Integrated micro-fluidic chip for joint detection of nucleic acid and protein and detection method

Abstract

The invention relates to the technical field of microfluidics and in-vitro diagnosis, in particular to an integrated micro-fluidic chip for joint detection of nucleic acid and protein and a detection method, the integrated micro-fluidic chip comprises a chip top layer, a functional structure layer and a substrate; the functional structure layer is provided with a biomarker enrichment and separation area, a nucleic acid detection area, a protein detection area and a visual reading area. Wherein the enrichment and separation region adopts a configuration that the flowing direction is vertical to the direction of an electric field, and dynamic electric enrichment and synchronous separation of nucleic acid and protein are realized through an ion concentration polarization effect; an amplification or immune reaction reagent is preset in the detection area; lFA test paper is arranged in the reading area, and liquid is pressed and driven to flow through an elastic film covering the observation window. The chip can realize the whole process integration of one-step sample adding, rapid enrichment, isothermal amplification and visual reading, obviously improves the detection sensitivity and multi-index joint detection efficiency of low-abundance targets, and is suitable for the fields of infectious disease diagnosis, cancer screening, on-site rapid detection and the like.

Description

Technical Field

[0001] This invention relates to the fields of microfluidics and in vitro diagnostics, specifically to an integrated microfluidic chip and detection method for nucleic acid and protein co-detection. Background Technology

[0002] Accurate and rapid biomarker detection is a core requirement for disease prevention, diagnosis, and treatment prognosis. Currently, the gold standard laboratory methods for nucleic acids and proteins are polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), but both still face challenges in achieving rapid, specific, and sensitive detection. PCR requires complex sample extraction and precise thermal cycling (taking 2-3 hours), while ELISA is cumbersome, time-consuming, and has relatively low sensitivity.

[0003] The methods described above are labor-intensive and rely on specialized equipment and personnel. Therefore, many researchers have subsequently developed integrated microfluidic devices for biomarker detection, reducing the detection process, making them user-friendly, and typically requiring no specialized personnel or equipment. However, microfluidic devices are usually designed for nucleic acid or protein targets only, limiting their application in the combined detection of nucleic acids and proteins.

[0004] Existing technology CN119082268 A discloses an enzyme cascade reaction system and its application for the detection of nucleic acid and protein molecules. Utilizing the synergistic enzyme activity of APE1 and PfAgo, this enzyme cascade signal amplification system enables the immediate detection of nucleic acid and protein biomarkers without pre-amplification. However, certain other proteins or biomolecules in clinical samples may interfere with the detection process, potentially leading to false negatives or reduced detection performance. This method requires additional purification steps and optimized buffer formulations, and suffers from low sensitivity and a limited range of detectable protein targets (FEN1 or APE1). Detecting ultra-low abundance molecular biomarkers in some clinical samples is challenging, especially when the target concentration is close to the detection limit, which may cause false negative detection blind spots. Furthermore, common on-chip solid-phase extraction methods suffer from target loss due to low adsorption efficiency and insufficient elution of adsorption columns, magnetic beads, or membranes. There is a lack of methods that can simultaneously separate and extract nucleic acid and protein molecules, and the cumbersome and time-consuming operation can easily lead to target degradation, further reducing detection sensitivity.

[0005] Therefore, there is an urgent need to develop a method that can rapidly and efficiently enrich, separate, and detect low-abundance nucleic acid and protein biomarkers in samples, thereby improving detection sensitivity and better adapting to diverse diagnostic needs. Summary of the Invention

[0006] One of the objectives of this invention is to provide an integrated microfluidic chip for the joint detection of nucleic acids and proteins, which solves the problems of existing microfluidic chips being unable to achieve efficient joint detection of nucleic acids and proteins simultaneously, the low separation efficiency of targets being easily trapped in traditional enrichment methods, and the insufficient sensitivity of low-abundance biomarkers.

[0007] To achieve the above objectives, an integrated microfluidic chip for nucleic acid and protein co-detection is provided, the chip comprising a three-layer structure: a top layer of the chip, a functional structure layer of the chip, and a chip substrate;

[0008] The chip functional structure layer includes:

[0009] The biomarker enrichment and separation region includes a Nafion cation membrane and electrodes located on both sides of the Nafion cation membrane. The biomarker enrichment and separation region is configured to perform dynamic electrokinetic enrichment and separation of nucleic acids and proteins by utilizing the ion concentration polarization effect when an electric field perpendicular to the sample flow direction is applied through the electrodes.

[0010] The nucleic acid molecule detection area and the protein molecule detection area are connected to the biomarker enrichment and separation area through branched microchannels, and the detection result visualization reading area equipped with LFA test strips is connected to the nucleic acid molecule detection area and the protein molecule detection area, respectively.

[0011] The chip has a result observation window on its top layer that corresponds to the detection result visualization reading area. The surface of the result observation window is covered with an elastic material film. The elastic material film is used to seal the detection result visualization reading area and generates air pressure by pressing and deforming to drive the liquid in the nucleic acid molecule detection area and the protein molecule detection area to flow into the corresponding LFA test strip.

[0012] Furthermore, the Nafion cation membrane is composed of multiple parallel-arranged plate-column structures, forming multiple parallel microchannels to constitute a nanochannel membrane for generating ion concentration polarization effect, and is disposed on the cathode side near the electrode; wherein, the plate-column structure is formed by curing a Nafion perfluorosulfonic acid resin solution.

[0013] Furthermore, downstream of the biomarker enrichment and separation zone, and located between the Nafion cation membrane and the anode and cathode, a Y-shaped nucleic acid separation channel and a protein separation channel are provided, which are connected to the nucleic acid molecule detection zone and the protein molecule detection zone through branched microchannels, respectively. The downstream flow channels of the protein separation channel and the nucleic acid separation channel are tortuously arranged.

[0014] Furthermore, the downstream channels of the protein separation channel and the nucleic acid separation channel are respectively provided with an enriched nucleic acid collection chamber and a protein collection chamber. The enriched nucleic acid collection chamber is connected to the nucleic acid molecule detection area through a microchannel, and the protein collection chamber is connected to the protein molecule detection area through a microchannel.

[0015] Furthermore, the nucleic acid molecular detection area includes multiple independent nucleic acid detection chambers, which are connected via microchannels after the enriched nucleic acid collection chamber and before the LFA test strip slot where the LFA test strip is placed; each nucleic acid detection chamber has multiple reaction wells designed in parallel.

[0016] Furthermore, the reaction wells store RPA amplification reaction components, specifically including primers, RPA enzyme reaction lyophilized powder, RPA reaction promoter, exonuclease IV, FAM, and biotin-labeled nfo probes.

[0017] Furthermore, the protein molecule detection area includes multiple independent protein detection chambers, which are connected via microchannels after the enriched protein collection chamber and before the LFA test strip slot where the LFA test strip is placed.

[0018] Furthermore, the chip substrate has chambers corresponding to the chip's functional structure layers to increase sample processing capacity.

[0019] Furthermore, the LFA test strip has a structure that, along the chromatography direction, includes a sample pad, a binding pad, an NC membrane coated with a detection line (T line) and a control line (C line), and an absorbent pad.

[0020] The second objective of this invention is to provide a detection method based on an integrated microfluidic chip, using an integrated microfluidic chip for nucleic acid and protein co-detection as described above, comprising the following steps:

[0021] The sample to be tested is injected into the biomarker enrichment and separation region of the integrated microfluidic chip, and the electrodes are connected to the power supply.

[0022] The samples achieve the enrichment and separation of nucleic acids and proteins through the ion concentration polarization effect in the biomarker enrichment and separation zone;

[0023] The enriched nucleic acids are then amplified at an isothermal temperature in the nucleic acid molecular detection area.

[0024] The enriched proteins enter the protein molecule detection area;

[0025] By pressing the elastic membrane on the observation window, the liquid in the nucleic acid molecule detection area and the protein molecule detection area is driven to flow into the corresponding LFA test strips respectively;

[0026] Read the test results on the LFA test strip through the results viewing window.

[0027] Principles and advantages:

[0028] 1. This solution integrates ion concentration polarization (ICP)-based enrichment and separation technology, nucleic acid detection technology, and protein detection technology on a single chip, overcoming the limitations of existing technologies that typically target only a single type of target. Through multi-channel and multi-detection chambers, along with corresponding LFA test strip design, multiple nucleic acid and protein biomarkers in the same sample can be processed and analyzed in parallel, greatly expanding the chip's multi-target diagnostic capabilities. It achieves efficient simultaneous detection of nucleic acids and proteins and multiple target analysis.

[0029] 2. This scheme achieves dynamic electrokinetic enrichment of charged biomarkers (nucleic acids and proteins) by employing a unique configuration where the electric field direction is perpendicular to the sample flow direction. This design avoids the problem of enriched molecules being "confined" to a fixed region in traditional parallel electric fields, allowing the enriched target analytes to be efficiently transported to the downstream detection area via the flow path. Simultaneously, by utilizing the difference in electrophoretic mobility between nucleic acids and proteins, spatial separation of the two can be achieved during the enrichment process, reducing cross-interference and laying the foundation for subsequent high-specificity detection.

[0030] 3. The dynamic enrichment process of this protocol can aggregate low-concentration target substances in a short time (e.g., within 5 minutes), playing a significant "pre-concentration" role and effectively increasing the initial concentration. Combined with signal amplification and visual readout from isothermal nucleic acid amplification (e.g., RPA) and immunochromatography (LFA), the system's limit of detection (LOD) for low-abundance biomarkers is significantly reduced, minimizing the risk of false negatives. It offers high detection sensitivity, making it particularly suitable for low-abundance targets.

[0031] 4. High system integration and extremely simple and fast operation: The chip adopts a fully integrated design with "one-step sample addition". The key liquid driving step is accomplished by pressing the elastic material membrane covering the observation window to generate air pressure, replacing the complex external pump and valve system. The entire process, from sample addition to visual result reading, can be completed in a short time without the need for professional instruments and personnel, making it ideal for rapid on-site testing and use in primary healthcare institutions.

[0032] 5. Strong platform versatility and adaptability: The core enrichment and detection modules of this chip platform have excellent versatility. By changing the pre-installed primers, probes, or antibodies in the detection chamber, it can be flexibly adapted to multiple fields such as collaborative diagnosis of infectious diseases (e.g., simultaneous detection of pathogen nucleic acids and specific antigens / antibodies), cancer biomarker screening, food safety monitoring, and life science research, enabling rapid and sensitive analysis of different target combinations. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of an integrated microfluidic chip for nucleic acid and protein co-detection according to an embodiment of the present invention;

[0034] Figure 2 for Figure 1 A schematic diagram of the functional structure layer of the chip;

[0035] Figure 3 A schematic diagram of the four functional zones of an integrated microfluidic chip;

[0036] Figure 4 A schematic diagram of the pressable elastic membrane on the observation window of the integrated microfluidic chip;

[0037] Figure 5 a is a schematic diagram showing the relationship between the electric field intensity applied to both ends of the Nafion membrane in the integrated microfluidic chip of this invention and the distance from the enrichment region. Figure 5 b is a schematic diagram showing the relationship between fluorescence intensity and the thickness of the Nafion film in an integrated microfluidic chip;

[0038] Figure 6 a is a graph showing the fluorescence quantitative results of samples of different concentrations before and after enrichment in the chip in an embodiment of the present invention. Figure 6 b shows the enrichment fold results of the chip used for samples of different concentrations;

[0039] Figure 7 a is a schematic diagram illustrating the process and time required for sample enrichment, amplification, and detection in an embodiment of the present invention. Figure 7 b is a schematic diagram of sample addition, voltage application, and fluid drive. Figure 7 c is a schematic diagram illustrating the principle of nucleic acid RPA amplification detection in the example. Figure 7 d is a schematic diagram of the LFA test strip detection results. Detailed Implementation

[0040] The following detailed description illustrates the specific implementation method:

[0041] Figure numbering explanation: 1. Chip top layer; 2. Chip functional structure layer; 3. Chip substrate; 4. First sample inlet; 5. First buffer inlet; 6. First anode electrode insertion chamber; 7. First cathode electrode insertion chamber; 8. First result observation window; 9. Second result observation window; 10. Third result observation window; 11. Fourth result observation window; 12. Fifth result observation window; 13. Sixth result observation window; 14. Second sample inlet; 15. Second buffer inlet; 16. Second anode electrode insertion chamber; 17. Second cathode electrode insertion chamber; 18. First waste liquid chamber; 19. Second waste liquid chamber; 20. Nafion cation membrane; 21. First waste liquid channel; 22. Second waste liquid channel; 23. Protein separation channel; 24. Nucleic acid separation channel; 25. Nucleic acid enrichment collection chamber; 26. Protein collection chamber. 26. Nucleic acid testing chamber 27. Nucleic acid testing chamber 28. Nucleic acid testing chamber 29. Nucleic acid testing chamber 20. Protein testing chamber 45. Protein testing chamber 46. Protein testing chamber 47. LFA test strip channel 48. LFA test strip channel 49. LFA test strip channel 50. LFA test strip channel 51. LFA test strip channel 52. LFA test strip channel 53. LFA test strip channel 54. Third sample inlet 55. Buffer inlet 56. Anode electrode insertion chamber 57. Cathode electrode insertion chamber 58. LFA test strip slot 59. LFA test strip slot 60. LFA test strip slot 61. LFA test strip slot 62. LFA test strip slot 63.

[0042] Example

[0043] An integrated microfluidic chip for nucleic acid and protein co-detection, basically as follows: Figure 1 As shown, the chip includes a three-layer structure: a top layer 1, a functional structure layer 2, and a substrate 3.

[0044] The top layer 1 of the chip is mainly used for sample addition, electrode insertion and result observation. Specifically, it is designed with a first sample inlet 4, a first buffer inlet 5, a first anode electrode insertion chamber 6, a first cathode electrode insertion chamber 7, and multiple result observation windows (first result observation window 8, second result observation window 9, third result observation window 10, fourth result observation window 11, fifth result observation window 12, and sixth result observation window 13).

[0045] like Figure 2As shown, the chip functional structure layer 2 includes: flow channels that fit into the chambers of the chip top layer 1, specifically including a second sample inlet 14, a second buffer inlet 15, a second anode electrode insertion chamber 16, a second cathode electrode insertion chamber 17, a first waste liquid chamber 18, a second waste liquid chamber 19, a Nafion cation membrane 20, a first waste liquid flow channel 21, a second waste liquid flow channel 22, a protein separation channel 23, a nucleic acid separation channel 24, multiple nucleic acid detection chambers, and multiple LFA test strip slots (including the first...). LFA test strip slots 48, 49, 50, 51, 52, and 53 correspond to the first result observation window 8, second result observation window 9, third result observation window 10, fourth result observation window 11, fifth result observation window 12, and sixth result observation window 13, respectively. These slots are used to place LFA test strips or to introduce the solution sample to be tested (depending on the thickness of the test strip).

[0046] like Figure 3 As shown, the integrated microfluidic chip is configured with four functional zones: a biomarker enrichment and separation zone A, a nucleic acid molecule detection zone B, a protein molecule detection zone C, and a detection result visualization and reading zone D.

[0047] (1) Biomarker enrichment and separation zone A, in which a Nafion cation membrane 20 and electrodes located on both sides of the Nafion cation membrane 20 are disposed; wherein, the biomarker enrichment and separation zone A is configured to: when an electric field perpendicular to the sample flow direction is applied through the electrodes, the dynamic electrokinetic enrichment and separation of nucleic acids and proteins is carried out by utilizing the ion concentration polarization effect.

[0048] The Nafion cation membrane 20 is composed of multiple parallel-arranged plate-column structures, forming multiple parallel microchannels to constitute a nanochannel membrane for generating ion concentration polarization effect, located near the cathode; wherein, the plate-column structure is formed by curing a Nafion perfluorosulfonic acid resin solution.

[0049] (2) Nucleic acid molecular detection area B and protein molecular detection area C, which are respectively connected to the biomarker enrichment and separation area A through branch microchannels, and detection result visualization reading area D, which is respectively connected to nucleic acid molecular detection area B and protein molecular detection area C and is equipped with LFA test strip;

[0050] like Figure 2As shown, downstream of the biomarker enrichment and separation zone A, and located between the Nafion cation exchange membrane 20 and the anode and cathode of the electrode, a Y-shaped nucleic acid separation channel 24 and a protein separation channel 23 are provided. These channels connect the nucleic acid molecule detection zone B and the protein molecule detection zone C via branched microchannels, respectively. The downstream flow channels of both the protein separation channel 23 and the nucleic acid separation channel 24 are tortuously arranged. The downstream flow channels of the first waste liquid flow channel 21 and the second waste liquid flow channel 22 are also tortuously arranged.

[0051] The downstream channels of the protein separation channel 23 and the nucleic acid separation channel 24 are respectively provided with a protein collection chamber 26 and a nucleic acid enrichment collection chamber 25. The nucleic acid enrichment collection chamber 25 is connected to the nucleic acid molecule detection area B (entering multiple nucleic acid detection chambers) through a microchannel, and the protein collection chamber 26 is connected to the protein molecule detection area C (entering multiple protein detection chambers) through a microchannel.

[0052] like Figure 2 As shown, the nucleic acid molecular detection area B includes three independent nucleic acid detection chambers (first nucleic acid detection chamber, second nucleic acid detection chamber, and third nucleic acid detection chamber). Each nucleic acid detection chamber has six reaction wells arranged in parallel, for a total of eighteen reaction wells, including reaction well 27, reaction well 28, reaction well 29, reaction well 4, reaction well 5, reaction well 6, reaction well 7, reaction well 8, reaction well 9, reaction well 10, reaction well 11, reaction well 12, reaction well 13, reaction well 14, reaction well 15, reaction well 16, reaction well 17, and reaction well 18. Multiple reaction wells are connected via microchannels after the enriched nucleic acid collection chamber 25 and before the LFA test strip slot where the LFA test strip is placed.

[0053] Each reaction well contains the raw materials for the RPA amplification reaction, specifically including primers, RPA enzyme reaction lyophilized powder, RPA reaction promoter, exonuclease IV (nfo), FAM, and biotin-labeled nfo probe.

[0054] The protein molecular detection area C includes multiple independent protein detection chambers (first protein detection chamber 45, second protein detection chamber 46, and third protein detection chamber 47), which are connected via microchannels after the enriched protein collection chamber 26 and before the LFA test strip slot where the LFA test strip is placed.

[0055] The LFA test strip has a structure that, along the chromatography direction, includes a sample pad, a binding pad, an NC membrane coated with a detection line (T line) and a control line (C line), and an absorbent pad.

[0056] The top layer 1 of the chip is provided with result observation windows corresponding to the detection result visualization reading area (LFA test strip slot). In this embodiment, there are six result observation windows (first result observation window 8, second result observation window 9, third result observation window 10, fourth result observation window 11, fifth result observation window 12, and sixth result observation window 13). The LFA test strips include first LFA test strip, second LFA test strip, third LFA test strip, fourth LFA test strip, fifth LFA test strip, and sixth LFA test strip, which are respectively set in the corresponding numbered result observation windows. The number can be designed according to requirements.

[0057] like Figure 4 As shown, the surface of the result observation window is covered with an elastic material membrane. This elastic material membrane is used to seal the visualization reading area of ​​the test results and generates air pressure by pressing and deforming it, driving the liquid in the nucleic acid molecule detection area and the protein molecule detection area to flow into the corresponding LFA test strips respectively. In this embodiment, the outlet of the nucleic acid detection chamber is connected to the sample inlet of the LFA test strip slot through a microchannel. After pressing the elastic membrane, the amplification product is driven by air pressure to flow through the test strip sample pad. The outlet of the protein detection chamber is connected to the sample inlet of the LFA test strip slot through a microchannel. After pressing the elastic membrane, the amplification product is driven by air pressure to flow through the test strip sample pad.

[0058] The chip substrate 3 has chambers corresponding to the chip functional structure layer 2 to increase sample processing capacity. For example... Figure 1 As shown, it includes: a third sample inlet 54 corresponding to the first sample inlet 4 and the second sample inlet 14; a third buffer inlet 55 corresponding to the first buffer inlet 5 and the second buffer inlet 15; a third anode electrode insertion chamber 56 corresponding to the first anode electrode insertion chamber 6 and the second anode electrode insertion chamber 16; a third cathode electrode insertion chamber 57 corresponding to the first cathode electrode insertion chamber 7 and the second cathode electrode insertion chamber 17; and LFA test paper slots including a first LFA test paper slot 58, a second LFA test paper slot 59, a third LFA test paper slot 60, a fourth LFA test paper slot 61, a fifth LFA test paper slot 62, and a sixth LFA test paper slot 63.

[0059] This scheme utilizes the principle of ionic concentration polarization (ICP) effect in the electrokinetic biomarker enrichment zone A of the biomarker enrichment and separation zone. Traditionally, the electric field direction (i.e., the enrichment direction) is the same as the liquid sample flow direction, which traps the enriched biomarkers and hinders downstream detection. This invention improves upon this technology by adopting a method where the flow direction x is perpendicular to the electric field direction y, achieving dynamic electrokinetic enrichment of biomarkers (mainly nucleic acids and proteins). Specifically, the nucleic acid and protein molecules to be captured flow with the liquid along the x-direction. Under the action of the applied electric field, cations in the solution can be transported through the nafion exchange membrane to the cathode and buffer channel, while anions cannot pass through. However, in the y-direction, anions in the fluid are subjected to both electroosmosis (EO) and electrophoresis (EP) within the microchannel. These contribute opposing electroosmotic and electrophoretic velocities, respectively. When electroosmosis and electrophoresis are in equilibrium in the y-direction, the net velocity in the y-direction becomes zero, enabling the electrokinetic capture of negatively charged nucleic acids and proteins, forming an ion enrichment zone (IEZ). At this point, the enriched biomolecules continue to move forward along the x-direction, and can be received downstream through the Y-shaped separation channel.

[0060] During ICP enrichment, the location where charged biomolecules accumulate in the microchannel is determined by the preset parameters of the ICP system (operating voltage and liquid ionic strength) and the electrophoretic mobility of the biomolecules. For example... Figure 5 As shown in figure 'a' (the vertical axis represents the electric field intensity E-field applied across the Nafion membrane, and the horizontal axis represents the distance (distance) from the enriched region to the Nafion membrane), 'eo' represents Electroosmotic Flo, and 'ep' represents Electrophoresis. Since the electrophoretic mobility of nucleic acids (DNA + RNA) is much higher than that of proteins (μDNA ≫ μpro), if appropriate ICP system parameters are applied, nucleic acid and protein molecules can be focused (enriched) at different locations, thus achieving the enrichment and separation of nucleic acid and protein molecules in one step. Figure 5Figure b (vertical axis: fluorescence intensity; horizontal axis: distance from Nafion membrane) shows the enrichment and separation results of DNA with a green fluorescent group and protein with a red label in the chip. With sample flow and the electrokinetic ICP effect, DNA and protein are enriched at different locations on the chip from the Nafion membrane. Specifically, proteins are concentrated in the region 100-400 μm from the Nafion cation membrane (red fluorescence), while DNA molecules show green fluorescence in the region 600-1200 μm.

[0061] Figure 6 The enrichment performance of the chip in the electrokinetic biomarker enrichment region was measured. Synthesized fluorescently tagged FAM-DNA was diluted to different concentrations (1E5-1 pmol / L) using TE buffer. The results showed a significant increase in the fluorescence intensity of the enriched DNA. The chip effectively enriched FAM-DNA samples of different concentrations, and the fluorescence intensity was positively correlated with the concentration. Figure 6 a). Figure 6 b. Further calculation of the enrichment fold of the electrobiomarker enrichment region (the ratio of the maximum sample concentration to the initial sample concentration) showed that at least 1000-fold enrichment was achieved for nucleic acid samples at each sample concentration.

[0062] This invention also discloses a detection method based on an integrated microfluidic chip, using an integrated microfluidic chip for nucleic acid and protein co-detection as described above, comprising the following steps:

[0063] like Figure 7 As shown, during operation, buffer solution is added to the chip's first buffer inlet 5 and electrode insertion chambers (first anode electrode insertion chamber 6 and first cathode electrode insertion chamber 7). A positive DC voltage (V) is applied to the first anode electrode insertion chamber 6, and the first cathode electrode insertion chamber 7 is connected to the negative terminal. The sample to be tested is injected through the first sample inlet 4, and the power supply is connected. Figure 7As shown in b, the sample enters the biomarker enrichment and separation zone A for enrichment and separation. With the flow of liquid, nucleic acid and protein molecules are focused and separated, and impurities and inhibitors from the DNA extraction process are removed. The enriched sample liquid is collected by the collection channels, and the remaining waste liquid is discharged into the waste liquid chambers (first waste liquid chamber 18, second waste liquid chamber 19). Pressing the elastic membrane on the result observation window allows the liquid in the protein collection chamber to enter the LFA test strip slot. The nucleic acid detection chamber reaction wells store RPA amplification reaction components, which are hydrated and activated by the inflow of sample liquid. The microfluidic chip is placed at a constant temperature for RPA amplification, initiating nucleic acid detection. Pressing the elastic membrane allows the liquid containing amplicon to enter the LFA test strip slot, ultimately resulting in a color reaction, and the detection result is read. Figure 7 'a' is a schematic diagram illustrating the process and time required for sample DNA enrichment, amplification, and detection, as well as protein labeling and detection. The principle of nucleic acid RPA amplification detection is as follows: Figure 7 As shown in Figure c, during the reaction, the forward primer and reverse primer, with the help of recombinase, bind to the two strands of the double-stranded target DNA, respectively, initiating DNA synthesis and undergoing exponential amplification to produce a large amount of double-stranded DNA containing the target sequence. One of the amplified single-stranded DNA strands (the target sequence located between the two primers) specifically hybridizes and binds. Exonuclease IV (nfo nuclease cleavage) recognizes and cleaves the THF site, amplifying to obtain FAM and biotin-labeled amplicon. Specifically, the nfo probe, also known as the LF probe, is labeled with biotin; FAM is a fluorescent group, serving as a reporter group at the 5' end of the LF probe; the THF site is a specific abasing site, the recognition and cleavage site for exonuclease IV (nfo); and the 3' blocker is a chemical modification that prevents polymerase from extending from the 3' end of the probe, ensuring that the probe is used only as a "probe" and not a "primer."

[0064] On the test strip, colloidal gold particles are coupled with an antibody against the anti-FAM fluorescent group. The T line is coated with an anti-biotin antibody. When a positive product is present, a sandwich structure of "anti-biotin antibody / biotin-nucleic acid target-FAM / anti-FAM antibody-colloidal gold" will form on the T line, appearing red. The C line is coated with an anti-FAM secondary antibody; a red color indicates normal operation. For protein detection visualization, the colloidal gold particles are labeled with the detection antibody for the target protein. The T line is coated with the detection antibody that captures the sample molecules, and the C line is coated with the detection antibody that captures the colloidal gold-labeled antibody. Specific LFA test strip results are as follows: Figure 7 As shown in d.

[0065] The “without IPC” curve represents the test results of a sample without an internal positive control (IPC), reflecting the binding or dissociation behavior of the target molecule (such as nucleic acid or protein) under uncontrolled conditions.

[0066] The "IPC" curve represents the test results of a sample with an internal positive control. IPC is used to monitor the reliability and efficiency of the experimental system (such as excluding false negatives), and its curve can serve as a reference benchmark to ensure that the testing process is under control.

[0067] The beneficial effects of this invention are as follows:

[0068] 1. Multiple detection at once: Immunoassay and nucleic acid detection technologies are integrated on the same microfluidic chip, enabling simultaneous detection of two different targets, nucleic acids and proteins. At the same time, multiple detection wells can achieve multiple target analysis.

[0069] 2. High sensitivity and specificity: By utilizing ion concentration polarization technology to enrich and separate nucleic acids and proteins, a signal amplification effect is achieved, significantly improving the sensitivity of biomarker detection without the need for complex extraction steps; the content of target molecules is increased, and the detection precision and accuracy are further improved when combined with detection technology.

[0070] 3. Fast, simple, and easy to operate: The entire enrichment process is completed within 5 minutes. The operation is simple, requiring only one step of sample addition. The test results are visible to the naked eye, making it very suitable for on-site rapid testing and use in primary healthcare institutions.

[0071] 4. Wide range of applications: This platform can be flexibly adapted to multiple fields such as infectious disease diagnosis (e.g., simultaneous detection of viral nucleic acids and antigens / antibodies), food safety (e.g., simultaneous detection of pathogenic genes and toxin proteins), and medical research (e.g., analysis of gene expression and secreted proteins in inflammatory responses). Furthermore, it has good adaptability for detecting both low-abundance and high-content biomarkers.

[0072] The above descriptions are merely embodiments of the present invention. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, based on the guidance provided in this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An integrated microfluidic chip for nucleic acid and protein co-detection, characterized in that, The chip comprises a three-layer structure: a top layer, a functional structure layer, and a substrate. The chip functional structure layer includes: The biomarker enrichment and separation region includes a Nafion cation membrane and electrodes located on both sides of the Nafion cation membrane. The biomarker enrichment and separation region is configured to perform dynamic electrokinetic enrichment and separation of nucleic acids and proteins by utilizing the ion concentration polarization effect when an electric field perpendicular to the sample flow direction is applied through the electrodes. The nucleic acid molecule detection area and the protein molecule detection area are connected to the biomarker enrichment and separation area through branched microchannels, and the detection result visualization reading area equipped with LFA test strips is connected to the nucleic acid molecule detection area and the protein molecule detection area, respectively. The chip has a result observation window on its top layer that corresponds to the detection result visualization reading area. The surface of the result observation window is covered with an elastic material film. The elastic material film is used to seal the detection result visualization reading area and generates air pressure by pressing and deforming to drive the liquid in the nucleic acid molecule detection area and the protein molecule detection area to flow into the corresponding LFA test strip.

2. The integrated microfluidic chip for nucleic acid and protein co-detection according to claim 1, characterized in that: The Nafion cation membrane is composed of multiple parallel-arranged plate-column structures, forming multiple parallel microchannels to constitute a nanochannel membrane for generating ion concentration polarization effect, and is disposed on the cathode side near the electrode; wherein, the plate-column structure is formed by curing a Nafion perfluorosulfonic acid resin solution.

3. An integrated microfluidic chip for nucleic acid and protein co-detection according to claim 2, characterized in that: Downstream of the biomarker enrichment and separation zone, and located between the Nafion cation membrane and the anode and cathode, a Y-shaped nucleic acid separation channel and a protein separation channel are provided. The nucleic acid molecule detection zone and the protein molecule detection zone are respectively connected by branched microchannels. The downstream flow channels of the protein separation channel and the nucleic acid separation channel are tortuously arranged.

4. An integrated microfluidic chip for nucleic acid and protein co-detection according to claim 3, characterized in that: The downstream channels of the protein separation channel and the nucleic acid separation channel are respectively equipped with a nucleic acid enrichment collection chamber and a protein collection chamber. The nucleic acid enrichment collection chamber is connected to the nucleic acid molecule detection area through a microchannel, and the protein collection chamber is connected to the protein molecule detection area through a microchannel.

5. An integrated microfluidic chip for nucleic acid and protein co-detection according to claim 4, characterized in that: The nucleic acid molecular detection area includes multiple independent nucleic acid detection chambers, which are connected via microchannels after the enriched nucleic acid collection chamber and before the LFA test strip slot where the LFA test strip is placed; each nucleic acid detection chamber has multiple reaction wells designed in parallel.

6. An integrated microfluidic chip for nucleic acid and protein co-detection according to claim 5, characterized in that: The reaction wells contain raw materials for RPA amplification reaction components, specifically including primers, RPA enzyme reaction lyophilized powder, RPA reaction promoter, exonuclease IV, FAM, and biotin-labeled nfo probes.

7. An integrated microfluidic chip for nucleic acid and protein co-detection according to claim 4, characterized in that: The protein molecule detection area includes multiple independent protein detection chambers, which are connected via microchannels after the enriched protein collection chamber and before the LFA test strip slot where the LFA test strip is placed.

8. An integrated microfluidic chip for nucleic acid and protein co-detection according to claim 1, characterized in that: The chip substrate has chambers corresponding to the chip's functional structure layers to increase sample processing capacity.

9. An integrated microfluidic chip for nucleic acid and protein co-detection according to claim 1, characterized in that: The LFA test strip has a structure that, along the chromatography direction, includes a sample pad, a binding pad, an NC membrane coated with a detection line (T line) and a control line (C line), and an absorbent pad.

10. A detection method based on an integrated microfluidic chip, characterized in that: Using an integrated microfluidic chip for nucleic acid and protein co-detection as described in any one of claims 1 to 9, the method includes the following steps: The sample to be tested is injected into the biomarker enrichment and separation region of the integrated microfluidic chip, and the electrodes are connected to the power supply. The samples achieve the enrichment and separation of nucleic acids and proteins through the ion concentration polarization effect in the biomarker enrichment and separation zone; The enriched nucleic acids are then amplified at an isothermal temperature in the nucleic acid molecular detection area. The enriched proteins enter the protein molecule detection area; By pressing the elastic membrane on the observation window, the liquid in the nucleic acid molecular detection area and the protein molecular detection area is driven to flow into the corresponding LFA test strips respectively; Read the test results on the LFA test strip through the results viewing window.

Images