic cell is composed of a cation exchange membrane and a cell body, and the negative electrode electrolytic cell is composed of an anion exchange membrane and a cell body. In this way, the target protein can be concentrated in the main channel while maintaining the stability of the pH value in the main channel. In addition, by adjusting the relative positions of the positive electrode electrolytic cell and the negative electrode electrolytic cell to control the overlap of the enrichment area and the detection line, the concentration and detection steps are carried out simultaneously, greatly shortening the time required for the overall detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematically shows a perspective view of a paper-based nanofluid concentrator provided according to some embodiments;

[0030] Figure 2 The following is a schematic structural diagram of a paper-based microfluidic chip according to some embodiments;

[0031] Figure 3 Schematic diagram of the structure of a paper-based nanofluid concentrator provided according to some embodiments is shown;

[0032] Figure 4 A schematic diagram of a scenario for detecting the fluorescence intensity of a test line and a control line according to some embodiments is exemplified;

[0033] Figure 5 A schematic diagram illustrating a concentration situation according to some embodiments is shown;

[0034] Figure 6 A schematic diagram exemplarily shows an experimental result provided according to some embodiments. DETAILED DESCRIPTION

[0035] In order to better understand the above technical solution, the technical solution of this application is described in detail below through specific implementation methods.

[0036] In order to solve the above technical problems, the embodiments of the present application provide a paper-based nanofluid concentrator and a preparation method and a use method thereof. The concentrator is provided with a positive electrode electrolytic cell and a negative electrode electrolytic cell. The positive electrode electrolytic cell is composed of a cation exchange membrane and a cell body, and the negative electrode electrolytic cell is composed of an anion exchange membrane and a cell body. In this way, the target protein can be concentrated in the main channel while maintaining the stability of the pH value in the main channel. In addition, by adjusting the relative positions of the positive electrode electrolytic cell and the negative electrode electrolytic cell to control the overlap of the enrichment area and the detection line, the concentration and detection steps are carried out simultaneously, which greatly shortens the time required for the overall detection.

[0037] Figure 1 A perspective view of a paper-based nanofluid concentrator according to some embodiments is exemplarily shown. Figure 2 A schematic structural diagram of a paper-based microfluidic chip provided according to some embodiments is exemplarily shown.

[0038] The concentrator includes a paper-based microfluidic chip and a concentrating module. In the embodiment of the present application, the concentrator includes a paper-based microfluidic chip for detecting target proteins using immunochromatography and a concentrating module. The paper substrate in the paper-based microfluidic chip is the primary reaction site for antigen-antibody immunological binding.

[0039] exist Figure 1 In the embodiment, the paper-based microfluidic chip includes a sample pad 1, a conjugation pad 2, a main channel 3, a water absorbent pad 10 and a bottom plate, and the sample pad 1, the conjugation pad 2, the main channel 3 and the water absorbent pad 10 are all located on the bottom plate.

[0040] One end of the sample pad 1 covers one end of the conjugate pad 2. In the present embodiment, the sample pad is made of glass fiber paper. The sample pad is laminated on the conjugate pad, and the sample solution to be tested is dripped onto the sample pad. The sample pad can promote uniform and controlled distribution of the sample solution on the conjugate pad 2 by controlling the rate at which the sample solution to be tested enters the conjugate pad 2.

[0041] The other end of the binding pad 2 covers one end of the main channel 3 .

[0042] In the embodiment of the present application, the bonding pad is made of glass fiber paper. The glass fiber paper is compressed into a thin pad to serve as the bonding pad. The bonding pad is laminated on the upstream end of the main channel.

[0043] The conjugate pad 2 contains a non-specifically adsorbed detection reagent, which includes a fluorescently labeled specific antibody. After the test sample solution passes through the conjugate pad, the detection reagent and the target protein in the test sample solution undergo specific immunological binding to form an immunoconjugate containing a fluorescent label. In the embodiment of the present application, when the test sample solution flows into the conjugate pad, the detection reagent dissolves and is evenly transferred to the main flow channel along with the test sample solution.

[0044] In embodiments of the present application, the main channel is made of a nitrocellulose membrane. In some embodiments, a hydrophobic film is disposed on the back of the nitrocellulose membrane. The hydrophobic film disposed on the back of the main channel allows the sample solution to flow laterally along the membrane and not through the bottom plate.

[0045] exist Figure 2 In the embodiment, a detection line 11 is provided on the main channel 3, and the detection line 11 is used to capture the protein and retain the immunoconjugate containing the target protein on the detection line.

[0046] One end of the absorbent pad 10 covers the other end of the main channel 3. In the embodiment of the present application, the absorbent pad is made of pure cotton linter pulp filter paper. The absorbent pad is laminated on the downstream end of the main channel to absorb the liquid after the reaction, forming a continuous flow and washing away the unbound molecules in the detection reagent from the test line and the control line.

[0047] In some embodiments, refer again to Figure 2 A control line 12 is provided on the main channel 3 at a predetermined distance from the detection line 11 and after the detection line 11 in the direction of flow of the sample solution to be tested. The control line is used to capture fluorescently labeled specific antibodies that are not bound to the target protein. In this embodiment, the excess specific antibodies from the conjugate pad 2 in the sample solution to be tested will be captured by the corresponding antibodies in the downstream control line. In this way, whether the detection reagent has entered the main channel can be determined by whether the control line has fluorescence intensity. If the control line has fluorescence intensity, it can be determined that the detection reagent has entered the main channel, and the target protein concentration obtained based on the detection line is reliable.

[0048] See again Figure 1 The concentration module includes a positive electrode electrolysis module and a negative electrode electrolysis module. The concentration module is a key component of the concentrator, which realizes the concentration of the target protein in the detection line area and can realize the in-situ detection of the target protein. It should be noted that Figure 1 This is only an example of the installation of the positive electrode electrolysis module and the negative electrode electrolysis module. In fact, if the target protein in the sample solution to be tested is a positively charged molecule, the negative electrode electrolysis module is pressed upstream of the main channel and the positive electrode electrolysis module is pressed downstream of the main channel, that is, Figure 1 The positive electrode electrolysis module and the negative electrode electrolysis module are installed in opposite positions; if the target protein in the sample solution to be tested is a loaded molecule, the positive electrode electrolysis cell is located upstream of the main channel and the negative electrode electrolysis cell is located downstream of the main channel, that is, Figure 1 The positive electrode electrolysis module and the negative electrode electrolysis module are installed in the same position.

[0049] The positive electrolysis module includes a positive electrolysis cell and a first inert metal electrode 6. One end of the first inert metal electrode 6 is inserted into the positive electrolysis cell, and the other end of the first inert metal electrode 6 is connected to a voltage source. In the embodiment of the present application, the positive electrolysis cell includes a cell body 5 and a cell bottom 4. The cell bottom of the positive electrolysis cell is made of a cation exchange membrane.

[0050] In the embodiment of the present application, the cell body 5 of the positive electrode electrolytic cell is made of polymethyl methacrylate (PMMA). The first inert metal electrode can be a platinum electrode.

[0051] The negative electrolysis module includes a negative electrolytic cell and a second inert metal electrode 9. One end of the second inert metal electrode 9 is inserted into the negative electrolytic cell, and the other end of the second inert metal electrode 9 is connected to the voltage source. In this embodiment of the present application, the negative electrolytic cell includes a cell body 8 and a cell bottom 7. The cell bottom of the negative electrolytic cell is made of an anion exchange membrane.

[0052] In the embodiment of the present application, the cell body 8 of the negative electrode electrolytic cell is made of polymethyl methacrylate (PMMA). The second inert metal electrode can be a platinum electrode.

[0053] When actually detecting the target protein in the sample solution to be tested, a certain amount of deionized water is injected into both the positive electrode electrolytic cell and the negative electrode electrolytic cell. One end of the first inert metal electrode and the second inert metal electrode are respectively inserted into the corresponding electrolytic cell, and the other end is connected to the voltage source to realize voltage supply and current monitoring.

[0054] The solution in the embodiment of the present application uses an electrolytic cell integrated with an ion exchange membrane to achieve concentration of target protein molecules in the main channel while maintaining the stability of the pH value in the channel. The surface charge of amphiphilic molecules widely used as biomarkers, such as proteins, will change with the pH value of the buffer solution in the sample solution to be tested, and the buffer solution in the sample solution to be tested is a phosphate buffer. Specifically, when the pH value of the buffer solution is greater than the isoelectric point of the protein, the surface of the protein is negatively charged; and when the pH value in the buffer solution is less than the isoelectric point of the protein, the surface of the protein is positively charged. Introducing an external electric field in a conventional paper-based chip will cause a change in the pH in the channel, thereby causing the surface charge of the protein molecules in the buffer solution to change, changing the direction of the Coulomb force on the protein, causing the force balance state of the protein in the ion concentration polarization system to be destroyed, and protein enrichment cannot be maintained. The electrolytic cell in the present application maintains the pH value in the main channel stable while introducing an external electric field, and can stably form protein enrichment for a long time, thereby increasing the target protein concentration in the detection line area in the main channel 3.

[0055] The present application also provides a method for preparing a paper-based nanofluid concentrator, comprising:

[0056] Bond the main channel to the base plate.

[0057] Specifically, the nitrocellulose membrane constituting the main channel 3 is adhered to the bottom plate, and a hydrophobic film is attached to the back of the nitrocellulose membrane. The liquid will flow laterally along the membrane and will not flow away through the bottom plate.

[0058] Adhere one end of the bonding pad to one end of the main channel, and adhere the other end of the bonding pad to the base plate;

[0059] Adhere one end of the sample pad to the other end of the conjugate pad, and adhere the other end of the sample pad to the base plate;

[0060] Specifically, the top half of conjugate pad 2 is bonded to the base plate, while the bottom half, which contacts the nitrocellulose membrane, is laminated onto the main flow channel 3, ensuring good contact between the two. The top half of sample pad 1 is bonded to the base plate, while the bottom half is laminated onto conjugate pad 2. The sample solution to be tested enters the paper-based microfluidic chip from sample pad 1, then passes through conjugate pad 2 and into the main flow channel 3.

[0061] Adhere one end of the water-absorbing pad to the other end of the main channel, and adhere the other end of the water-absorbing pad to the bottom plate;

[0062] Specifically, the lower half of the absorbent pad 10 is bonded to the base plate, while the upper half is laminated onto the main channel 1, covering the lower half of the main channel 1. This ensures that liquid flowing out of the main channel 3 is absorbed by the absorbent pad and then directed out of the main channel 3. After assembly, the paper-based microfluidic chip is compacted to ensure that the entire system maintains structural stability without the application of external forces.

[0063] Processing and preparing a cell body and a cell bottom of a positive electrode electrolytic cell, and bonding the cell body and the cell bottom of the positive electrode electrolytic cell to form a positive electrode electrolytic cell;

[0064] The cell body and cell bottom of the negative electrode electrolytic cell are processed and prepared, and the cell body and cell bottom of the negative electrode electrolytic cell are bonded together to form the negative electrode electrolytic cell.

[0065] Specifically, the positive and negative electrolytic cell bodies 5 and 8 of the concentration module are machined using a laser cutter. The shape of the cell bodies is cut out of a PMMA sheet using the laser cutter, with a wall thickness of approximately 0.3 mm. The cation exchange membranes 4 and anion exchange membranes 7 are cut to the same dimensions as the corresponding cell bottoms and bonded to the bottoms to prevent leakage.

[0066] The present application also provides a method for using a paper-based nanofluid concentrator, comprising:

[0067] The sample solution to be tested is injected into the sample pad so that the sample pad absorbs the sample solution to be tested to an oversaturated state, and then the sample solution to be tested flows from the sample pad into the conjugate pad. The target protein in the sample solution to be tested is specifically immunologically bound to the detection reagent in the conjugate pad to form an immunoconjugate containing a fluorescent marker. After the conjugate pad absorbs the sample solution to be tested to an oversaturated state, the immunoconjugate will flow into the main channel along with the sample solution to be tested. The detection line on the main channel captures the target protein and retains the immunoconjugate containing the target protein on the detection line, causing the detection line to exhibit a certain level of fluorescence intensity. After flowing through the main channel, the sample solution to be tested is absorbed by the absorbent pad and is led out of the channel. In the embodiment of the present application, the sample solution to be tested will continue to flow laterally from the sample pad to the absorbent pad.

[0068] Figure 3A schematic diagram of the structure of a paper-based nanofluid concentrator provided according to some embodiments is exemplified. After the main channel is completely soaked by the test sample solution, the positive and negative electrolysis modules are pressed onto the main channel at a certain distance. When the target protein in the test sample solution is pre-concentrated to be a positively charged molecule, the negative electrolysis module is pressed upstream of the main channel, and the positive electrolysis module is pressed downstream of the main channel. When the target protein in the test sample solution is pre-concentrated to be a charged molecule, the positive electrolysis cell is located upstream of the main channel, and the negative electrolysis cell is located downstream of the main channel.

[0069] applying power to the first inert metal electrode and the second inert metal electrode using a voltage source to introduce a potential difference between the positive electrolytic cell and the negative electrolytic cell in the main channel;

[0070] In the embodiment of the present application, after the ion exchange membrane at the bottom of the electrolytic cell is in full contact with the main channel, a potential difference is introduced between the two electrolytic cells in the main channel 3 by a voltage source through an inert electrode. Due to the directional movement of ions in the sample solution to be tested through the ion exchange membrane and the electrical neutrality on both sides of the membrane, the positive and negative ions at the upstream end of the downstream ion exchange membrane gradually decrease, forming an ion depletion zone. The generation of the ion depletion zone will cause the field strength in the main channel to be nonlinearly distributed. In particular, the potential drop in the depletion zone will increase significantly, forming an invisible barrier that hinders the passage of ions or biomolecules with the same electrical properties as the ion exchange membrane. The immunoconjugate in the solution will be subject to the drag force exerted by the lateral flow of the liquid along the main channel 3 and the Coulomb force exerted on it by the electric field. When the two forces reach equilibrium, the immunoconjugate will remain in the channel and will not move with the liquid, thereby forming an immunoconjugate enrichment area.

[0071] In the embodiment of the present application, detection is carried out while achieving concentration. By controlling the position of the enrichment area to overlap with the detection line area, the concentration and detection links are carried out simultaneously, which greatly shortens the time required for the overall detection and avoids the loss of target molecules. The number of target molecules captured by the detection line is positively correlated with the concentration of the target molecules in the liquid in this area. The target protein molecule enrichment area formed in the main channel 3 in the present application is covered on the detection line area of ​​the main channel 3. The concentration of target protein molecules in this area is greatly improved compared to the sample solution without pre-concentration, so more target protein molecules will be captured by the detection line. On a macroscopic scale, it is manifested as a significant improvement in the fluorescence intensity of this area compared to the case without pre-concentration. Due to the limitations of detection technology and the influence of interference, the instrument cannot distinguish the fluorescence intensity of the detection line area of ​​the detection card from the blank value when the low-concentration sample solution is injected, and the specific concentration of the low-concentration sample solution cannot be detected. The blank value is the fluorescence intensity of the detection line measured when there is no target protein in the sample solution to be tested. After the low-concentration sample solution is concentrated by the present invention, the fluorescence intensity of the detection line is enhanced, and the detection instrument can distinguish it from the blank value, thereby determining the specific concentration of the low-concentration sample solution and reducing the detection limit of the target protein by the reagent card.

[0072] Adjusting the positions of the positive and negative electrolytic cells on the main channel and / or the voltage of the voltage source to form an immunoconjugate enrichment region on the main channel, and to make the enrichment region coincide with the detection line;

[0073] In the embodiments of the present application, adjusting the position of the electrolytic cell relative to the main channel 3 and the magnitude of the external electric field changes the position of the enrichment region, causing it to coincide with the detection line in the main channel 1, thereby enabling simultaneous in-situ detection while achieving concentration. After the immunoconjugate-enriched region is formed, the immunoconjugate concentration in the detection line is significantly increased, increasing the probability of the immunoconjugate being trapped by specific antibodies. Compared to the case without the enrichment process, the fluorescence intensity in the detection line region is significantly enhanced.

[0074] In the embodiment of the present application, under the action of the sample pad 1 and the absorbent pad 10, there will be a continuous lateral flow from the sample pad 1 to the absorbent pad 10 in the main channel 3. The Coulomb force on the target protein molecule is related to its own surface charge and charge amount, and the drag force it is subjected to is related to its own volume and mass. Different protein molecules have different charge amounts and charge-to-mass ratios, resulting in different enrichment areas formed in the working area. At this time, adjusting the direction or strength of the external electric field can change the Coulomb force on the target protein, change the position of the protein in the main channel 3 when it reaches equilibrium, and then adjust the position of the protein enrichment area so that it coincides with the detection line in the paper-based chip. For example, when pre-concentrating cytochrome C, since it has a negative surface charge in a conventional buffer solution environment, the positive electrode electrolytic cell needs to be upstream of the main channel 3 during pre-concentration to provide the protein molecules with a Coulomb force to counteract the flow drag force. When pre-concentrating phycocyanin with a positive surface charge in a conventional buffer solution, it is necessary to change the direction of the electric field and adjust the positive electrode electrolytic cell to the downstream. Without changing the structure of the device, pre-concentration of multiple proteins is achieved, which greatly improves the universality of the invention.

[0075] After the voltage source is powered on for a preset time, power is turned off, the fluorescence intensity of the detection line is detected, and the concentration of the target protein is determined based on the fluorescence intensity and a predetermined curve, wherein the predetermined curve includes different concentrations of the target protein and the fluorescence intensities corresponding to the concentrations.

[0076] In this embodiment, the external voltage is maintained for ten minutes. After the voltage is removed, the enriched area is dispersed by the continuous lateral flow, eliminating any interference from impurities in the detection line region on the final fluorescence intensity measurement. After the flow ends, the fluorescence intensity of the detection line is measured, and the data is processed to determine the concentration of the target protein molecule in the sample solution.

[0077] In some embodiments, the fluorescence intensities corresponding to different target protein concentrations can be measured and stored in advance. When the fluorescence intensity of the test line is measured during actual testing, the measured value is compared with the pre-stored data to find a specific value that matches the measured fluorescence intensity. Through this comparison, the target protein concentration corresponding to the measured fluorescence intensity can be determined. This found concentration value is used as the quantitative result of the target protein in the test sample. This process not only improves the accuracy of the test, but also makes the quantitative analysis more efficient and reliable.

[0078] In some embodiments, the method further comprises: detecting the fluorescence intensity of the control line.

[0079] In this embodiment, the excess specific antibodies from conjugate pad 2 in the sample solution are captured by the corresponding antibodies in the downstream control line. If the control line shows fluorescence intensity, it can be detected that the reagent has entered the main channel, thereby confirming that the target protein concentration obtained based on the test line is reliable. Figure 4 A schematic diagram of a scenario for detecting the fluorescence intensity of a test line and a control line according to some embodiments is exemplified.

[0080] The present application detects while achieving concentration, and by controlling the position of the enrichment area to overlap with the detection line area, the concentration and detection links are carried out simultaneously, which greatly shortens the time required for the overall detection and avoids the loss of target molecules. The number of target molecules captured by the detection line is positively correlated with the concentration of the target molecules in the liquid in this area. The target molecule enrichment area formed in the main channel 3 in the present application is covered on the detection line area of ​​the main channel 3. The concentration of target protein molecules in this area is greatly improved compared to the sample solution without pre-concentration, so more target protein molecules will be captured by the detection line. On a macroscopic scale, the fluorescence intensity of this area is significantly improved compared to the case without pre-concentration. Due to the limitations of detection technology and the influence of interference, the instrument cannot distinguish the fluorescence intensity of the detection line area of ​​the detection card from the blank value when the low-concentration sample solution is injected, and the specific concentration of the low-concentration sample solution cannot be detected. After the low-concentration sample solution is pre-concentrated by the present application, the fluorescence intensity of the detection line is enhanced, and the detection instrument can distinguish its fluorescence intensity from the blank value, thereby determining the specific concentration of the low-concentration sample solution, reducing the detection limit of the reagent card for detecting target proteins.

[0081] Under the action of sample pad 1 and absorbent pad 10, a continuous lateral flow from sample pad 1 to absorbent pad 10 occurs in main channel 3. The Coulomb force acting on the target protein molecule is related to its surface charge and charge amount, while the drag force it experiences is related to its volume and mass. Different protein molecules have different charge amounts and charge-to-mass ratios, resulting in different enrichment zones within the working area. Adjusting the direction or strength of the external electric field can alter the Coulomb force acting on the target protein, changing its equilibrium position in main channel 3 and subsequently adjusting the position of the protein enrichment zone to coincide with the detection line on the paper-based chip. For example, when pre-concentrating cytochrome c, due to its negative surface charge in a conventional buffer solution, the positive electrode electrolytic cell needs to be upstream of main channel 3 to provide the protein molecules with a Coulomb force that counteracts the drag force. However, when pre-concentrating phycocyanin, which has a positive surface charge in a conventional buffer solution, the direction of the electric field needs to be changed, and the positive electrode electrolytic cell needs to be moved downstream. Without changing the structure of the device, pre-concentration of multiple proteins is achieved, which greatly improves the universality of the invention.

[0082] Effect after implementation: Thanks to the electrolytic cell with a special structure, the present application introduces ion concentration polarization into the paper-based microfluidic chip without interference. In the main channel 3, the drag force applied to the target protein molecules by the lateral flow and the Coulomb force on the target molecules reach a dynamic balance, so that the protein molecules are enriched in the working area in the main channel 3, and the protein molecules enriched in this area are stacked to form an enrichment plug. After the enrichment plug is formed, as the sample solution to be processed enters the main channel 3 from the sample pad 1, the target protein molecules will be captured by the enrichment plug, and the treated sample solution will be led out of the main channel 3 by the absorbent pad 10, thereby forming a stable liquid circulation. The target protein molecules in the sample solution will be continuously enriched in the flow channel, forming an "ion sieve" in the main channel 3.

[0083] When the low-abundance target molecule solution is not concentrated, the fluorescence intensity of the detection line usually cannot reach the minimum threshold of spectral quantitative detection, and cannot be distinguished from the interference value of the blank test group. Therefore, the specific concentration of the low-abundance target molecule solution cannot be measured. Under the same circumstances, after pre-concentration of the present application, the fluorescence intensity of the detection line reaches the feasible range of spectral quantitative detection. When testing a sample solution with a lower concentration, the result obtained can be distinguished from the interference value of the blank test group. With the help of specific data fitting processing, the concentration of the low-abundance target molecule solution can be detected and specific data can be obtained. The present application has good compatibility with paper-based chips, does not require additional structural support, and can be widely used in various protein molecule detection test cards. At the same time, the present application has the general characteristics of paper-based microfluidic chips, low cost, simple structure, and easy operation; it has broad application prospects in the fields of biomedicine and chemical analysis.

[0084] In order to facilitate the observation of pre-concentration in the flow channel under natural conditions, the color-developing protein molecules cytochrome C (red) and phycocyanin (blue) were first tested. The isoelectric point of cytochrome C is greater than the pH value of the conventional buffer and is negatively charged in the buffer. The isoelectric point of phycocyanin is less than the pH value of the conventional buffer and is positively charged in the buffer. Figure 5 As shown, it can be clearly observed that a distinct pigment strip is formed in front of the working area in the main channel 3, indicating that the chromogenic protein is enriched in this area. This is consistent with the theoretical results of the present application and proves the feasibility of the present application.

[0085] The present application was then applied to a commercial cardiac troponin test card. When detecting a low concentration (lower than the nominal minimum detection concentration) of cardiac troponin solution, the detection instrument was unable to detect an intensity signal from the reagent card that could be distinguished from the blank group. The present application was integrated into a commercial test card to pre-concentrate and detect a lower concentration of cardiac troponin. The pre-concentration process lasted for ten minutes. After the pre-concentration was completed, the signal detected by the detection instrument was significantly different from that of the blank group (e.g., Figure 6). The experimental results show that the present application can increase the target protein concentration in the detection line area by more than ten times within ten minutes, thereby improving the minimum detection limit of the test card and proving the practical operability of the present application.

[0086] In summary, the embodiments of the present invention provide a paper-based nanofluid concentrator and a preparation method and a use method thereof. The concentrator is provided with a positive electrode electrolytic cell and a negative electrode electrolytic cell. The positive electrode electrolytic cell is composed of a cation exchange membrane and a cell body, and the negative electrode electrolytic cell is composed of an anion exchange membrane and a cell body. In this way, the target protein can be concentrated in the main channel while maintaining the stability of the pH value in the main channel. In addition, by adjusting the relative positions of the positive electrode electrolytic cell and the negative electrode electrolytic cell to control the overlap of the enrichment area and the detection line, the concentration and detection links are carried out simultaneously, greatly shortening the time required for the overall detection.

[0087] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed. The above is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application. The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present application. These improvements and variations should also be regarded as the scope of protection of the present application.

Claims

1. A paper-based nanofluid concentrator, characterized in that: Includes paper-based microfluidic chip and concentration module; The paper-based microfluidic chip comprises a sample pad, a conjugation pad, a main channel, a water-absorbing pad and a bottom plate, wherein the sample pad, the conjugation pad, the main channel and the water-absorbing pad are all located on the bottom plate; one end of the sample pad covers one end of the conjugation pad; the other end of the conjugation pad covers one end of the main channel; the conjugation pad contains a non-specifically adsorbed detection reagent, wherein the detection reagent includes a fluorescently labeled specific antibody; after the sample solution to be tested passes through the conjugation pad, the detection reagent and the target protein in the sample solution to be tested undergo specific immune binding to form an immunoconjugate containing a fluorescent label; a detection line is provided on the main channel, wherein the detection line is used to capture the target protein; one end of the water-absorbing pad covers the other end of the main channel; The concentration module includes a positive electrode electrolysis module and a negative electrode electrolysis module, the positive electrode electrolysis module includes a positive electrode electrolysis cell and a first inert metal electrode, one end of the first inert metal electrode is inserted into the positive electrode electrolysis cell, the other end of the first inert metal electrode is connected to a voltage source, and the material of the bottom of the positive electrode electrolysis cell is a cation exchange membrane; the negative electrode electrolysis module includes a negative electrode electrolysis cell and a second inert metal electrode, one end of the second inert metal electrode is inserted into the negative electrode electrolysis cell, the other end of the second inert metal electrode is connected to the voltage source, and the material of the bottom of the negative electrode electrolysis cell is an anion exchange membrane.

2. The concentrator according to claim 1, characterized in that A control line is set on the main channel at a position after the detection line and at a preset distance from the detection line according to the flow direction of the sample solution to be tested. The control line is used to capture fluorescently labeled specific antibodies that are not bound to the target protein.

3. The concentrator according to claim 1, characterized in that The material of the sample pad is glass fiber paper.

4. The concentrator according to claim 1, characterized in that The material of the bonding pad is glass fiber paper.

5. The concentrator according to claim 1, characterized in that The material of the main channel is nitrocellulose membrane.

6. The concentrator according to claim 5, characterized in that A hydrophobic film is provided on the back side of the main channel.

7. The concentrator according to claim 1, characterized in that The material of the water-absorbing pad is pure cotton linter pulp filter paper.

8. A method for preparing a paper-based nanofluid concentrator, applied to the paper-based nanofluid concentrator according to any one of claims 1 to 7, characterized in that: include: Bond the main channel to the base plate; Adhere one end of the bonding pad to one end of the main channel, and adhere the other end of the bonding pad to the base plate; Adhere one end of the sample pad to the other end of the conjugate pad, and adhere the other end of the sample pad to the base plate; Adhere one end of the water-absorbing pad to the other end of the main channel, and adhere the other end of the water-absorbing pad to the bottom plate; Processing and preparing a cell body and a cell bottom of a positive electrode electrolytic cell, and bonding the cell body and the cell bottom of the positive electrode electrolytic cell to form a positive electrode electrolytic cell; The cell body and cell bottom of the negative electrode electrolytic cell are processed and prepared, and the cell body and cell bottom of the negative electrode electrolytic cell are bonded together to form the negative electrode electrolytic cell.

9. A method for using a paper-based nanofluid concentrator, applied to the paper-based nanofluid concentrator according to any one of claims 1 to 7, characterized in that: include: The sample solution to be tested is injected into the sample pad, so that the sample pad absorbs the sample solution to be tested to a supersaturated state, and then the sample solution to be tested flows from the sample pad into the conjugate pad, and the target protein in the sample solution to be tested is specifically immunologically bound to the detection reagent in the conjugate pad to form an immunoconjugate containing a fluorescent marker; After the binding pad absorbs the test sample solution to a supersaturated state, the immunoconjugate flows into the main channel along with the test sample solution. The detection line on the main channel captures the target protein and retains the immunoconjugate containing the target protein on the detection line, causing the detection line to exhibit a certain level of fluorescence intensity. After flowing through the main channel, the test sample solution is absorbed by the absorbent pad and is led out of the channel. After the main channel is completely soaked by the test sample solution, the positive electrode electrolysis module and the negative electrode electrolysis module are pressed onto the main channel, wherein when the target protein in the test sample solution is concentrated into positively charged molecules, the negative electrode electrolysis module is pressed upstream of the main channel, and the positive electrode electrolysis module is pressed downstream of the main channel; when the target protein in the test sample solution is pre-concentrated into loaded molecules, the positive electrode electrolysis cell is located upstream of the main channel, and the negative electrode electrolysis cell is located downstream of the main channel; applying power to the first inert metal electrode and the second inert metal electrode using a voltage source to introduce a potential difference between the positive electrolytic cell and the negative electrolytic cell in the main channel; Adjusting the positions of the positive and negative electrolytic cells on the main channel and / or the voltage of the voltage source to form an immunoconjugate enrichment region on the main channel, and to make the enrichment region coincide with the detection line; After the voltage source is powered on for a preset time, power is turned off, the fluorescence intensity of the detection line is detected, and the concentration of the target protein is determined based on the fluorescence intensity and a predetermined curve, wherein the predetermined curve includes different concentrations of the target protein and the fluorescence intensities corresponding to the concentrations.

10. The method of use according to claim 9, characterized in that: Also includes: The fluorescence intensity of the control line was detected.

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