POCT (point-of-care testing) microfluidic device for trace detection of nucleic acid from whole blood sample and application of POCT microfluidic device

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a POCT (point-of-care testing) micro-fluidic device for trace detection of nucleic acid from a whole blood sample and application of the POCT micro-fluidic device. The device comprises an ICP (inductively coupled plasma) paper-based preconcentrator, a plurality of PDMS (polydimethylsiloxane) micro-fluidic chip reaction chambers for nucleic acid reaction and a lateral flow test strip for signal reading. The detection process comprises the following steps: loading a whole blood sample into a preconcentrator, and carrying out blood cell separation and protein digestion; applying low direct-current voltage to realize pre-concentration of DNA; the enriched DNA is introduced into a reaction chamber for nucleic acid amplification, a nucleic acid amplification product flows into the reaction chamber through gravity for CRISPR-mediated reporter molecule activation, and the reporter molecule is combined with the mesoporous gold nano-enzyme material; finally, the reaction mixture generates a visual result on the lateral flow test strip. The device can realize rapid detection, has the advantages of high sensitivity, high specificity, low cost, simplicity and convenience in operation and the like, and is suitable for clinical and field screening application with limited resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a POCT microfluidic device for detecting trace amounts of nucleic acid from a whole blood sample and an application thereof. Background Art

[0002] Cell-free DNA (cfDNA) refers to DNA fragments present in body fluids such as blood, and its sources include release during apoptosis, necrosis, or cell secretion. In recent years, the detection of cfDNA has shown great potential in clinical diagnosis, disease monitoring, and personalized treatment. Since cfDNA comes from cells in various tissues throughout the body, its content and changes can reflect the physiological and pathological state of an individual, and are particularly important in the early diagnosis and monitoring of cancer, infectious diseases, and genetic diseases. In the field of cancer, cfDNA helps to detect tumors early, evaluate treatment effects, and monitor recurrence. For example, by detecting specific cancer-related gene mutations, methylation status, and copy number variations in cfDNA, accurate analysis of tumors can be achieved. In addition, cfDNA also plays an important role in the detection and monitoring of pathogens of infectious diseases. By identifying specific pathogen cfDNA in the blood, rapid and accurate pathogen identification and drug sensitivity analysis can be achieved, thereby providing guidance for the formulation of clinical treatment plans. In terms of genetic diseases, cfDNA testing has been widely used in prenatal screening, and early detection of fetal chromosomal abnormalities can be achieved by analyzing fetal cfDNA in maternal blood.

[0003] Although cfDNA testing has many advantages over traditional tissue biopsy, such as simple operation, high reproducibility and low risk, it still faces multiple challenges in practical application. First, the concentration of cfDNA in the blood is extremely low, usually containing only thousands to hundreds of copies per milliliter of blood, which places extremely high demands on the sensitivity and specificity of the detection technology. Secondly, traditional DNA separation and purification methods are complex and costly, usually requiring centrifugation and the use of commercial nucleic acid extraction kits, which limits its feasibility in clinical and field applications with limited resources. In addition, the complex components in blood samples, such as red blood cells, bilirubin and hemolysis products, may cause cfDNA to degrade before separation, further affecting the accuracy of liquid biopsy. Therefore, it is urgent to develop a rapid, simple and cost-effective detection platform that can effectively integrate nucleic acid separation, pre-concentration and detection steps to achieve high-sensitivity detection of trace nucleic acids in whole blood samples.

[0004] Among the existing nucleic acid detection technologies, lateral flow assay (LFA), as a widely used point-of-care testing (POCT) technology, has attracted much attention due to its simple operation, low cost and rapid results. At present, most commercial and research test strips are mainly for antigens and proteins in whole blood. For example, common rapid diagnostic tests (such as rapid antigen test kits) are widely used in the screening of infectious diseases, such as new coronavirus antigen detection and influenza virus antigen detection. However, compared with antigen and protein detection, nucleic acid detection has higher sensitivity and specificity, but there are currently few test strip products for nucleic acids in whole blood, mainly due to the low concentration of nucleic acid molecules in whole blood and the complex extraction process. Ion concentration polarization (ICP) is an efficient method for pre-concentration of charged molecules. It can efficiently pre-concentrate target analytes under low electric fields and has minimal effect on their denaturation. It has become one of the effective strategies for extracting and enriching analytes from complex body fluids. ICP systems are usually composed of surface-patterned cation exchange membranes and low-voltage power supplies, do not require dedicated buffers, and can be implemented in paper-based LFA or microfluidic devices. However, existing ICP devices are mostly used for analyte enrichment and lack effective integration with downstream nucleic acid detection steps, which limits their application in actual detection.

[0005] To solve the above problems, there is an urgent need for a fast, simple and cost-effective platform that can effectively integrate DNA separation, pre-concentration and detection steps to achieve high-sensitivity detection of trace nucleic acids in whole blood samples. Taking cfDNA in Epstein-Barr Virus (EBV), which is highly correlated with nasopharyngeal carcinoma, as an example, the present invention has developed a POCT device for the detection of trace nucleic acids in whole blood that integrates ICP enrichment and nanozyme catalysis. This integrated microfluidic platform improves sensitivity through two signal enhancement modes, including ICP enrichment of target cfDNA and MGNS@Pt nanozyme enhancement of colorimetric signals in LFA. The device not only simplifies the operating process and reduces the cost of detection, but also significantly improves the sensitivity and specificity of detection, demonstrating its strong potential in clinical and field applications with limited resources. In addition, the design of this method is highly versatile, not only suitable for the detection of EBV cfDNA, but also can be extended to other types of nucleic acid detection, with broad application prospects and market potential. Summary of the invention

[0006] The object of the present invention is to provide a POCT microfluidic device for detecting trace amounts of nucleic acids from whole blood samples with low detection limit, high detection sensitivity and fast detection speed, and its application.

[0007] The POCT microfluidic device for detecting trace amounts of nucleic acid from a whole blood sample provided by the present invention has an overall structure as follows: Figure 1 As shown, it is mainly divided into four parts: ICP-assisted paper-based pre-concentrator, microfluidic chip reaction chamber, lateral flow test strip and packaging support assembly; among which:

[0008] (1) ICP-assisted paper-based pre-concentrator (100-104) is a three-layer membrane. Through the synergistic effect of the multiple membranes, plasma is first separated, then protein is digested, and finally cfDNA is efficiently concentrated under the action of the electric field, providing a high concentration of target molecules for subsequent nucleic acid amplification and detection. The specific structure includes: a plasma separation membrane (100) at the top of the concentrator, a protein digestion membrane (102) in the middle, and a DNA pre-concentration membrane (104) at the bottom; the upper plasma separation membrane (100) and the middle protein digestion membrane (102) are connected by an arc-shaped conductive tape (101); the protein digestion membrane (102) and the DNA pre-concentration membrane (104) are connected by an arc-shaped conductive tape (103); wherein the plasma separation membrane (100) is rectangular, has a high-efficiency plasma filtration capability, and is responsible for separating blood cells from plasma in whole blood; the protein digestion membrane (102) in the middle layer is used to digest proteins in plasma to prevent them from interfering with the subsequent DNA concentration process; the bottom DNA pre-concentration membrane (104), after Nafion patterning treatment, is responsible for enriching cfDNA from the sample by electric field driving to enhance the capture efficiency of DNA. The arc-shaped conductive tapes (101, 103) are used to ensure a stable connection between the membrane layers and the conduction of the electric field.

[0009] (2) Microfluidic chip reaction chambers (200-202), which integrate nucleic acid amplification, CRISPR reaction, and nanozyme coupling functions; specifically include: an RPA reaction chamber (200) located on one side of the chip, a CRISPR reaction chamber (201) adjacent to the RPA reaction chamber, and a functionalized nanozyme coupling reaction chamber (202) connected to the CRISPR reaction chamber; the RPA reaction chamber (200) is used to amplify enriched cfDNA; the CRISPR reaction chamber (201) is used to specifically identify the target DNA and cut the nucleic acid reporter molecule, and the functionalized nanozyme coupling reaction chamber (202) is used to couple the nanozyme with the cut reporter molecule.

[0010] (3) A lateral flow test strip (300), including a control line (C line) fixed with anti-FAM antibody and a test line (T line) fixed with biotin-labeled anti-IgG, for detecting nucleic acids and providing fast and readable visual results. The test strip transfers the reaction mixture to the test area by capillary action, and generates a visual signal in combination with the nanozyme catalytic reaction. Functionally, the lateral flow test strip moves the mixed solution after the coupling reaction along the test strip, and the nanozyme catalyzes the substrate reaction to enhance the detection signal.

[0011] (4) The packaging support component (400-405) is responsible for fixing, sealing and controlling the flow of liquid in the entire device. Its main components include a plastic cover (400), a PDMS layer (401), a plastic barrier (402-404), and a PDMS layer (405); wherein the plastic cover (400) is used to cover the RPA reaction chamber to prevent contamination; the PDMS layer (401) is used to support the ICP-assisted paper-based preconcentrator; the plastic barrier (402-404) is used to prevent cross-contamination of liquids between different reaction chambers. Each barrier is a circular hole structure. The barrier can be removed manually to allow the liquid to flow to the lower reaction chamber as needed; the PDMS layer (405) is used to support the lateral flow test strip. In terms of overall function, the packaging support component achieves isolation and control between the various components through the design of the PDMS layer and the plastic barrier.

[0012] In the overall device, the ICP-assisted paper-based pre-concentrator is located on the top layer and is tightly integrated on the microfluidic chip. Its three-layer membrane structure is used to efficiently separate plasma, digest proteins and concentrate cfDNA. The reaction chambers of the microfluidic chip are arranged vertically, including the RPA reaction chamber, the CRISPR reaction chamber and the functionalized nanozyme coupling reaction chamber. The lateral flow test strip is placed at the bottom layer for the visualization of the final test results.

[0013] The present invention also provides a method for preparing the above-mentioned POCT microfluidic device for detecting trace amounts of nucleic acids from whole blood samples, the specific steps of which are:

[0014] (1) Prepare an ICP-assisted paper-based preconcentrator. Use Fusion 5 chromatography paper to precisely cut the plasma separation membrane (i.e., plasma separation membrane), protein digestion membrane, and DNA preconcentrator membrane. Add an appropriate amount (e.g., 5 to 8 uL) of 20-40% Nafion solution to the DNA preconcentrator membrane, and let it stand to solidify after processing according to the designed pattern. Cut the protein separation membrane to an appropriate size, add a solution containing proteinase K and Triton X-100, and then dry it under vacuum conditions. After completion, perform Nafion patterning treatment, and use conductive tape to fix and assemble the various membrane layers to form an ICP-assisted paper-based preconcentrator. The workflow of the ICP-assisted paper-based preconcentrator is as follows: Figure 2 As shown. The ICP-assisted concentrator can effectively enrich the sample and improve the detection sensitivity. The results are shown in Figure 3 shown.

[0015] (2) Prepare lateral flow test strips. Dilute anti-FAM antibody and biotin-labeled IgG with PBS to appropriate concentrations. Use a membrane scriber to spray the antibody on the control line (C line) and test line (T line) of the NC membrane and dry at room temperature. Assemble the NC membrane, absorption pad and sample pad onto the support card and cut into test strips with a width of 3 to 7 mm.

[0016] (3) Prepare an integrated microfluidic chip. Mix PDMS and curing agent in a mass ratio of (8-12):1 (preferably 10:1), degas, and then cure at 65-85°C. Cut PDMS sheets into the required size and punch circular holes with a diameter of 4-6 mm on them as RPA, CRISPR and functionalized nanozyme coupling reaction chambers. Assemble PDMS with ICP-assisted preconcentrators, lateral flow test strips and isolation plates, activate the surface by plasma treatment and bond them. Use sealant to prevent leakage, install plastic barriers to control solution flow, and remove the barriers by gently pulling them out manually, allowing the solution to flow into the lower reaction chamber, such as Figure 5 shown.

[0017] (4) Preparation and functionalization of MGNS@Pt. First, the liposome components were dissolved in chloroform and the solvent was removed. Subsequently, a chloroauric acid solution was slowly added under controlled conditions to form gold nanoparticles. Platinum nanoclusters were loaded on the surface of gold nanoparticles by reducing chloroplatinic acid to obtain MGNS@Pt nanozymes with peroxidase activity. Next, MGNS@Pt was functionalized by shaking streptavidin at room temperature for 3.5-4.5 hours, and the unbound streptavidin was removed by centrifugation and washing to finally obtain functionalized MGNS@Pt nanozymes.

[0018] The POCT microfluidic device of the present invention can be used for the detection of trace nucleic acids in whole blood samples. The detection process includes loading the whole blood sample onto the plasma separation membrane on the top layer of the preconcentrator, separating blood cells by capillary action, and then passing through a protein digestion membrane for protein digestion; subsequently, passing through a DNA preconcentration membrane and applying a low DC voltage, so that the negatively charged DNA migrates to the positively charged region of the enrichment channel under the action of electrophoresis and electroosmosis, thereby achieving DNA preconcentration; then, the enriched DNA is introduced into the microfluidic chip reaction chamber; first passes through the nucleic acid amplification reaction chamber; the plastic barriers are removed in sequence, so that the nucleic acid amplification product flows into the downstream CRISPR reaction chamber by gravity for CRISPR-mediated reporter molecule activation, and the reporter molecule enters the functionalized nanozyme coupling reaction chamber and combines with the mesoporous gold nanozyme material pre-modified with platinum nanoclusters; finally, the reaction mixture is made to generate a visual result on a lateral flow test strip.

[0019] The specific steps are:

[0020] (1) After the whole blood sample is mixed with the buffer in an appropriate proportion, a plasma separation membrane is added for blood cell aggregation. Through capillary action, the plasma is filtered to the protein digestion membrane, and an appropriate amount of protease (such as proteinase K) and surfactant (such as Triton X-100) are added for protein digestion. Subsequently, an appropriate amount of 0.25×TAE buffer is added to the DNA pre-concentration membrane, and an appropriate voltage (such as 20-40V) is applied and maintained for 20-30 minutes to start the ICP pre-concentration process, so that cfDNA migrates to the enrichment area under the drive of the electric field. The specific buffer volume, voltage, etc. can be adjusted within the above range according to actual needs to optimize the DNA enrichment efficiency.

[0021] (2) RPA reaction: Use a pipette to transfer the enriched DNA to the nucleic acid amplification reaction chamber of the microfluidic chip. Pre-add nucleic acid amplification reagents including primers, buffer, dNTPs, enzymes, etc. into the reaction chamber to perform the RPA reaction. The reaction volume and reaction conditions (such as temperature and time) can be adjusted within the recommended range according to the kit instructions and actual needs. The RPA amplification step provides the present invention with highly sensitive detection capabilities, but not all application scenarios require its participation. In some cases, it can be selected whether to include the RPA amplification step based on specific detection sensitivity requirements.

[0022] (3) CRISPR reaction and functionalized nanozyme coupling reaction: gradually remove the plastic barrier and add the previous reaction solution to the CRISPR reaction chamber and the coupling reaction chamber in turn. Preload the CRISPR reaction solution containing LbCas12a enzyme, crRNA, buffer, reporter nucleic acid molecule and MGNS@Pt-SA nanozyme solution into each reaction chamber. When the target DNA is present, the CRISPR-Cas12a system is activated, cutting the reporter molecule and fixing it on the nanozyme surface to complete the signal amplification. This step is usually performed at 37°C for about 30 minutes, but can be optimized and adjusted according to the actual reaction system.

[0023] (4) Flow chromatography experiment: PBST buffer is added to the mixed solution after the coupling reaction, and it flows along the test strip through capillary action. Then substrate solution is added to the T line and C line. The substrate solution consists of about 15-25% CN / DAB mixture, 5-7% hydrogen peroxide and an appropriate amount of buffer. CN / DAB is added to enhance the color signal, thereby improving the sensitivity and visualization of the detection. CN / DAB reacts with hydrogen peroxide under the catalysis of MGNS@Pt to generate a gray precipitate, which enhances the color signal of the T line and C line on the test strip. The results are as follows: Figure 5After adding CN / DAB, the color of T line and C line changes from light to dark, and the signal becomes clearer. Qualitative or quantitative analysis can be performed by colorimetric analysis or image processing software (such as ImageJ) to achieve rapid and accurate detection of EBV cfDNA. The composition ratio and volume of the substrate solution can be adjusted within the above range.

[0024] The POCT microfluidic device for integrated ICP enrichment and nanozyme catalysis for trace nucleic acid detection in whole blood samples constructed by the present invention can achieve ICP pre-concentration without RPA reaction to increase the signal intensity by 4.0 to 8.4 times, and reduce the detection limit from 40pM to 4pM. After the integrated RPA reaction, the detection limit is further reduced to 4aM, meeting the needs of high-sensitivity detection. In whole blood samples, its detection limit reaches 100aM, and only 100μL whole blood sample is required. In addition, the entire detection process is completed within 75 minutes, and the cost is significantly lower than traditional commercial DNA extraction kits and PCR methods. The estimated detection cost is only US$4.72. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the POCT microfluidic device integrating ICP enrichment and nanozyme catalysis.

[0026] Figure 2 Schematic diagram of the working process of ICP-assisted paper-based preconcentrator.

[0027] Figure 3 To compare the C / T ratio values ​​and their corresponding photos under different concentrations of EBV cfDNA pre-concentration and non-pre-concentration conditions.

[0028] Figure 4 Flow chart of the step-by-step liquid mixing reaction in each PDMS microfluidic reaction chamber by extracting the plastic barrier.

[0029] Figure 5 The figure is a comparison of the color change of the test strip before and after adding CN / DAB.

[0030] Figure 6 This is a schematic diagram of the dimensions of the entire POCT device prepared in Example 1.

[0031] Figure 7 This is a physical diagram of the dimensions of the entire POCT device prepared in Example 1.

[0032] Figure 8 This is the standard curve of C / T ratio value and logarithm of EBV cfDNA concentration in the non-amplification mode of Example 2.

[0033] Fig. 9 The figure is a comparison of the C / T ratio values ​​between the nasopharyngeal carcinoma group and the healthy group of rats in Example 3.

[0034] Numbers in the figure: 100 is a plasma separation membrane, 102 is a protein digestion membrane, 104 is a DNA pre-concentration membrane, 101 and 103 are conductive tapes; 200 is an RPA reaction chamber, 201 is a CRISPR reaction chamber, 202 is an enzyme coupling reaction chamber; 300 is a lateral flow test strip; 400 is a plastic cover, 401 is a PDMS layer, 402-404 are plastic barriers, and 405 is a PDMS layer. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1: Construction of a POCT microfluidic device integrating ICP enrichment and nanozyme catalysis, the dimensions of each component of which are as follows: Figure 6 As shown in the actual picture Figure 7 The specific steps are:

[0037] (1) Preparation of an ICP-assisted paper-based preconcentrator. The concentrator is composed of a plasma separation membrane, a protein digestion membrane, and a DNA preconcentration membrane. Each layer of the membrane is made of Fusion 5 chromatography paper with a thickness of 0.37 mm and cut into a suitable shape with scissors. About 10 μL of 30% Nafion solution is dripped on the DNA preconcentration membrane, and after patterning according to the design, it is left to stand overnight to ensure sufficient solidification. Next, the protein separation membrane is cut into 27.5 mm long and 10.0 mm wide, and 40 μL of a solution containing proteinase K and Triton X-100 is added, and then dried under vacuum conditions for 1 hour. After drying, the protein separation membrane is subjected to Nafion patterning. Finally, two layers of arc-shaped conductive tape with a width of 8.0 mm are used to connect the Nafion pattern, thereby fixing and assembling the three layers of membrane to complete the preparation of the ICP-assisted paper-based preconcentrator.

[0038] (2) Preparation of lateral flow test strips. Anti-FAM antibody was diluted to 0.55 mg / mL with PBS, and biotin-labeled IgG was diluted at different concentrations. Antibodies were sprayed on the C and T lines of the NC membrane using a membrane scriber, and dried overnight at room temperature. Subsequently, the NC membrane, absorption pad, and sample pad were assembled onto a support card and cut into strips with a width of 4 mm.

[0039] (3) Preparation of microfluidic chip. The PDMS substrate and curing agent were mixed in a ratio of 10:1 and vacuum degassed for 5 minutes. Subsequently, the mixture was poured into two culture dishes, cured at 80°C for 4 hours, and cut into rectangles of 10 mm width and different lengths after removal. A hole puncher was used to punch holes with a diameter of 4 mm in the PDMS as reaction chambers for RPA, CRISPR and coupling reactions. Next, the PDMS was assembled with an ICP-assisted paper-based preconcentrator, a lateral flow test strip, and a plastic isolation plate, and its surface was activated and bonded using a plasma cleaner. Finally, glue was used to seal the device to prevent liquid leakage. The plastic barrier was inserted between the PDMS layers.

[0040] Example 2: Use the device constructed in Example 1 to detect standard EBV cfDNA.

[0041] (1) Sample enrichment.

[0042] Prepare a series of EBV cfDNA standard solutions with a concentration gradient. Since the standard does not contain non-nucleic acid substances in the blood such as plasma and blood cells, 100uL of samples of each concentration can be directly loaded into the DNA pre-concentration membrane, 20uL of 0.25×TAE buffer is added, and a low DC voltage of 30V is applied for about 25 minutes to promote the migration of cfDNA from the enrichment channel of the DNA pre-concentration membrane through the ICP effect.

[0043] (2) CRISPR reaction and flow cytometry test strip experiments.

[0044] After sample enrichment, the DNA pre-concentration membrane of the enrichment channel is directly moved to the reaction chamber containing the CRISPR reaction reagent. Since the RPA amplification step is not performed in this embodiment, the RPA reaction chamber is omitted. Subsequently, the Cas12a-crRNA complex is added to the reaction chamber, allowing it to bind to the target EBV cfDNA and activate the collateral cleavage activity of Cas12a. The activated Cas12a cuts the nucleic acid reporter molecule with a biotin group and releases the biotin end. The cut nucleic acid reporter molecule is coupled to the MGNS@Pt-SA nanozyme, and then 30μL PBST buffer is added to flow along the test strip by capillary action. In the reporting area of ​​the test strip, the cut reporter molecule is combined with the anti-FAM antibody (C line) and the biotin-labeled anti-IgG (T line) to form a visible color band. Subsequently, 5μL of a substrate solution consisting of 19% CN / DAB, 6% hydrogen peroxide and buffer was added to each of the T line and the C line. CN / DAB reacts with hydrogen peroxide under the catalysis of MGNS@Pt to generate a gray precipitate, which enhances the color signals of the T line and C line. Finally, the gray value ratio of the C line to the T line is calculated by visual colorimetry or quantitative analysis using ImageJ software. This method can accurately detect EBV cfDNA of different concentration gradients without amplification, verifying the sensitivity and specificity of the device. The results are shown in Figure 2. Figure 8 Shown is a standard curve of C / T ratio value and logarithm of EBV cfDNA concentration in the non-amplification mode.

[0045] Example 3: Use the device constructed in Example 1 to detect EBV cfDNA in rat whole blood.

[0046] (1) Establishment of rat model and blood sample collection.

[0047] Healthy adult rats were selected and a nasopharyngeal carcinoma model was established in accordance with ethical requirements. Human nasopharyngeal carcinoma cells were injected into nude mice to induce the formation of nasopharyngeal carcinoma. After the model was established, the growth of the tumor was monitored regularly. After the nasopharyngeal carcinoma tumor grew significantly, about 100 μL of whole blood was collected from the rat's tail vein or heart using sterile techniques. A total of 16 whole blood samples were collected in this experiment, including 8 in the disease group (nasopharyngeal carcinoma model) and 8 in the control group (healthy rats).

[0048] (2) Sample enrichment.

[0049] A 100 μL rat whole blood sample was loaded onto the left side of the plasma separation membrane, and blood cells were filtered by capillary action. The sample then passed through the protein digestion layer. Proteinase K and Triton X-100 were added to the protein digestion membrane and incubated for five minutes to digest the protein. Next, 0.25×TAE buffer was added to the DNA pre-concentration membrane, and a low DC voltage of 30V was applied for about 25 minutes to promote the migration of cfDNA from the protein digestion membrane to the enrichment channel of the DNA pre-concentration membrane through the ICP effect.

[0050] (3) Sample amplification and detection.

[0051] The membrane of the enrichment channel was lightly punctured with a pipette tip to immerse the pre-concentrated cfDNA in the reaction chamber containing the RPA amplification reagent. The RPA reaction was performed in the reaction chamber at 37°C for 20 to 30 minutes to amplify the cfDNA. The plastic barrier was extracted to allow the amplified product to flow by gravity into the downstream CRISPR-mediated reporter activation region. In this region, the Cas12a-crRNA complex binds to the target cfDNA, activating the collateral cleavage activity of Cas12a, which in turn cleaves the nucleic acid reporter with a biotin group and releases the biotin end. The cleaved nucleic acid reporter was coupled to the MGNS@Pt-SA nanozyme, and 30 μL of PBST buffer was added to allow it to flow along the test strip by capillary action. Visible color bands were formed by binding to anti-FAM antibodies (C line) and biotin-labeled anti-IgG (T line). Subsequently, 5 μL of substrate solution consisting of 19% CN / DAB, 6% hydrogen peroxide and buffer was added to each of the T and C lines. CN / DAB reacts with hydrogen peroxide under the catalysis of MGNS@Pt to generate gray precipitate, which enhances the color signal of T line and C line. The gray value ratio of C line to T line is calculated by visual colorimetry or quantitative analysis using ImageJ software. The results are shown as follows Fig. 9 As shown, the C / T ratio of mice in the disease group was higher than that in the healthy group, indicating that this method has a high specificity in distinguishing NPC models from healthy rats.

Claims

1. A POCT microfluidic device for detecting trace amounts of nucleic acids from whole blood samples, characterized in that: It is divided into four parts: ICP-assisted paper-based pre-concentrator, microfluidic chip reaction chamber, lateral flow test strip and packaging support assembly; among them: (1) An ICP-assisted paper-based preconcentrator (100-104) is a three-layer membrane structure, specifically comprising: a plasma separation membrane (100) at the top of the concentrator, a protein digestion membrane (102) in the middle, and a DNA preconcentration membrane (104) at the bottom; the upper plasma separation membrane (100) and the middle protein digestion membrane (102) are connected by an arc-shaped conductive tape (101); the protein digestion membrane (102) and the DNA preconcentration membrane (104) are connected by an arc-shaped conductive tape (103); wherein the plasma separation membrane (100) is rectangular and has high-efficiency plasma filtration capability, and is responsible for separating blood cells from plasma in whole blood; the protein digestion membrane (102) in the middle layer is used to digest proteins in plasma to prevent them from interfering with the subsequent DNA concentration process; the DNA pre-concentration membrane (104) at the bottom layer is treated with Nafion patterning and is responsible for enriching cfDNA from the sample by electric field driving to enhance the DNA capture efficiency; the arc-shaped conductive tape (101, 103) is used to ensure a stable connection between the membrane layers and the conduction of the electric field; (2) Microfluidic chip reaction chambers (200-202), which integrate nucleic acid amplification, CRISPR reaction, and nanozyme coupling functions; specifically, they include: an RPA reaction chamber (200) located on one side of the chip, a CRISPR reaction chamber (201) adjacent to the RPA reaction chamber, and a functionalized nanozyme coupling reaction chamber (202) connected to the CRISPR reaction chamber; the RPA reaction chamber (200) is used to amplify enriched cfDNA; the CRISPR reaction chamber (201) is used to specifically identify target DNA and cut nucleic acid reporter molecules, and the functionalized nanozyme coupling reaction chamber (202) is used to couple the nanozyme with the reporter molecules after cutting; (3) A lateral flow test strip (300), comprising a control line (C line) fixed with anti-FAM antibody and a test line (T line) fixed with biotin-labeled anti-IgG, for detecting nucleic acid and providing a fast and readable visual result; the lateral flow test strip transfers the reaction mixture to the test area by capillary action, and generates a visual signal in combination with the nanozyme catalytic reaction; the lateral flow test strip moves the mixed solution after the coupling reaction along the test strip, and the nanozyme catalyzes the substrate reaction to enhance the detection signal; (4) A packaging support component (400-405), comprising a plastic cover (400), a PDMS layer (401), a plastic barrier (402-404), and a PDMS layer (405); wherein the plastic cover (400) is used to cover the RPA reaction chamber to prevent contamination; the PDMS layer (401) is used to support the ICP-assisted paper-based pre-concentrator; the plastic barrier (402-404) is used to prevent cross-contamination of liquids between different reaction chambers, and each barrier is a circular hole structure. The barrier can be manually removed to allow the liquid to flow to the lower reaction chamber as needed; the PDMS layer (405) is used to support the lateral flow test strip.

2. The method for preparing a POCT microfluidic device for detecting trace amounts of nucleic acids from whole blood samples according to claim 1, characterized in that: The specific steps are: (1) Preparation of ICP-assisted paper-based preconcentrator: using Fusion 5 chromatography paper to precisely cut plasma separation membranes, i.e., plasma separation membranes, protein digestion membranes, and DNA preconcentration membranes; 20-40% Nafion solution is added to the DNA pre-concentration membrane, and the membrane is treated according to the designed pattern and then allowed to stand and solidify; the protein separation membrane is cut into an appropriate size, and a solution containing proteinase K and Triton X-100 is added and then dried under vacuum conditions; after completion, Nafion patterning is performed, and each layer of the membrane is fixed and assembled using conductive tape to form an ICP-assisted paper-based pre-concentrator; (2) Preparation of lateral flow test strips: Anti-FAM antibody and biotin-labeled IgG were diluted to appropriate concentrations with PBS; the antibody was sprayed on the control line (C line) and the test line (T line) of the NC membrane using a membrane scriber and dried at room temperature; the NC membrane, the absorption pad and the sample pad were assembled on a support card and cut into test strips with a width of 3 to 7 mm; (3) Preparation of an integrated microfluidic chip: PDMS and a curing agent are mixed in a ratio of (8 to 12):1, degassed, and then cured at 65 to 85°C; PDMS sheets are cut into the required size, and circular holes with a diameter of 4 to 6 mm are punched thereon as RPA, CRISPR, and functionalized nanozyme coupling reaction chambers; PDMS is assembled with an ICP-assisted preconcentrator, a lateral flow test strip, and an isolation plate, and the surface is activated and bonded by plasma treatment; a sealant is used to prevent leakage, and a plastic barrier is installed to control the flow of the solution. The barrier can be removed by gently pulling it out manually, thereby allowing the solution to flow into the lower reaction chamber; (4) Preparation and functionalization of MGNS@Pt: First, the liposome components were dissolved in chloroform and the solvent was removed. Subsequently, a chloroauric acid solution was added under controlled conditions to form gold nanoparticles. Platinum nanoclusters were loaded on the surface of gold nanoparticles by reducing chloroplatinic acid to obtain MGNS@Pt nanozyme with peroxidase activity. Next, MGNS@Pt was functionalized by shaking with streptavidin at room temperature for 3.5-4.5 hours. Unbound streptavidin was removed by centrifugation and washing to finally obtain functionalized MGNS@Pt nanozyme.

3. The method for detecting trace amounts of nucleic acids using a POCT microfluidic device according to claim 1, wherein: The process includes: first, loading the whole blood sample onto the plasma separation membrane on the top layer of the preconcentrator to separate blood cells by capillary action, and then passing through the protein digestion membrane for protein digestion; subsequently, passing through the DNA preconcentration membrane and applying a low DC voltage, the negatively charged DNA migrates to the positively charged region of the enrichment channel under the action of electrophoresis and electroosmosis to achieve DNA preconcentration; then, the enriched DNA is introduced into the microfluidic chip reaction chamber; first passing through the nucleic acid amplification reaction chamber; removing the plastic barriers in turn, so that the nucleic acid amplification product flows into the downstream CRISPR reaction chamber by gravity for CRISPR-mediated reporter molecule activation, and the reporter molecule enters the functionalized nanozyme coupling reaction chamber and combines with the mesoporous gold nanozyme material pre-modified with platinum nanoclusters; finally, the reaction mixture generates a visual result on the lateral flow test strip.

4. The method according to claim 3, characterized in that The specific steps are: (1) After mixing the whole blood sample with the buffer, a plasma separation membrane is added to aggregate the blood cells; through capillary action, the plasma is filtered to the protein digestion membrane, and an appropriate amount of protease and surfactant are added to digest the protein; Subsequently, an appropriate amount of 0.25×TAE buffer was added to the DNA pre-concentration membrane, and a voltage of 20-40V was applied and maintained for 20-30 minutes to start the ICP pre-concentration process, so that the cfDNA migrated to the enrichment area under the drive of the electric field; (2) RPA reaction: Use a pipette to transfer the enriched DNA to the nucleic acid amplification reaction chamber of the microfluidic chip. Primers, buffer, dNTPs, and enzyme nucleic acid amplification reagents are pre-added in the reaction chamber for RPA reaction. (3) CRISPR reaction and functionalized nanozyme coupling reaction: gradually remove the plastic barrier and add the previous reaction solution to the CRISPR reaction chamber and the coupling reaction chamber in turn; preload the CRISPR reaction solution containing LbCas12a enzyme, crRNA, buffer, reporter nucleic acid molecule and MGNS@Pt-SA nanozyme solution into each reaction chamber; when the target DNA is present, the CRISPR-Cas12a system is activated, cutting the reporter molecule and fixing it on the nanozyme surface to complete signal amplification; (4) Flow chromatography experiment: PBST buffer is added to the mixed solution after the coupling reaction, and it flows along the test strip through capillary action; then substrate solution is added to the T line and C line respectively; the substrate solution is composed of 15-25% CN / DAB, 5-7% hydrogen peroxide and buffer solution; CN / DAB is added to enhance the color signal, thereby improving the sensitivity and visualization effect of detection; CN / DAB reacts with hydrogen peroxide under the catalysis of MGNS@Pt to generate a gray precipitate, which enhances the color signal of the T line and C line on the test strip; after adding CN / DAB, the color of the T line and C line changes from light to dark, and the signal becomes clearer; qualitative or quantitative analysis is performed through colorimetric analysis or image processing software to achieve rapid and accurate detection of EBV cfDNA.

5. The method according to claim 4, characterized in that: The buffer volume and voltage in step (1) are adjusted as needed to optimize the DNA enrichment efficiency.

Images