Paper-based micro-fluidic chip device and method for immediately detecting blood metabolism markers by using paper-based micro-fluidic chip device

Abstract

The invention discloses a paper-based micro-fluidic chip device ([mu] PAD) and a method for instantly detecting a blood metabolism marker by using the paper-based micro-fluidic chip device ([mu] PAD). The micro-PAD is a paper-based micro-fluidic chip device (MOG-micro-PAD) based on metal organic gel (MOG), and the micro-PAD is a paper-based micro-fluidic chip device (MOG-micro-PAD) based on metal organic gel (MOG). According to the device, MOG with peroxidase-like activity is used as a catalyst, and the detection activity can still be maintained after the device is placed at room temperature for 90 days, so that the device is convenient to store and transport. According to the method, the blood metabolism marker is detected based on MOG-[mu] PAD, a Cu / Co-MOG dispersion liquid with the concentration of 3-9 mg / mL is firstly added as a filtering medium, then a whole blood or plasma sample is added, red blood cells and colored macromolecular substances in the blood sample can be blocked and filtered in situ, detection can be completed without an additional separation process, operation is easy and convenient, cost is low, and the method is suitable for large-scale popularization and application. The method is suitable for instantly detecting the blood metabolism marker in a resource-deficient region.

Description

Technical Field

[0001] This invention belongs to the field of biomedical and blood metabolism biomarker detection technology, specifically relating to a paper-based microfluidic chip device based on metal-organic gel and its method for the real-time detection of blood metabolism biomarkers. Background Technology

[0002] Detecting disease-related metabolic biomarkers in the blood is an essential step in the accurate diagnosis and treatment of diseases. Dynamic changes in metabolic biomarkers can objectively reflect the body's metabolic state, helping to determine an individual's health status and providing timely intervention. For example, measuring glucose helps manage diabetes, measuring uric acid helps monitor gout, measuring xanthine helps assess kidney failure, and measuring creatinine helps assess chronic kidney disease and muscle diseases. Currently, methods for analyzing blood metabolic biomarkers mainly include nuclear magnetic resonance (NMR), chromatography, mass spectrometry (MS), spectroscopy, and electrochemical techniques. However, NMR, chromatography, mass spectrometry, and spectroscopy are not suitable for point-of-care testing (POCT). In contrast, electrochemical techniques (such as amperometric and impedance methods) offer advantages such as high sensitivity, rapid response, and ease of miniaturization, making them theoretically suitable for POCT. However, these methods are susceptible to coexisting ions (such as Ca²⁺ and Mg²⁺) in complex biological samples. 2 It suffers from several drawbacks in POCT applications, including non-specific protein adsorption, interference from endogenous electrochemically active substances (such as ascorbic acid and uric acid), and reliance on a stable external power supply and signal processing system. Furthermore, the long-term stability and reproducibility of the electrodes, as well as the cost associated with single-use, are also shortcomings that need to be overcome.

[0003] Colorimetric reaction-based detection technologies have become an important analytical method widely used in the POCT field due to their advantages such as ease of operation, intuitive results, and low cost. In recent years, POCT sensing technology has shown a diversified development trend, with the main technology platforms including lateral flow chromatography test strips and microfluidic chips. Microfluidic chips can be further divided into traditional microfluidic devices and paper-based analytical devices (μPADs). Traditional microfluidic technology uses glass, polymers, etc. as substrates. Although it has advantages such as precise fluid control, high efficiency, and good integration, its high manufacturing cost, dependence on external equipment, and complex operation limit its application in POCT. Due to the inherent white background of μPADs, the inherent dark matrix characteristics of undiluted plasma and whole blood samples often strongly interfere with the detection process during colorimetric reactions; at the same time, red blood cells can directly pass through the pores of the filter paper (approximately 10-20 μm in diameter). Therefore, when using μPADs for POCT of blood samples, it is necessary to separate and remove red blood cells first, and such pretreatment steps are not suitable for point-of-care testing in resource-scarce areas. In summary, existing blood sample separation strategies are still limited by specific assembly steps, operational complexity, or the cost of separation modules.

[0004] Furthermore, in traditional blood metabolic biomarker analysis, the specific recognition and signal transduction of metabolites typically require the synergistic action of two natural enzymes (peroxidase and oxidase), such as horseradish peroxidase (HRP) mediating subsequent signal generation. However, natural peroxidase preparations are expensive and exhibit poor enzyme stability at room temperature, making long-term storage difficult and severely limiting the practical application of this technology in point-of-care testing of blood metabolic biomarkers. Therefore, researching a low-cost method that allows for direct point-of-care testing of blood samples without the need for additional red blood cell separation would be more conducive to widespread application in resource-scarce regions, meeting the demand for rapid on-site diagnosis of diseases. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a paper-based microfluidic chip device and a method for the point-of-care detection of blood metabolic markers. This invention discovers that the metal-organic gel Cu / Co-MOG itself possesses peroxidase-like activity, and uses it as a catalyst combined with a paper chip to prepare a paper-based microfluidic chip device (MOG-μPAD). Adding a Cu / Co-MOG dispersion at a concentration of 3-9 mg / mL to the front of the MOG-μPAD serves as a filter medium. Adding a whole blood or plasma sample allows for in-situ blocking and separation of erythrocytes and / or colored macromolecules from the whole blood or plasma sample, achieving point-of-care detection of blood metabolic markers without separation. This solves the technical problems of existing technologies requiring separate separation of blood samples to remove erythrocytes and colored macromolecules, and the high cost and inability to store natural peroxidases for long periods at room temperature.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a paper-based microfluidic chip device based on a metal-organic gel, which is prepared according to the following method: (1) Preparation of three-layer laminated high-throughput μPAD: The design pattern is printed on the front side of the filter paper using a wax printer. The wax is heated to make the wax diffuse evenly into the interior of the filter paper base to form a hydrophobic area. The area without wax treatment is used as the hydrophilic detection area. After the treatment, it is folded into three layers so that the hydrophilic detection areas on each layer correspond one-to-one. The two ends are fixed to form a three-layer laminated high-throughput μPAD. (2) Preparation of paper-based microfluidic chip device based on metal organogel: The hydrophilic detection area on the back of the three-layer laminated high-throughput μPAD obtained in step (1) is uniformly coated with metal organogel as a catalyst, and heated and dried to make it uniformly deposited on the hydrophilic detection area on the back of the μPAD, which is the paper-based microfluidic chip device MOG-μPAD. The metal-organic gel includes one or more of Cu / Co-MOG, Cu-MOG, and Co-MOG that have peroxidase-like activity.

[0007] Preferably, in the paper-based microfluidic chip device, the metal-organic gel used as a catalyst in step (2) is Cu / Co-MOG, and its coating concentration is 0.5~3 mg / mL.

[0008] Preferably, the paper-based microfluidic chip device detects blood metabolic markers based on colorimetry, and the hydrophilic detection area on the back of the MOG-μPAD of the paper-based microfluidic chip device is further enriched with oxidase and / or chromogenic solution. In some embodiments, the oxidase includes one or more of glucose oxidase, uricase oxidase, xanthine oxidase, and a combination of creatinine oxidase + creatine oxidase + sarcosine oxidase; the chromogenic solution includes a solution of the chromogenic agent 3,3',5,5'-tetramethylbenzidine (TMB), i.e., TMB chromogenic solution.

[0009] Secondly, the present invention also provides a blood metabolic marker detection kit based on colorimetry, which includes a paper-based microfluidic chip device as described in the present invention, as well as an oxidase and / or a colorimetric solution.

[0010] Preferably, for the immediate detection of metabolic markers in whole blood or plasma samples without separation, the detection kit further includes a Cu / Co-MOG dispersion at a concentration of 3-9 mg / mL; the Cu / Co-MOG dispersion serves as a filter medium, which is a stable or semi-stable colloidal system formed by mixing micro / nano-scale Cu / Co-MOG particles with water, used for in-situ blocking and separation of erythrocytes and / or colored macromolecules in the sample.

[0011] Preferably, in the detection kit, the Cu / Co-MOG dispersion has a hierarchical porous structure with a pore size of 1~80nm.

[0012] Preferably, the detection kit is used for the immediate detection of one or more metabolic markers, such as glucose, uric acid, xanthine, and creatinine, in whole blood or plasma samples without separation, using a Cu / Co-MOG dispersion at a concentration of 6 mg / mL as the filter medium.

[0013] Preferably, in the detection kit, the oxidase includes one or more of glucose oxidase, uricase oxidase, xanthine oxidase, and a combination of creatinine oxidase + creatine oxidase + sarcosine oxidase; the colorimetric solution is selected from TMB colorimetric solution.

[0014] Thirdly, the present invention also provides a method for immediate detection of blood metabolic markers without separation, which employs the paper-based microfluidic chip device as described in the present invention to detect metabolic markers in whole blood or plasma samples without separation, comprising the following steps: (1) Add oxidase solution and TMB colorimetric solution to the hydrophilic detection area on the back of the paper-based microfluidic chip device MOG-μPAD; (2) In-situ separation and instant detection In the hydrophilic detection area on the front of the paper-based microfluidic chip device MOG-μPAD, a Cu / Co-MOG dispersion with a concentration of 3~9 mg / mL is first added as a filter medium, and then a whole blood or plasma sample is added. The red blood cells and / or macromolecular colored substances in the whole blood or plasma sample are blocked and separated in situ, so that the target metabolic markers in the sample migrate to the corresponding detection area on the back. The target metabolic marker produces hydrogen peroxide (H2O2) under the action of oxidase. H2O2 reacts with colorless TMB under the catalysis of Cu / Co-MOG to generate blue oxidized TMB (ox-TMB), which then shows color. (3) Quantitative analysis Colorimetric images are captured by taking photos with a smartphone. Image analysis software is used to measure the grayscale intensity of the red (R) channel in the captured image area. Based on the relationship between the grayscale intensity I0-I in the R channel and the concentration of the target metabolic biomarker standard, the concentration of the target metabolic biomarker in the sample is quantitatively analyzed. Here, I and I0 represent the grayscale intensities with and without the target metabolic biomarker, respectively; I is the grayscale intensity corresponding to the sample, and I0 is the grayscale intensity corresponding to the blank control group. TMB itself is oxidized by air, resulting in slight color development. Even without color development, there will still be a corresponding grayscale intensity; that is, I is the grayscale intensity corresponding to the sample, and I0 is the grayscale intensity corresponding to the blank control group. The darker the color, the smaller the grayscale intensity value.

[0015] Preferably, in the method, the target metabolic marker is grape, and the relationship is I0-I=k1×log 10 (C) 葡萄糖 )+b1; The target metabolic marker is uric acid, and the relationship is I0-I=k2×C. 尿酸 +b2; The target metabolic marker is xanthine, and the relationship is log 10 (I0-I) = k3×log 10 (C) 黄嘌呤 )+b3; The target metabolic marker is creatinine, and the relationship is I0-I=k4×log 10 (C) 肌酐 )+b4.

[0016] Preferably, in the method, the Cu / Co-MOG dispersion contains Cu / Co-MOG with a hierarchical porous structure having a pore size of 1~80 nm.

[0017] Preferably, the method is used for immediate detection of one or more metabolic markers, such as glucose, xanthine, uric acid, and creatinine, in whole blood or plasma samples without separation. In step (2), Cu / Co-MOG dispersion with a concentration of 6 mg / mL is added as a filter medium, and then whole blood or plasma samples are added.

[0018] Preferably, the method is used for the immediate detection of glucose, xanthine, or creatinine in whole blood or plasma samples without separation, wherein the pH of the TMB chromogenic solution is 4 and the concentration of TMB is 4.5 mg / mL; and for the immediate detection of uric acid in whole blood or plasma samples without separation, wherein the pH of the TMB chromogenic solution is 5 and the concentration of TMB is 4.5 mg / mL.

[0019] Preferably, in the method, the oxidase is glucose oxidase, and the reaction pH is 6; the oxidase is uric acid oxidase, and the reaction pH is 8; the oxidase is xanthine oxidase or a combination enzyme of creatinine oxidase + creatine oxidase + sarcosine oxidase, and the reaction pH is 7.

[0020] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: The paper-based microfluidic chip device provided by this invention uses one or more of the metal organogel Cu / Co-MOG, Cu-MOG, and Co-MOG as catalysts. In particular, Cu / Co-MOG as a catalyst has better enzyme activity than horseradish peroxidase (HRP), and it retains its original detection activity after being placed at room temperature for 90 days. It has good stability and is therefore easy to store and transport.

[0021] The present invention provides a method for the immediate detection of blood metabolic markers. First, a Cu / Co-MOG dispersion with a concentration of 3-9 mg / mL is added as a filter medium to the hydrophilic detection area on the front side of a paper-based microfluidic chip device. Then, whole blood or plasma samples are added for immediate detection. The Cu / Co-MOG dispersion contains Cu / Co-MOG with a hierarchical porous structure with pore sizes ranging from 1 to 80 nm. The Cu / Co-MOG is fully swollen and forms a hydrated gel state with water, which can effectively block and separate erythrocytes and colored macromolecules in situ. Using this method to detect blood samples enables immediate detection of blood metabolic markers without separation. The method is simple to operate, low in cost, and suitable for immediate detection of blood metabolic markers in resource-scarce areas. Attached Figure Description

[0022] Figure 1 A schematic diagram of a paper-based microfluidic chip device based on metal-organic gels and the detection of metabolic biomarkers.

[0023] Figure 2This is a characterization of Cu / Co-MOG. A is a schematic diagram of the Cu / Co-MOG synthesis method, B and C are SEM characterization morphologies of Cu / Co-MOG at different multiples, and D is TEM characterization of elemental distribution.

[0024] Figure 3 These are the infrared spectra of H3TATAB (blue line) and Cu / Co-MOG (red line).

[0025] Figure 4 This is the TGA curve of Cu / Co-MOG in an N2 environment.

[0026] Figure 5 In the diagram, A represents the N2 adsorption-desorption isotherm; B represents the pore size distribution of Cu / Co-MOG.

[0027] Figure 6 A represents the design and fabrication of the high-throughput μPAD, while B represents the optimization of the paraffin melting time.

[0028] Figure 7 In the diagram, A represents the assembly of a high-throughput μPAD, and B represents a high-throughput μPAD.

[0029] Figure 8 In the diagram, A represents the original hydrophilic region of the untreated filter paper, B represents the hydrophilic region modified by Cu / Co-MOG, C represents the effect of filtering a whole blood sample through ordinary filter paper, and D represents the effect of filtering a whole blood sample through MOG-μPAD.

[0030] Figure 9 These are scanning electron microscope images of the hydrophobic region (wax-modified region).

[0031] Figure 10 These are optical microscope images of whole blood samples that have not undergone any processing.

[0032] Figure 11 In the diagram, A represents the Michaelis-Menten curve of the reaction between Cu / Co-MOG catalyst and TMB, B represents the Michaelis-Menten curve of the reaction between Cu / Co-MOG catalyst and H2O2, C represents the Lineweaver-Burk curve of the reaction between Cu / Co-MOG catalyst and TMB, and D represents the Lineweaver-Burk curve of the reaction between Cu / Co-MOG catalyst and H2O2.

[0033] Figure 12 This is a diagram illustrating smartphone signal acquisition.

[0034] Figure 13 This describes the effect of different color channels on the MOG-μPAD analysis signal.

[0035] Figure 14In Figure A, the peroxidase properties of MOG-μPAD are verified, and in Figure B, a schematic diagram of its catalytic mechanism is shown.

[0036] Figure 15 Electron spin resonance spectra of different systems.

[0037] Figure 16 In the diagram, A represents the high-resolution XPS spectra of Cu / Co-MOG before (green line) and after (red line) H2O2 treatment; B represents the broad and high-resolution Cu2p spectra of Cu / Co-MOG without H2O2 treatment; C represents the broad and high-resolution Cu2p spectra of Cu / Co-MOG after H2O2 treatment; D represents the CuLMMAuger spectra of Cu / Co-MOGs before (black line) and after (red line) H2O2 treatment; E represents the broad and high-resolution Co2p spectra of Cu / Co-MOG without H2O2 treatment; and F represents the broad and high-resolution Co2p spectra of Cu / Co-MOG after H2O2 treatment.

[0038] Figure 17 The optimization of experimental conditions for H2O2 detection includes: A) optimization of Cu / Co-MOG concentration, B) optimization of TMB concentration, C) optimization of pH value, and D) optimization of reaction time.

[0039] Figure 18 This is a standard curve for detecting H2O2 based on Cu / Co-MOG peroxidase-like activity. The inset shows the linear calibration curve of H2O2 (10~200μM).

[0040] Figure 19 In the figures A to D, the optimal pH for oxidase detection of glucose (A), uric acid (B), xanthine (C), and creatinine (D) is optimized.

[0041] Figure 20 In the table, A through D represent the optimal pH for TMB detection of glucose (A), uric acid (B), xanthine (C), and creatinine (D), respectively.

[0042] Figure 21 The reaction time optimizations for glucose (A), uric acid (B), xanthine (C), and creatinine (D) are shown in sections A through D.

[0043] Figure 22 In the diagram, A represents the standard curve for glucose detection, B represents the standard curve for uric acid detection, C represents the standard curve for xanthine detection, and D represents the standard curve for creatinine detection.

[0044] Figure 23In the diagram, A represents the linear calibration curve for glucose detection, C represents the linear calibration curve for uric acid detection, E represents the linear calibration curve for xanthine detection, G represents the linear calibration curve for creatinine detection, B represents the glucose selectivity assessment, D represents the uric acid selectivity assessment, F represents the xanthine selectivity assessment, and H represents the creatinine selectivity assessment.

[0045] Figure 24 These are the results of long-term storage stability tests for MOG-μPAD.

[0046] Figure 25 In Figure A, the long-term storage stability assessment of MOG-μPAD after incubation at room temperature for 48 hours is presented, and in Figure B, the long-term storage stability assessment of HRP after incubation at room temperature for 48 hours is presented.

[0047] Figure 26 In the table, A represents the consistency between this method and the conventional clinical method for measuring glucose in healthy individuals (n=6), and B represents the consistency between this method and the conventional clinical method for measuring uric acid in healthy individuals (n=6).

[0048] Figure 27 In the diagram, A and B represent the simultaneous detection of glucose (a) and uric acid (b) in whole blood, while C and D represent the simultaneous detection of glucose (c), uric acid (d), xanthine (e), and creatinine (f) in whole blood at simulated pathological concentrations. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0050] This invention provides a paper-based microfluidic chip device based on metal-organic gel, which is prepared according to the following method: (1) Preparation of a three-layer laminated high-throughput μPAD: A wax printer is used to print the design pattern on the front side of the filter paper. Heating allows the wax to diffuse evenly into the filter paper base, forming hydrophilic and hydrophobic zones. The hydrophilic zones are the untreated areas. After processing, the paper is folded three times to ensure each layer has a corresponding detection area, and the ends are fixed to form a three-layer laminated high-throughput μPAD. In some embodiments, the heating temperature is 70°C. o Heating at C for 55~110s results in a circular hydrophilic detection area.

[0051] (2) Preparation of paper-based microfluidic chip device: The hydrophilic detection area on the back of the three-layer laminated high-throughput μPAD obtained in step (1) is uniformly coated with a metal-organic gel as a catalyst, and heated and dried to make it uniformly deposited on the hydrophilic detection area on the back, which is the paper-based microfluidic chip device MOG-μPAD; the metal-organic gel includes one or more of Cu / Co-MOG, Cu-MOG, and Co-MOG with peroxidase-like activity.

[0052] In some embodiments, the metal-organic gel used as a catalyst is Cu / Co-MOG. 5 μL of Cu / Co-MOG dispersion (0.5~3 mg / mL) is added to a 6 mm diameter circular hydrophilic detection area. Heating and drying are then performed to ensure the Cu / Co-MOG catalyst is uniformly distributed throughout the hydrophilic detection area, resulting in a paper-based microfluidic chip device, MOG-μPAD. The side coated with Cu / Co-MOG is the back side, and the side without Cu / Co-MOG coating is the front side. The Cu / Co-MOG itself is in a gel state. The Cu / Co-MOG dispersion mentioned in this invention refers to a stable or semi-stable colloidal system containing micro / nano-scale metal-organic gel (Cu / Co-MOG) particles, formed by dispersing Cu / Co-MOG in a solvent after mixing with water and using mechanical force (such as stirring or ultrasound).

[0053] Preferably, 5 μL Cu / Co-MOG dispersion (1.5 mg / mL, water as solvent) is added to the back detection area of ​​the μPAD, and then dried (50°C). o (C, 5 min) to uniformly deposit Cu / Co-MOG on μPAD, thus obtaining the paper-based microfluidic chip device MOG-μPAD.

[0054] Based on colorimetric detection of blood metabolic markers, the hydrophilic detection area on the back of the MOG-μPAD paper-based microfluidic chip device is further enriched with oxidase and / or chromogenic solution. In some embodiments, the oxidase includes one or more of glucose oxidase, uricase oxidase, xanthine oxidase, and a combination of creatinine oxidase + creatine oxidase + sarcosine oxidase; the chromogenic solution includes a 3,3',5,5'-tetramethylbenzidine (TMB) solution, i.e., TMB chromogenic solution.

[0055] In addition, the present invention also provides a blood metabolic marker detection kit based on colorimetry, which includes a paper-based microfluidic chip device as described in the present invention, as well as an oxidase and / or a colorimetric solution.

[0056] Furthermore, for the immediate detection of metabolic markers in whole blood or plasma samples without separation, the detection kit also includes a Cu / Co-MOG dispersion at a concentration of 3-9 mg / mL; the Cu / Co-MOG dispersion serves as a filter medium for in-situ blocking and separating erythrocytes and / or colored macromolecules in the sample.

[0057] Excessive Cu / Co-MOG concentration in Cu / Co-MOG dispersions can clog paper-based microfluidic chip devices, hindering the passage of target metabolites from the sample to the back detection area for reaction. Conversely, insufficient Cu / Co-MOG concentrations fail to completely block and separate red blood cells, allowing some to pass through and accumulate in the back detection area, thus affecting the detection results. Experiments revealed that Cu / Co-MOG dispersions with concentrations of 3–9 mg / mL fully swell and form a hydrated gel state with water, effectively blocking and separating red blood cells and colored macromolecules from the sample. This allows it to be used as a filter medium for in-situ blocking and separation of red blood cells and colored macromolecules in whole blood or plasma samples. However, the three-dimensional network structure of Cu / Co-MOG collapses after heating and drying, and even subsequent addition of water cannot restore its original structure. Therefore, dried organometallic gels cannot effectively block and separate red blood cells and colored macromolecules in situ.

[0058] In some embodiments, the Cu / Co-MOG dispersion described herein has a hierarchical porous structure with pore sizes ranging from 1 to 80 nm. For the immediate, separation-free detection of one or more metabolic markers, such as glucose, xanthine, uric acid, and creatinine, in whole blood or plasma samples, a Cu / Co-MOG dispersion with a concentration of 6 mg / mL is preferred as the filter medium, as this concentration is more suitable for the permeation of blood metabolites.

[0059] Furthermore, the kit is used to detect glucose, and the oxidase is selected from glucose oxidase (GOD); the kit is used to detect uric acid, and the oxidase is selected from uricase (UO); the kit is used to detect xanthine, and the oxidase is selected from xanthine oxidase (XO); the kit is used to detect creatinine, and the oxidase is selected from a combination enzyme of creatinine amicohydrolase (CAH), creatinase (CI), and sarcosine oxidase (SOX). The chromogenic solution is selected from the chromogenic reagent 3,3',5,5'-tetramethylbenzidine (TMB) solution, i.e., TMB chromogenic solution.

[0060] The kit is used for the immediate detection of metabolic biomarkers in whole blood or plasma samples without separation. First, a Cu / Co-MOG dispersion, acting as a filter medium, is added to the hydrophilic detection area on the front side of a paper-based microfluidic chip device. Then, a whole blood or plasma sample is added. The Cu / Co-MOG dispersion, acting as a filter medium, blocks large colored molecules such as erythrocytes from the whole blood or plasma sample in the front detection area, allowing metabolic biomarkers to migrate to the back side. The target metabolic biomarker reacts with a specific oxidase added to the back detection area to produce H2O2. The Cu / Co-MOG coated on the back detection area acts as a peroxidase-like enzyme, catalyzing the H2O2 to oxidize colorless TMB to blue ox-TMB, thus developing color. A colorimetric image is captured by taking a picture with a smartphone. Commercial software such as ImageJ is used to measure the grayscale intensity of the red channel in the detection area of ​​the captured image. Based on the relationship between the grayscale intensity (I0-I) in the R channel and the concentration of the metabolic biomarker standard, the concentration of the metabolic biomarker in the sample can be analyzed. Where I and I0 represent the grayscale intensities with and without the metabolic biomarker, respectively. Since TMB itself is oxidized by air and will show slight color, it will still have a corresponding grayscale intensity even if it does not show color. Therefore, a relationship is constructed between the grayscale intensity (I0-I) and the concentration of the metabolic biomarker standard, that is, I is the grayscale intensity corresponding to the sample, and I0 is the grayscale intensity corresponding to the blank control group.

[0061] In addition, the present invention also provides a method for real-time detection of metabolic markers in blood samples without separation, which is based on the paper-based microfluidic chip device or reagent kit described in the present invention for real-time detection, and specifically includes the following steps: (1) Add oxidase solution and colorimetric solution to the detection area on the back of the paper-based microfluidic chip device MOG-μPAD; the back of the paper-based microfluidic chip device is the side coated with metal-organic gel as a catalyst. (2) In situ isolation of red blood cells and immediate detection of metabolic markers First, a Cu / Co-MOG dispersion with a concentration of 3~9 mg / mL is added to the detection area on the front of the paper-based microfluidic chip device MOG-μPAD as a filter medium, and then a whole blood or plasma sample is added. Red blood cells and / or macromolecular colored substances in the whole blood or plasma sample are separated in situ, so that the target metabolic markers in the sample can permeate and migrate to the corresponding detection area on the back. The target metabolic marker produces H2O2 under the action of oxidase. H2O2 reacts with colorless TMB under the catalysis of Cu / Co-MOG catalyst to generate blue ox-TMB, which then shows color. (3) Quantitative analysis Colorimetric images are captured by taking photos with a smartphone. Commercial software such as ImageJ is used to measure the gray intensity of the red channel (R channel) of the captured image. Based on the relationship between the gray intensity (I0-I) in the R channel and the concentration of the metabolic biomarker standard, the concentration of the metabolic biomarker in the sample can be analyzed. Here, I and I0 are the gray intensities of the sample containing the metabolic biomarker and the sample without the metabolic biomarker, respectively. That is, I is the gray intensity corresponding to the sample, and I0 is the gray intensity corresponding to the blank control group.

[0062] The metabolic marker is glucose, and the relationship is I0-I=k1×log 10 (C) 葡萄糖 In some embodiments, the grape concentration is 0.4~2.5 mM, and the relationship is I0-I=13.44×log 10 (C) Glucose +8.71, the grape concentration is 3~12mM, and the relationship is I0-I=27.72×log 10 (C) Glucose )+2.75.

[0063] The metabolic marker is uric acid, and the relationship is I0-I=k2×C. 尿酸 +b2, in some embodiments the relationship is I0-I=5.02×C Uricacid +1.52.

[0064] The metabolic marker is xanthine, and the relationship is log 10 (I0-I) = k3×log 10 (C) 黄嘌呤 In some embodiments, the relation is log + b3, where b3 is the logarithm. 10 (I0-I) = 0.40 × log 10 (C) Xanthine )+1.13.

[0065] The metabolic marker is creatinine, and the relationship is I0-I=k4×log 10 (C) 肌酐 ) + b4; In some embodiments, the creatinine concentration is 0.4~2.5mM, and the relationship is I0-I=10.09×log 10 (C) Creatinine +11.56; creatinine concentration is 3~12mM, the relationship is I0-I=24.70×log 10 (C) Creatinine +4.93.

[0066] The Cu / Co-MOG dispersion contains Cu / Co-MOG with a hierarchical porous structure and a pore size of 1~80 nm.

[0067] In some embodiments, for the immediate detection of one or more metabolic markers among glucose, xanthine, uric acid, and creatinine in whole blood or plasma samples without separation, step (2) involves first adding a Cu / Co-MOG dispersion with a concentration of 6 mg / mL as a filter medium, and then adding the whole blood or plasma sample.

[0068] In some embodiments, for the detection of glucose, xanthine, or creatinine, the pH of the TMB chromogenic solution is preferably 4, and the concentration of the TMB chromogenic reagent is 4.5 mg / mL; for the detection of uric acid, the pH of the TMB chromogenic solution is preferably 5, and the concentration of the TMB chromogenic reagent is 4.5 mg / mL.

[0069] The oxidase is GOD, and its reaction pH is 6; the oxidase is UO, and its reaction pH is 8; the oxidase is XO or CAH+CI+SOX, and its reaction pH is 7.

[0070] The following are examples. The following examples illustrate the detection of blood metabolic biomarkers using a paper-based microfluidic chip device based on metal-organic gels. Figure 1 As shown.

[0071] Example 1: Preparation of metal-organic gels 1.1 Preparation of Cu / Co-MOG A schematic diagram of the preparation of Cu / Co-MOG is shown below. Figure 2 As shown in Figure A, Cu / Co-MOG was synthesized according to the following steps: 200 mg of 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (H3TATAB) was dissolved in 10 mL of ultrapure water (containing 200 μL of triethylamine as a co-solvent). The mixture was sonicated at 200 W for 10 min until the solution became clear, thus obtaining the H3TATAB stock solution. 79 mg of CuCl2·2H2O and 109 mg of CoCl2·6H2O were dissolved in ultrapure water to prepare Cu... 2+ and Co 2+ The reserve liquid.

[0072] First, according to Cu 2+ / Co 2+ A mixed metal ion solution with a molar ratio of 1:1 and a volume of 250 µL was prepared, maintaining a total molar concentration of 0.046 M. Then, an equal volume of 0.046 M H3TATB was added to the mixed metal ion solution. After thorough mixing, the solution was allowed to stand at room temperature (25°C). oC) After standing for 30 min, the metal-organic gel Cu / Co-MOG is obtained. The obtained gel is centrifuged (3200×g, 5 min) and then dispersed and washed in ultrapure water by sonication (100W, 5 min). This process is repeated three times to remove free metal ions and ligands.

[0073] This embodiment can also use a single metal ion Cu. 2+ or Co 2+ Cu-MOG or Co-MOG can be prepared by mixing the ligand H3TATAB with metal ions in a simple one-step reaction. This method is simple, efficient, and requires no heating. It is foreseeable that other ions with oxidizing properties can also be used in the aforementioned sol (MOG).

[0074] 1.2 Material Characterization The morphology of Cu / Co-MOG was characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 2 As shown in B and C, SEM images of Cu / Co-MOG show that Cu / Co-MOG is in the form of nanofibers. TEM elemental distribution maps show that copper (Cu), cobalt (Co), carbon (C), nitrogen (N), and oxygen (O) are uniformly distributed in the Cu / Co-MOG nanofibers. Figure 2 (D).

[0075] Fourier transform infrared (FT-IR) spectra of H3TATAB (blue line) and Cu / Co-MOG (red line) are as follows: Figure 3 As shown, the stretching vibration of C=O in H3TATAB starts from 1706 cm⁻¹. -1 Move to 1604cm -1 This indicates that the metal ion and the carboxyl group are linked through a coordinate bond. For example... Figure 4 Thermogravimetric analysis (TGA) curves of Cu / Co-MOG in N2 environment show that the prepared Cu / Co-MOG has high thermal stability, and the weight loss rate is slow (1.06%) within the experimental temperature range (25~50°C) of this method. The N2 adsorption-desorption isotherms of Cu / Co-MOG are shown below. Figure 5 As shown in Figure A, the Brunauer-Emmett-Teller (BET) isotherm reveals that Cu / Co-MOG exhibits typical Type III isotherm characteristics, indicating that Cu / Co-MOG possesses a hierarchical porous structure. The pore size distribution of Cu / Co-MOG is shown in Figure A. Figure 5 As shown in Figure B, the results indicate that the pore size distribution of Cu / Co-MOG is in the range of 1~80 nm, with an average pore size of 21.82 nm.

[0076] Example 2: Cu / Co-MOG as a filter medium for filtering red blood cells in whole blood. This embodiment uses a three-layer laminated high-throughput μPAD and a Cu / Co-MOG dispersion to study the effect of Cu / Co-MOG dispersion on filtering red blood cells from whole blood, as detailed below: (1) Preparation of three-layer laminated high-throughput μPAD and morphology characterization Preparation of a three-layer laminated high-throughput μPAD: High-throughput μPADs are prepared using a standardized process: A4-sized (210mm × 297mm) filter paper (pore size 10~20μm) is used. A wax printer (ProJet MJP3600W) is used to print a specified pattern on one side of the filter paper (each sheet contains 3 arrays, such as...). Figure 6 (As shown in A); respectively via 70 o Heating at C for 55s, 60s, 65s, 70s, 80s, 90s, 100s, and 110s creates a hydrophobic barrier, as shown below. Figure 6 As shown in Figure B, the white circular hydrophilic region serves as the detection area; the hydrophilic region has a diameter of 6 mm, surrounded by a wax-treated hydrophobic region. 70 o Heating at 65 seconds allows the wax to penetrate evenly and form a complete hydrophobic barrier.

[0077] After the wax has fully and evenly diffused into the paper base, it is precisely cut and then aligned accurately across the three layers using pre-made creases. The purpose of these pre-made creases is to ensure that all detection areas are perfectly aligned. Figure 7 As shown in Figure A, the assembly is completed using double-sided adhesive, which constitutes the fabrication of a three-layer laminated high-throughput μPAD, as shown in Figure A. Figure 7 As shown in Figure B, the three-layer design is used to form a tiny reaction vessel, allowing for more uniform color development in the later stages.

[0078] Select 70 o A three-layer laminated high-throughput μPAD was prepared by heating at C for 65 s, with an untreated μPAD as a control. In the modified group, 10 μL of Cu / Co-MOG dispersion (Cu / Co-MOG concentration 6 mg / mL, water as solvent) was added to the front detection area of ​​the μPAD. The morphological characteristics of both were observed using scanning electron microscopy, and the results are as follows: Figure 8 As shown in Figures A and B, A represents the untreated μPAD detection area (the original hydrophilic region of the filter paper), and B represents the μPAD-modified detection area. The morphology of the hydrophobic region of the wax-modified filter paper in both cases is as follows: Figure 9 As shown.

[0079] The hydrophobic region treated with wax became smooth, with no obvious pores observed. Compared to untreated μPAD, the porosity of the detection region (hydrophilic region) was significantly reduced after modification with Cu / Co-MOG dispersion, indicating that the presence of Cu / Co-MOG dispersion provides a physical barrier. The Cu / Co-MOG dispersion is a stable or semi-stable colloidal system containing micro / nano-sized Cu / Co-MOG particles. In the dispersion, Cu / Co-MOG swells fully, forming a hydrated gel state with water. This not only maintains the three-dimensional network structure of Cu / Co-MOG (Cu / Co-MOG itself has nanoscale pores, with pore sizes ranging from 1 to 80 nm), but also further reduces the nanopore size in the hydrated gel state. It is speculated that the Cu / Co-MOG dispersion can effectively prevent the passage of red blood cells and large colored molecules through a size sieving effect, thereby filtering and separating red blood cells and colored macromolecules from whole blood samples.

[0080] (2) Verify the filtration of red blood cells from whole blood using Cu / Co-MOG dispersion Ordinary filter paper filtration: Add 10 μL of whole blood sample to the hydrophilic area on the front side of the μPAD; Cu / Co-MOG dispersion filtration: First, add 10 μL of Cu / Co-MOG dispersion (Cu / Co-MOG concentration of 6 mg / mL, with water as solvent) to the hydrophilic front side of the μPAD as the filtration medium, and then add 10 μL of whole blood sample.

[0081] The effects of filtering whole blood samples using ordinary filter paper and Cu / Co-MOG dispersion on the removal of red blood cells were compared and analyzed using optical microscopy. The results are as follows: Figure 8 As shown in C and D, C represents the effect of filtering a whole blood sample through ordinary filter paper, and D represents the effect of filtering a whole blood sample through Cu / Co-MOG dispersion. The circular pattern on the left side of the image represents the hydrophilic region of the front side of the μPAD, and the pattern on the right side represents the hydrophilic region of its back side. An optical microscopic image of untreated whole blood is shown below. Figure 10 As shown.

[0082] Depend on Figure 8 The results from the C-cell assay show that even after filtration with ordinary filter paper, significant red blood cell residue remains in whole blood samples, indicating that traditional μPADs cannot completely remove red blood cells from whole blood. However, filtration using Cu / Co-MOG dispersion completely removes red blood cells and colored macromolecules from whole blood, thus preventing their migration and appearance on the back of the μPAD. This experiment fully demonstrates that the Cu / Co-MOG dispersion can effectively filter and eliminate interference from red blood cells and colored macromolecules in blood samples through in-situ blocking. Figure 8 (D). It should be noted that, Figure 8The artifacts in D that are significantly larger than red blood cells are from the glass slide, but they do not affect the experimental results.

[0083] Example 3: Study on the catalytic activity of Cu / Co-MOG This embodiment uses the typical TMB-H2O2 reaction as a model reaction to systematically evaluate the peroxidase-like activity (POD-like activity) of Cu / Co-MOG. Based on the colorimetric reaction after TMB is oxidized by H2O2, the reaction process was monitored in real time at 652 nm using a multi-functional microplate reader to obtain time-absorbance kinetic curves. K was calculated by nonlinearly fitting the experimental data based on the Michaelis-Mentene equation. m and V max Key kinetic parameters were obtained to quantitatively characterize the catalytic activity of the enzyme mimic.

[0084] In this embodiment, the reaction conditions were (0.05M acetate-sodium acetate (HAc-NaAc) buffer, pH 4.0, 25) o The steady-state kinetic parameters of Cu / Co-MOG peroxidase-like compounds were determined using a system with a reaction time of 30 min (C reaction). The total reaction volume was 250 μL, containing: HAc-NaAc buffer (pH 4.0), 20 μL Cu / Co-MOG (1.5 mg / mL) as a catalyst, and H2O2 and TMB as substrates. Kinetic determination of Cu / Co-MOG using TMB as a substrate was performed by adding 30 μL of 1M H2O2 and different amounts (0, 1, 2, 4, 8, 12, 16, 20 μL) of 4.5 mg / mL TMB solution. Kinetic determination of Cu / Co-MOG using H2O2 as a substrate was performed by adding 20 μL of 4.5 mg / mL TMB and different amounts (0, 2, 4, 8, 12, 16, 24, 32 μL) of 1% H2O2 solution.

[0085] Michaelis-Mentenconstant (K) was calculated using Lineweaver-Burkplot and according to the following equation. m This constant represents the substrate affinity of MOG as a peroxidase: ; In this equation, v represents the initial velocity, V max [S] represents the maximum reaction rate, [S] represents the substrate concentration, and K represents the maximum reaction rate. m This represents the Michaelis-Menten constant, which is an indicator of enzyme-substrate affinity: K mThe smaller the value, the stronger the affinity between the enzyme and the substrate.

[0086] Furthermore, this embodiment reveals the catalytic characteristics of Cu / Co-MOG through apparent steady-state kinetic analysis. Under the experimental conditions, concentration gradient experiments were conducted using TMB and H2O2 as substrates, respectively, to construct Michaelis-Menten curves for the reactions of Cu / Co-MOG catalyst with TMB (A) and H2O2 (B). Figure 11 (A, B), construct a Lineweaver-Burk double reciprocal curve ( Figure 11 (C, D), the Michaelis constant (K) is calculated. m ) and maximum reaction rate (V max Experimental data show that when TMB is a variable substrate, K m The value is 0.5mM, corresponding to V max Reaching 3.1×10 -8 Ms -1 When H2O2 is a variable substrate, K m With V max The values ​​were 1.4 mM and 5.4 × 10, respectively. -8 Ms -1 Comparative analysis showed that these kinetic parameters were comparable to those of HRP and other artificial enzyme systems reported in the literature (Table 1), confirming that Cu / Co-MOG has excellent enzyme-like catalytic activity and can be used for the detection of blood metabolic markers.

[0087] Table 1 Comparison of reaction kinetic constants for Cu / Co-MOG, other artificial enzymes, and HRP Document 1: Hong, C.; Zhang, X.; Wu, C.; Chen, Q.; Yang, H.; Yang, D.; Huang, Z.; Cai, R.; erolBasedonPolypyrroleNanoparticles.ACSAppl.Mater.Interfaces2020,12(49),54426–54432.DOI:10.1021 / acsami.0c15900. Literature 2: Wang, J.; Hu, Y.; Zhou, Q.; Hu, L.; Fu, W.; Wang, Y. Peroxidase-likeActivityofMetal–OrganicFramework[Cu (PDA) (DMF)] and ItsAppl icationforColorimetricDetectionofDopamine.ACSAppl.Mater.Interfaces2019,11(47),44466–44473.DOI:10.1021 / acsami.9b17488. Document 3: Wu, Y.; Jiao, L.; Luo, X.; Xu, W.; Wei, X.; Wang, H.; Yan, H.; Gu, W.; Xu, BZ; Du, D. zymesfortheDetectionofAcetylcholinesteraseActivity.Small2019,15(43),1903108.DOI:10.1002 / smll.201903108. Example 4: Fabrication of a paper-based microfluidic chip device The paper-based microfluidic chip device is prepared according to the following method: (1) Preparation of Cu / Co-MOG: The preparation of the metal organogel Cu / Co-MOG is the same as in Example 1. The obtained gel-like Cu / Co-MOG is diluted with water to a concentration of 1.5 mg / mL, and stirred or sonicated to be uniformly dispersed in the solvent to prepare Cu / Co-MOG dispersion.

[0088] (2) Preparation of three-layer laminated high-throughput μPAD High-throughput μPADs were prepared using a standardized process: A4-sized (210mm × 297mm) filter paper (pore size 10~20μm) was used, and a specified pattern (each sheet containing 3 arrays) was printed on one side of the filter paper using a wax printer (ProJet MJP3600W); after 70... o Heating at C for 65 seconds forms a hydrophobic barrier. The white, circular hydrophilic area serves as the detection zone (6mm in diameter), surrounded by a wax-treated hydrophobic area. After the wax has fully and evenly diffused into the paper base, precise cutting is performed, and pre-made creases ensure accurate alignment of the three layers. The purpose of these pre-made creases is to guarantee perfect alignment of all detection zones. Figure 7 As shown in Figure A, the assembly is completed using double-sided adhesive, which is the fabrication of a three-layer laminated high-throughput μPAD. Figure 7As shown in B, the three-layer design is intended to form a tiny reaction vessel, allowing for more uniform color development in the later stages.

[0089] (3) Preparation of paper-based microfluidic chip device: Add 5 μL Cu / Co-MOG (1.5 mg / mL) to the back of μPAD and dry (50) o (C, 5 min) The Cu / Co-MOG catalyst is uniformly deposited on the μPAD to obtain the paper-based microfluidic chip device, abbreviated as MOG-μPAD, wherein the side coated with Cu / Co-MOG is the back side and the side not coated with Cu / Co-MOG is the front side.

[0090] Example 5: The Influence of Different Color Channels on the Analysis Signal of MOG-μPAD This example, based on a paper-based microfluidic chip device and a Cu / Co-MOG dispersion as the filter medium, compares the effects of different color channels on the MOG-μPAD analysis signal by taking the detection of the metabolic biomarker glucose standard solution as an example.

[0091] Add 2 μL of oxidase solution (GOD pH 6, UO pH 8, XO pH 7 or CAH+CI+SOX pH 7, 18000 U / L) and 2 μL of TMB chromogenic solution (4.5 mg / mL, TMB chromogenic solution pH 4 for glucose, xanthine or creatinine detection, TMB chromogenic solution pH 5 for uric acid detection) sequentially to the detection area on the back of the MOG-μPAD.

[0092] 10 μL of 6 mg / mL Cu / Co-MOG dispersion was first added to the detection area on the front of the MOG-μPAD as a filter medium, followed by 10 μL of glucose standard solution. After reacting at room temperature for 30 min, an image of the back of the detection area was captured using a smartphone under standardized light conditions. To effectively prevent interference from ambient light in contrast color detection, a suitably sized box was constructed from black cardboard, completely darkened to prevent ambient light from entering. An LED light was then fixed to the top of the box using double-sided tape to provide a stable light source. A hole the same size as the camera lens of a smartphone, such as an iPhone XR, was made in the top of the box to accommodate the smartphone for image capture. Figure 12 As shown.

[0093] By comparing the signal responses of different color channels (R+G+B combined channel, R channel, G channel, and B channel), the most sensitive color channel is selected. Figure 13 The effect of different color channels on the MOG-μPAD analysis signal is shown.

[0094] Depend on Figure 13The results show that the signal response of the R channel is significantly stronger than that of other channels (R+G+B combined channel, G channel, and B channel), indicating that the R channel has the most sensitive signal response. Specifically, the gray intensity (I0-I) in the R channel corresponds to the concentration of the metabolic marker, where I and I0 are the gray intensities with and without the metabolic marker, respectively. Therefore, the R channel was selected for subsequent experiments. The circular selection tool was used to select the detection area after the MOG-μPAD reaction color development in the R channel, and then the average intensity of the R channel in this area was quantified. The gray intensity of the red channel in the detection area was measured using commercial software such as ImageJ in the acquired images.

[0095] Example 6: Performance Validation and Mechanism Exploration of MOG-μPAD-based Detection of Metabolic Biomarkers Cu / Co-MOG, acting as a peroxidase, converts colorless TMB into colored ox-TMB for the quantitative analysis of metabolic biomarkers. To demonstrate this, we first investigated the color signals generated under different reaction conditions using standard metabolic biomarker solutions (such as glucose, uric acid, xanthine, and creatinine solutions). The specific steps were as follows: 5 μL of 1.5 mg / mL Cu / Co-MOG dispersion was added to the back detection area (a circular detection area with a diameter of 6 mm) of the μPAD, followed by heating in a 50°C drying oven for 5 min to ensure uniform deposition of Cu / Co-MOG as the peroxidase. After cooling, 2 μL of oxidase solution (GOD pH 6, UO pH 8, XO pH 7 or CAH+CI+SOX pH 7, 18000 U / L) and 2 μL of TMB chromogenic solution (4.5 mg / mL; the pH of the TMB chromogenic solution was 4 for glucose, xanthine, or creatinine detection, and the pH of the TMB chromogenic solution was 5 for uric acid detection) were added sequentially.

[0096] 10 μL of 6 mg / mL Cu / Co-MOG was first added to the detection area on the front of the MOG-μPAD as a filter medium, followed by 10 μL of metabolic marker standard solutions (glucose, uric acid, xanthine, or creatinine standard solutions) of different concentrations. After reacting at room temperature for 30 min, the back image of the detection area was captured using a smartphone under standardized light source conditions. The R channel gray value (0~255 scale) was analyzed using ImageJ software to achieve quantitative detection.

[0097] like Figure 14As shown in section A: a1, b1, c1, d1: Cu / Co-MOG (as peroxidase) + oxidase + Cu / Co-MOG (as filter medium) + metabolic marker standard solution; a2, b2, c2, d2: oxidase + TMB + Cu / Co-MOG (as filter medium) + metabolic marker standard solution; a3, b3, c3, d3: Cu / Co-MOG (as peroxidase) + oxidase + TMB + Cu / Co-MOG (as filter medium) + H2O; a4, b 4. c4, d4: Cu / Co-MOG (as peroxidase) + oxidase + TMB + Cu / Co-MOG (as filter medium) + metabolic marker standard solution; a5, b5, c5, d5: Cu / Co-MOG (as peroxidase) + oxidase + TMB + Cu / Co-MOG (as filter medium) + plasma; a6, b6, c6, d6: Cu / Co-MOG (as peroxidase) + oxidase + TMB + Cu / Co-MOG (as filter medium) + whole blood. Therefore, the colorimetric signal in the oxidase-TMB-metabolic marker reaction system using Cu / Co-MOG as the peroxidase is significantly higher than that in reaction systems lacking either Cu / Co-MOG, TMB, or one of the metabolic markers.

[0098] This invention proposes a hypothesis for a colorimetric detection mechanism of metabolic biomarkers based on MOG-μPAD, such as... Figure 14 As shown in Figure B, metabolic markers (such as glucose, uric acid, xanthine, or creatinine) are catalytically converted to H₂O₂ by their respective oxidases. Subsequently, the Cu / Co-MOG on the back of the MOG-μPAD, in the presence of H₂O₂, can convert colorless TMB into blue oxidized TMB (ox-TMB) through its peroxidase activity. Next, the peroxidase-like activity of Cu / Co-MOG was investigated by catalyzing TMB in the presence of H₂O₂. In the presence of Cu / Co-MOG, the mixture of H₂O₂ and TMB exhibits a deep blue color, which is significantly opposite to the control experiment. In the absence of Cu / Co-MOG, the color change is negligible, indicating that Cu / Co-MOG possesses peroxidase-like activity. We hypothesize that the colorless TMB is actually blue oxidized TMB (ox-TMB) formed by the oxidation of •OH. Therefore, we directly verified the generation of •OH using the electron spin resonance (ESR) method. Figure 15 The experiment used BMPO as a free radical scavenger. The BMPO / •OH adduct formed by BMPO and •OH exhibited a characteristic quartet signal (peak intensity ratio of 1:2:2:1) in the ESR spectrum, confirming that Cu / Co-MOG can effectively catalyze the decomposition of H2O2 to produce •OH.

[0099] To further explore the excellent catalytic mechanism of Cu / Co-MOG, X-ray photoelectron spectroscopy (XPS) was used to characterize the valence state changes of the metal components before and after the reaction with H2O2. Figure 16 ), where the high-resolution XPS spectra of Cu / Co-MOG before (green line) and after (red line) treatment with H2O2 ( Figure 16 (A) High-resolution Cu2p XPS spectra of Cu / Co-MOG before and after treatment with H2O2 ( Figure 16 Both B and C show two peaks at approximately 934.5 eV and 954.3 eV, which are mainly attributed to Cu(II) in the Cu2p-O bond. XPS data show that the Auger peak of Cu in Cu / Co-MOG shifted from 571.0 eV to 571.7 eV after H2O2 treatment, confirming the formation of Cu(I) after the reaction with H2O2. Figure 16 The XPS spectra and data analysis of Co are shown in [reference 1]. Figure 16 See E, F and Table 2.

[0100] Table 2. XPS characteristics of untreated and H2O2-treated Cu / Co-MOG: binding energy (eV) of different peaks, relative intensity within brackets (sati=Isati / Imain), spin-orbit splitting (ΔE=Co2p). 1 / 2 -Co2p 3 / 2 ) After treatment with H2O2, the spin orbital splitting ΔE(2p) 1 / 2 -2p 3 / 2 The voltage changes from 16.3 eV to 15.7 eV, and Σsat / ICo2p 3 / 2 The values ​​decreased significantly, indicating the presence of Co(III) components. Clearly, both Cu(II) and Co(II) exhibit catalytic activity towards H₂O₂, and their valence states also changed accordingly.

[0101] In summary, this invention proposes a catalytic mechanism for Cu / Co-MOG: metabolic markers react with corresponding oxidases to generate H2O2, and the active sites on the catalyst surface, including Cu(II) and Co(II), can catalyze the generation of •OH and Cu(I) and Co(III) from H2O2. TMB can be oxidized by •OH to ox-TMB.

[0102] This embodiment describes a method for detecting blood metabolic biomarkers based on a MOG-μPAD device. The reagents required for the colorimetric reaction (oxidases such as GOD, UO, XO, or CAH+CI+SOX, and the colorless indicator TMB) are pre-deposited in the detection area (the circular hole in the hydrophilic region) on the back of the MOG-μPAD device. A Cu / Co-MOG dispersion is first added to the detection area on the front of the MOG-μPAD device as a filter medium, followed by the sample to be tested. Glucose, uric acid, xanthine, or creatinine in the sample solution migrate through the front filter medium to the back detection area. In the presence of GOD, UO, XO, or CAH+CI+SOX on the back, they are selectively oxidized to form H2O2. The Cu / Co-MOG on the back acts as a peroxidase, catalyzing the H2O2 reaction and converting the colorless indicator TMB into its blue oxidation product (ox-TMB), which appears blue in the back detection area. The blue signal is captured by a smartphone and analyzed using commercial colorimetric analysis software (ImageJ, etc.) to achieve real-time quantitative determination of the metabolic biomarkers.

[0103] Example 7: Colorimetric detection of H2O2 solution Using H₂O₂ standard solution as the substrate, Cu / Co-MOG as the peroxidase, and TMB as the colorimetric reagent, the colorimetric reaction conditions were optimized as follows: The concentrations of Cu / Co-MOG as a catalyst (0.5, 1, 1.5, 2, 2.5, 3 mg / mL), TMB concentrations (2, 3, 3.5, 4, 4.5, 5 mg / mL), pH values ​​(pH 2, 3, 4, 5, 6, 7, 8, 9), and room temperature (25°C) were compared. o C) The reaction time (2, 6, 10, 15, 20, 25, 30, 35, 40 min) was optimized. The results showed that ( Figure 17 The preferred concentration of Cu / Co-MOG as a catalyst is 1.5 mg / mL, and the preferred concentration of TMB in the TMB colorimetric solution is 4.5 mg / mL. The reaction is carried out at room temperature (25°C) under pH=3 conditions. o C) The preferred reaction time is 30 min.

[0104] In this embodiment, the analytical performance of MOG-μPAD was evaluated using H2O2 solution: Add 5 μL Cu / Co-MOG (1.5 mg / mL) to the back of the μPAD and dry (50) o (C, 5 min) The Cu / Co-MOG catalyst is uniformly deposited on the μPAD to obtain the paper-based microfluidic chip device, abbreviated as MOG-μPAD, wherein the side coated with Cu / Co-MOG is the back side and the side not coated with Cu / Co-MOG is the front side.

[0105] Add 2 μL of TMB solution (4.5 mg / mL) dissolved in HAc-NaAc buffer (0.05 M, pH 4.0) and 2 μL of ultrapure water (to replenish the volume of the solution without enzyme) to the detection area on the back of the MOG-μPAD. Add 10 μL of Cu / Co-MOG dispersion (6 mg / mL) and H2O2 standard solutions of different concentrations (0.01~0.2 mM) to the detection area on the front of the MOG-μPAD and react at room temperature for 30 min.

[0106] Under standard lighting conditions, a smartphone captures the color reaction image of the MOG-μPAD detection area, and uses ImageJ software to quantify and analyze the pixel intensity of the R channel (0~255 scale) to achieve digital quantitative detection of color signals. Figure 18 The relationship between the analytical signal (I0-I) and H2O2 concentration is shown. Within the H2O2 concentration range of 0.01–0.2 mM, the signal (I0-I) and C... H2O2 There is a good linear relationship between them (I0-I=0.07C). H2O2 +1.07, r 2 =0.998). For H2O2 solution, the limit of detection (LOD) is 0.68 μM, which is superior to previously reported work (see Table 3).

[0107] Table 3. Results of H2O2 detection in solution and comparison with existing methods. Document 1: Hu, Y.; Cheng, H.; Zhao, X.; Wu, J.; Muhammad, F.; Lin, S.; He, J.; Zhou, L.; Zhang, C.; Deng, Y.; Wang, P.; Zhou, Z.; Nie, S.; Wei, H.; eGoldNanoparticleswithEnzyme-MimicckingActivitiesforMeasuringGlucoseandLactateinLivingTissues.ACSNano2017;11(6),5558–5566.DOI:10.1021 / acsnano.7b00905. Document 2: Jiang, S.; Chen, Document 3: Cai, L.; Wang, Y.; Yang, Y.; Wu, H.; Alow-cost, enzyme-coupled fluorescent assay for rapid quantification ofglycolysisrateofcells.Anal.Bioanal.Chem.2022;414(5),1987–1997.DOI:10.1007 / s00216-021-03834-2. Document 4: Liu,Y.;Yao,Y.;Yang,B.;Liu,Y.;LiuB.In-situmonitoringofcell-secretedlactatebyelectrochemiluminescencesensin gunderbiomimeticmicrofluidicconfinement.ChineseChem.Lett.2022;33(5),2705–2707.DOI:10.1016 / j.cclet.2021.09.074. Example 8: Validation of this method using metabolic biomarker standard solutions Add 5 μL Cu / Co-MOG (1.5 mg / mL) to the back detection area (a circular detection area with a diameter of 6 mm) of the μPAD, and then heat it in a 50 °C drying oven for 5 min to ensure uniform deposition of Cu / Co-MOG as a catalyst. After cooling, add 2 μL of oxidase solution (GOD, UO, XO or CAH+CI+SOX, 18000 U / L, diluted with buffer at the optimal pH) and 2 μL of LMB colorimetric solution (4.5 mg / mL, dissolved in buffer at the optimal pH).

[0108] In this method, 10 μL of 6 mg / mL Cu / Co-MOG dispersion was first added to the front detection area as a filter medium, followed by the addition of standard solutions of glucose, uric acid, xanthine, or creatinine at different concentrations. After reacting at room temperature for 30 min, images of the back of the detection area were acquired using a smartphone under standardized light source conditions. Quantitative detection was achieved by analyzing the R channel grayscale values ​​(0–255 scale) using ImageJ software. This method ensures high sensitivity and specificity by optimizing the enzyme reaction conditions and colorimetric system.

[0109] Following the above method, the pH and reaction time (5-40 min) of the oxidase (pH 3-10) and TMB (pH 3-8) were optimized. Figures 19 to 21 As shown, all reactions were completed within 30 minutes under optimal pH conditions: for glucose, the GOD reaction was at pH 6 and the TMB chromogenic solution was at pH 4; for uric acid, the UO reaction was at pH 8 and the TMB chromogenic solution was at pH 5; for xanthine, the XO reaction was at pH 7 and the TMB chromogenic solution was at pH 4; for creatinine, the (CAH+CI+SOX) reaction was at pH 7 and the TMB chromogenic solution was at pH 4.

[0110] Taking glucose, uric acid, xanthine, and creatinine as examples, GOD, UO, XO, and CAH+CI+SOX can effectively convert glucose, uric acid, xanthine, and creatinine into H2O2, respectively. Under Cu / Co-MOG catalysis, H2O2 oxidizes TMB to generate a deep blue ox-TMB product. Based on the colorimetric signal changes (I0-I), we established quantitative calibration relationships between glucose (A), uric acid (B), xanthine (C), and creatinine (D) and response values. Figure 22 Illustration: Image of color change of MOG-μPAD. Reaction conditions: [TMB] = 4.5 mg / mL, GOD, UO, XO, CAH+CI+SOX 18 U / mL; colorimetric signal (I0-I) and log 10 (C) 代谢标志物,Analyte A good linear relationship was also obtained between them. Figure 23 (Table 4), reaction conditions: [TMB]=4.5mg / mL, [GOD]=[UO]=[XO]=[CAH+CI+SOX]=18U / mL, linear calibration curves for the detection of glucose (A), uric acid (C), xanthine (E) and creatinine (G) based on MOG-μPAD; selective evaluation of the detection of glucose (B), uric acid (D), xanthine (F) and creatinine (H) by this method.

[0111] Table 4 shows the detection results of glucose, uric acid, xanthine, and creatinine in the solution. The analytical performance of the proposed MOG-μPAD was also compared with that of previously reported colorimetric sensors for glucose detection (see Table 5).

[0112] Table 5. Detection results of H2O2 in solution and comparison with existing methods. Document 1: Dai, J.; Zhang, H.; Huang, C.; Chen, Z.; Han, A. AGel-BasedSeparation-FreePoint-of-CareDevice for WholeBloodGlucoseDetection. Anal. Chem. 2020, 92 (24), 16122–16129. DOI: 10.1021 / acs.analchem.0c03801. Document 2: Ma, C.; Zhang, Y.; Liu, Q.; Du, Y.; Wang, E. Enhanced Stability of Enzyme Immobilized in Rationally Designed Amphiphilic Aerog elandItsApplicationforSensitiveGlucoseDetection.Anal.Chem.2020,92(7),5319–5328.DOI:10.1021 / acs.analchem.9b05858. Document 3: Xu, J.; Khan, H.; Yang, L. HydrogelPaper-BasedAnalyticalDevices:Separation-FreeInSituAssayofSmall -MoleculeTargetsinWholeBlood.Anal.Chem.2021,93(44),14755–14763.DOI:10.1021 / acs.analchem.1c03347. Reference 4: Zheng, J.; Zhu, M.; Kong, J; Li, Z.; Jiang, J.; Xi, Y.; Li, F. Microfluidic paper-based analytical device by using Pt nanoparticles as highly active peroxidase mimics for simultaneous detection of glucose and uric acid with use of a smartphone. Talanta. 2022, 237, 122954. DOI: 10.1016 / j.talanta.2021.122954. Reference 5: Kou, X.; Tong, L.; Shen, Y.; Zhu, W.; Yin, L.; Huang, S.; Zhu, F.; Chen, J.; Ouyang, G. Smartphone-assisted robust enzymes@MOFs-based paper biosensor for point-of-care detection. Biosens. Bioelectron. 2020, 156, 12095. DOI: 10.1016 / j.bios.2020.112095. Reference 6: Li, N.; Chen, Y.; H, Y.; Pang, H.; Huang, C.; Shiue, Y.; Wei, K.; Yang, H. Mobile healthcare system based on the combination of a lateral flow pad and smartphone for rapid detection of uric acid in whole blood. Biosens. Bioelectron. 2020, 164, 112309. DOI: 10.1016 / j.bios.2020.112309. Reference 7: Yang, X.; Jin, C.; Yang, S., Tian, M. Paper-based enzyme-linked biosensor combined with smartphone for simultaneous colorimetric sensing of xanthines and sarcosine. Sens. Actuators, B. 2024, 412, 135849. DOI: 10.1016 / j.snb.2024.135849.10.1016 / j.snb.2024.135849. Reference 8: Guo, Z.; Tian, Z.; Qu, Y. One-step reagentless colorimetric analysis platform of biomineralized Ce-UiO-66 for universal detection of biomarkers. Sens. Actuators, B. 2023, 397, 134705. DOI: 10.1016 / j.snb.2023.134705. Reference 9: Li, Z.; Liu, X.; Liang, X.; Zhong, J.; Guo, L.; Fu, F. Colorimetric determination of xanthine in urine based on peroxidase-like activity of WO3 nanosheets. Talanta. 2019, 204, 278–284. DOI: 10.1016 / j.talanta.2019.06.003. Reference 10: Jeon, S.; Lee, H.; Ha, C.; Kim, D.; Seong, G. Colorimetric and Photothermal Creatinine Determination Using MoS2 Nanosheet Cocatalyst with Cu 2+ / Creatinine Complexes. ACS Applied Nano Materials. 2024, 8(1), 169–178. DOI: 10.1021 / acsanm.4c05373. Literature 11: Lu, Y.; Shen, N.; Xi, Y; Zhu, T.; Peng, H.; Zhong, L.; Li, F. anoparticlescatalyzedoxidationof3,3',5,5'-tetramethylbenzidine.Microchim.Acta.2023,191(1),44.DOI:10.1007 / s00604-023-06129-8. Document 12: Guan, J.; Xiong, Y.; Wang, M.; Liu, Q.; Chen, X. AnovelfunctionalizedCdTe@MOFsbasedfluorometricandcolorimetric biosensorfordual-readoutassayofcreatinine.Sens.Actuators,B.2024,399,134842.DOI:10.1016 / j.snb.2023.134842. Blood glucose is the gold standard for diagnosing diabetes. The quantitative calibration relationship between glucose concentration and response value is as follows: Figure 22 As shown in Table A and Table 4, when the glucose concentration range is 0.4–2.5 mM or 3–12 mM, the colorimetric signal (I0–I) and log0 are compared. 10 (C) Glucose A good linear relationship was also obtained between them, such as Figure 23 As shown in Table A, the performance of our developed MOG-μPAD is comparable to previously reported methods, meeting the needs of clinical point-of-care blood glucose testing (see Table 5). More importantly, some potential interfering substances in blood samples, including fructose, galactose, mannose, uric acid, lactic acid, EDTA, citric acid, β-hydroxybutyrate (β-HB), glutamate, threonine, lysine, leucine, arginine, alanine, phenylalanine, valine, and glutamine, have no significant effect on glucose detection. Figure 23(B) These results demonstrate that the detection method established in this study exhibits significant advantages in colorimetric analysis of blood glucose, and that the method possesses high specificity.

[0113] Uric acid is a key biomarker in the pathological process of gout, and abnormally elevated uric acid levels have a clear dose-response relationship with the pathogenesis of gouty arthritis. Accurate detection of blood uric acid concentration is crucial for the clinical diagnosis and monitoring of gout treatment. Within the uric acid concentration range of 0.2–4 mM, the colorimetric signal (I0–I) and C... Uricacid A good linear relationship was also obtained between them. Figure 22 (See Tables B, C, and 4). The detection limit for uric acid is 0.14 mM. The analytical performance of the proposed MOG-μPAD was also compared with reported colorimetric sensors for uric acid detection (see Table 5). The performance of our developed MOG-μPAD is comparable to reported methods and meets the needs of clinical point-of-care uric acid detection. Importantly, several potential interfering substances in blood samples, including glucose, fructose, galactose, mannose, cholesterol, lactic acid, EDTA, citric acid, β-hydroxybutyric acid, glutamic acid, threonine, lysine, leucine, arginine, alanine, phenylalanine, valine, and glutamine, did not show significant effects on uric acid detection. Figure 23 (D). This method has high specificity for uric acid detection in blood samples.

[0114] Xanthine testing can reflect the functional status of the liver and kidneys and is closely related to diseases such as hyperuricemia, gout, nephritis, and liver necrosis. We... Figure 22 The figure in C shows a logarithmic plot of the analytical signal (I0-I) versus xanthine concentration. Figure 23 (E). For xanthine, accurate determination can be achieved within the detection range of 0.2–4 mM, with a detection limit of 0.07 mM (Table 4). The analytical capabilities of the proposed MOG-μPAD were also compared with published colorimetric sensors for xanthine detection (Table 5). Furthermore, certain potential interfering factors in blood samples, including glucose, galactose, uric acid, lactic acid, β-hydroxybutyric acid, creatinine, EDTA, citric acid, glutamic acid, threonine, lysine, leucine, arginine, alanine, phenylalanine, valine, and glutamine, had no significant effect on xanthine detection. Figure 23 (F). The results showed that the detection of xanthine in blood samples was highly specific.

[0115] Creatinine is an important biomarker for the clinical diagnosis and treatment of chronic kidney disease (CKD), diabetic nephropathy, and muscle diseases. At creatinine concentrations ranging from 0.4 to 2.5 mM or 3 to 12 mM, the signal intensity is significantly higher than that of lg[C]. creatinine A good linear relationship was also obtained between them. Figure 22 (Table 4). The detection limit for creatinine is 0.11 mM. This paper provides a comprehensive comparison of the analytical performance of the proposed MOG-μPAD with existing colorimetric creatinine sensors (Table 5). It is noteworthy that some potential interfering substances in blood samples, including glucose, fructose, galactose, uric acid, lactic acid, β-hydroxybutyric acid, EDTA, citric acid, glutamic acid, threonine, lysine, leucine, arginine, alanine, phenylalanine, valine, and glutamine, have no significant effect on creatinine detection. Figure 23 (H). The results show that the proposed method exhibits excellent specificity in the colorimetric determination of creatinine in blood samples.

[0116] Example 9: MOG-μPAD Stability Test Add 5 μL Cu / Co-MOG (1.5 mg / mL) to the back detection area (a circular detection area with a diameter of 6 mm) of the μPAD, and then heat it in a 50°C drying oven for 5 min to ensure uniform deposition of Cu / Co-MOG as a catalyst to form MOG-μPAD. After 90 days, it is used to detect blood metabolic markers as follows: Add 2 μL of oxidase solution (GOD pH 6, UO pH 8, XO pH 7 or CAH+CI+SOX pH 7, 18000 U / L) and 2 μL of TMB chromogenic solution (4.5 mg / mL; TMB chromogenic solution pH 4 is used for glucose, xanthine, or creatinine detection, and TMB chromogenic solution pH 5 is used for uric acid detection) sequentially to the back detection area of ​​the MOG-μPAD.

[0117] 10 μL of 6 mg / mL Cu / Co-MOG dispersion was first added to the front detection area of ​​the MOG-μPAD as a filter medium, followed by standard solutions of glucose, uric acid, xanthine, or creatinine at different concentrations. After reacting at room temperature for 30 min, images of the back of the detection area were acquired using a smartphone under standardized light source conditions. Quantitative detection was achieved by analyzing the R channel grayscale values ​​(0~255 scale) using ImageJ software. The MOG-μPAD device system maintained stable detection signals during 0-day and 90-day storage periods. Figure 24 ).

[0118] Furthermore, taking glucose detection as an example, the enzyme activities of Cu / Co-MOG and horseradish peroxidase (HRP) as catalytic enzymes were compared, as follows: Experimental group: 5 μL of 1.5 mg / mL Cu / Co-MOG, which had been stored at room temperature for 0 / 24 / 48 h, was added to the detection area (circular detection area with a diameter of 6 mm) on the back side of the μPAD. Then, the sample was placed in a 50 °C drying oven and heated for 5 min to ensure uniform deposition of Cu / Co-MOG to form MOG-μPAD. Then, 2 μL of glucose oxidase solution (GOD, 18000 U / L, pH 6) and 2 μL of LTMB colorimetric solution (4.5 mg / mL, pH 4) were added sequentially.

[0119] Control group: 5 μL of horseradish peroxidase (HRP) was added to the detection area (circular detection area with a diameter of 6 mm) on the back of the μPAD (and incubated at room temperature for 0, 24, and 48 h, respectively). Then, 2 μL of glucose oxidase solution (GOD, 18000 U / L, pH 6) and 2 μL of TMB colorimetric solution (4.5 mg / mL, pH 4) were added sequentially.

[0120] 10 μL of 6 mg / mL Cu / Co-MOG dispersion was first added to the front detection area of ​​the MOG-μPAD as a filter medium, followed by glucose standard solutions of different concentrations. After reacting at room temperature for 30 min, images of the back of the detection area were acquired using a smartphone under standardized light source conditions. Quantitative detection was achieved by analyzing the R channel grayscale values ​​(0~255 scale) using ImageJ software. Cu / Co-MOG retained over 90% of its enzyme catalytic activity after being placed at room temperature for 48 h, significantly outperforming traditional horseradish peroxidase (HRP). Figure 25 ), Figure 25 In the diagram, A stands for MOG-μPAD and B stands for HRP; this characteristic greatly improves the convenience of material storage and transportation.

[0121] Example 10: Real-time detection of metabolic biomarkers in blood samples without separation based on MOG-μPAD (1) Preparation of blood samples: Whole blood samples were provided to healthy adult participants in the human phenotype project with the approval of the local ethics committee (FE21087) and with informed consent. Plasma samples were separated from whole blood by centrifugation at 3000 r / min for 5 min.

[0122] (2) Determination of metabolic markers in whole blood and plasma The specific steps are as follows: First, add standard solutions of glucose (1, 3, 5, 7 mM), uric acid (0.35, 0.5, 0.75, 1.25 mM), xanthine (0.3, 0.5, 1 mM) and creatinine (0.5, 1, 2 mM) at different concentrations to the plasma sample.

[0123] Add 5 μL of 1.5 mg / mL Cu / Co-MOG dispersion to the back detection area (a circular detection area with a diameter of 6 mm) of the μPAD, and then heat it in a 50 °C drying oven for 5 min to ensure uniform deposition of Cu / Co-MOG. After cooling, add 2 μL of oxidase solution (GOD pH 6, UO pH 8, XO pH 7 or CAH+CI+SOX pH 7, 18000 U / L) and 2 μL of TMB chromogenic solution (4.5 mg / mL; TMB chromogenic solution pH 4 is used for glucose, xanthine, or creatinine detection; TMB chromogenic solution pH 5 is used for uric acid detection). 10 μL of 6 mg / mL Cu / Co-MOG was first added to the front detection area of ​​the MOG-μPAD as a filter medium, followed by 10 μL of plasma / whole blood (spike-added / unspike-added). After reacting at room temperature for 30 min, the back image of the detection area was acquired using a smartphone under standardized light conditions. Quantitative detection was achieved by analyzing the R channel grayscale values ​​(0~255 scale) using ImageJ software. Specifically, the concentrations of xanthine and creatinine in samples without added standards were determined using a commercial diagnostic kit (Beyotime, China).

[0124] The detection procedures for glucose, uric acid, xanthine, and creatinine in whole blood samples are the same as those for the spiked detection in human plasma described above. Clinical plasma and whole blood samples from different healthy subjects were tested. To verify the accuracy and reliability of this method, a standard addition method was used. Samples in which a certain amount of glucose, uric acid, xanthine, or creatinine was added to plasma and whole blood were called spiked samples. The detection results for glucose, uric acid, xanthine, and creatinine in plasma samples are shown in Table 6, and the detection results for glucose, uric acid, xanthine, and creatinine in whole blood samples are shown in Table 7.

[0125] Table 6. Detection results of glucose, uric acid, xanthine, and creatinine in plasma samples. Table 7. Detection results of glucose, uric acid, xanthine, and creatinine in whole blood samples. Tables 6 and 7 show the acceptable spiked recoveries (97–110%). This indicates that the method can be used to detect a variety of metabolic biomarkers in plasma and whole blood samples.

[0126] Example 11: Consistency assessment of this method with conventional clinical methods for measuring glucose and uric acid in healthy individuals The specific steps are as follows: Add 5 μL of 1.5 mg / mL Cu / Co-MOG dispersion to the back detection area (a circular detection area with a diameter of 6 mm) of the μPAD, and then heat it in a 50℃ drying oven for 5 min to ensure uniform deposition of Cu / Co-MOG. After cooling, add 2 μL of oxidase solution (GOD pH 6 or UO pH 8, 18000 U / L) and 2 μL of TMB colorimetric solution (4.5 mg / mL, TMB pH 4 for glucose detection, TMB pH 5 for uric acid detection).

[0127] 10 μL of 6 mg / mL Cu / Co-MOG was first added to the front detection area of ​​the MOG-μPAD, followed by 10 μL of plasma. After reacting at room temperature for 30 min, the back image of the detection area was acquired using a smartphone under standardized light source conditions. Quantitative detection was achieved by analyzing the gray values ​​of the R channel (0~255 scale) using ImageJ software.

[0128] We also compared the results obtained from our MOG-μPAD device with clinical test results. Bland-Altman analysis showed that the results obtained from the MOG-μPAD device were consistent with those obtained using routine clinical methods. Figure 26 (A in the figure represents glucose, and B represents uric acid). Analysis showed that the average deviation between the two methods was -0.08 mM for glucose detection and 12.33 μM for uric acid detection. The 95% boundary of agreement (LOA) ranged from -0.53 to 0.37 mM for glucose detection and from -6.59 to 31.26 μM for uric acid detection. In conclusion, the results of this study confirm that MOG-μPAD has clinically applicable glucose detection performance and demonstrates its application potential in uric acid screening. Due to a lack of clinical data, xanthine and creatinine were not analyzed in this study.

[0129] Example 12: Simultaneous detection of glucose, uric acid, xanthine, and creatinine in whole blood samples based on MOG-μPAD Add 5 μL of 1.5 mg / mL Cu / Co-MOG dispersion to the back detection area (a circular detection area with a diameter of 6 mm) of the μPAD, and then heat it in a 50℃ drying oven for 5 min to ensure uniform deposition of Cu / Co-MOG. After cooling, add 2 μL of oxidase solution (GOD pH 6, UO pH 8, XO pH 7 or CAH+CI+SOX pH 7, 18000 U / L) and 2 μL of TMB chromogenic solution (4.5 mg / mL, TMB pH 4 for glucose, xanthine, or creatinine detection, TMB pH 5 for uric acid detection). Add 10 μL of 6 mg / mL Cu / Co-MOG as a filter medium to the front detection area of ​​the MOG-μPAD, followed by 10 μL of whole blood. After reacting at room temperature for 30 min, use a smartphone to capture an image of the back of the detection area under standardized light conditions. Quantitative detection is achieved by analyzing the R channel grayscale values ​​(0~255 scale) using ImageJ software.

[0130] Figure 27 A (front) and B (back) show an example with six detection areas, where the first and second rows are used to measure glucose (a) and uric acid (b), respectively. The concentrations of the two targets in the same whole blood sample (n=3) were simultaneously determined (30 min), with glucose at 4.64 mM and uric acid at 0.32 mM. Furthermore, we simulated pathological levels using the standard spiking method and detected glucose (c), uric acid (d), xanthine (e), and creatinine (f) in whole blood using the MOG-μPAD. Figure 27 The linear reflection artifacts observed in the sample loading holes (A and C) on the front side (C and D) originate from light scattering at the blood-Cu / Co-MOG interface, and these optical phenomena do not affect the analytical results.

[0131] After 30 minutes of incubation, the concentrations of the target metabolic markers in the same whole blood sample (n=3) were as follows: glucose 10.82 mM (indicating hyperglycemia / diabetes), uric acid 0.78 mM (consistent with the risk of hyperuricemia / gout), xanthine 0.31 mM (elevated in xanthineuria or kidney disease), and creatinine 0.59 mM (suggesting early kidney dysfunction or muscle damage). This method, based on MOG-μPAD, has broad applicability for detecting metabolic markers (glucose, uric acid, xanthine, and creatinine) in whole blood samples and has practical application value in the real-time detection of multiple biomolecules in blood samples, enabling screening for diabetes, kidney disease, etc.

[0132] Example 13 Blood Metabolic Marker Detection Kit The kit includes a paper-based microfluidic chip device and a Cu / Co-MOG dispersion as a filter medium. The dispersion is a stable or semi-stable colloidal system containing micro / nano-scale Cu / Co-MOG particles, used for in-situ barrier separation of erythrocytes and colored macromolecules in whole blood or plasma samples. The Cu / Co-MOG particles have a hierarchical porous structure with a pore size of 1~80 nm.

[0133] The kit preferably further includes an oxidase and / or a TMB chromogenic solution; the oxidase includes one or more of glucose oxidase (GOD), uric acid oxidase (UO), xanthine oxidase (XO), and creatinineamidohydrolase (CAH) + creatinase (CI) + sarcosine oxidase (SOX).

[0134] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A paper-based microfluidic chip device based on metal-organic gel, characterized in that, Prepared according to the following method: (1) Preparation of three-layer laminated high-throughput μPAD: The design pattern is printed on the front side of the filter paper using a wax printer. The wax is heated to make the wax diffuse evenly into the interior of the filter paper base to form a hydrophobic area. The area without wax treatment is the hydrophilic detection area. After the treatment, it is folded into three layers so that the hydrophilic detection areas on each layer correspond one-to-one. The two ends are fixed to form a three-layer laminated high-throughput μPAD. (2) Preparation of paper-based microfluidic chip device based on metal organogel: The hydrophilic detection area on the back of the three-layer laminated high-throughput μPAD obtained in step (1) is uniformly coated with metal organogel as a catalyst, and heated and dried to make it uniformly deposited on the hydrophilic detection area on the back, which is the paper-based microfluidic chip device MOG-μPAD. The metal-organic gel includes one or more of Cu / Co-MOG, Cu-MOG, and Co-MOG that have peroxidase-like activity.

2. The paper-based microfluidic chip device as described in claim 1, characterized in that, The metal-organic gel used as the catalyst is Cu / Co-MOG, and its coating concentration is 0.5~3 mg / mL.

3. The paper-based microfluidic chip device as described in claim 2, characterized in that, The paper-based microfluidic chip device detects blood metabolic markers using a colorimetric method, and its hydrophilic detection area on the back is also enriched with oxidase and / or chromogenic solution.

4. A colorimetric-based blood metabolic marker detection kit, characterized in that, Includes the paper-based microfluidic chip device as described in any one of claims 1 or 2, and oxidase and / or colorimetric solution.

5. The detection kit as described in claim 4, characterized in that, This device is used for the immediate detection of metabolic markers in whole blood or plasma samples without separation. It also includes a Cu / Co-MOG dispersion with a concentration of 3-9 mg / mL. The Cu / Co-MOG dispersion serves as a filter medium to block and separate erythrocytes and / or colored macromolecules in the sample in situ.

6. The detection kit as described in claim 5, characterized in that, The Cu / Co-MOG dispersion contains Cu / Co-MOG with a hierarchical porous structure having a pore size of 1~80 nm.

7. A method for the immediate detection of blood metabolic markers without separation, characterized in that, Using the paper-based microfluidic chip device as described in claim 1 or 2, metabolic markers in whole blood or plasma samples can be detected instantly without separation, comprising the following steps: (1) Add oxidase solution and TMB colorimetric solution to the hydrophilic detection area on the back of the paper-based microfluidic chip device MOG-μPAD; (2) In-situ separation and instant detection In the hydrophilic detection area on the front of the paper-based microfluidic chip device MOG-μPAD, a Cu / Co-MOG dispersion with a concentration of 3~9 mg / mL is first added as a filter medium, and then a whole blood or plasma sample is added. The red blood cells and / or macromolecular colored substances in the whole blood or plasma sample are blocked and separated in situ, so that the target metabolic markers in the sample migrate to the corresponding detection area on the back. The target metabolic marker produces hydrogen peroxide under the action of oxidase. The hydrogen peroxide reacts with colorless TMB under the catalysis of Cu / Co-MOG to generate blue oxidized TMB, which then shows color. (3) Quantitative analysis A colorimetric image of the detection area on the back is captured by taking a picture with a smartphone. The gray intensity of the R channel in the detection area is measured using image analysis software. Based on the relationship between the gray intensity I0-I in the R channel and the concentration of the metabolic biomarker standard, the concentration of the target metabolic biomarker in the sample is quantitatively analyzed, where I is the gray intensity corresponding to the sample and I0 is the gray intensity corresponding to the blank control group.

8. The method as described in claim 7, characterized in that, The target metabolic marker is glucose, and the relationship is I0-I=k1×log 10 (C) 葡萄糖 )+b1; The target metabolic marker is uric acid, and the relationship is I0-I=k2×C. 尿酸 +b2; The target metabolic marker is xanthine, and the relationship is log 10 (I0-I) = k3×log 10 (C) 黄嘌呤 )+b3; The target metabolic marker is creatinine, and the relationship is I0-I=k4×log 10 (C) 肌酐 )+b4.

9. The method as described in claim 8, characterized in that, The Cu / Co-MOG dispersion contains Cu / Co-MOG with a hierarchical porous structure having a pore size of 1~80 nm.

10. The method as described in claim 9, characterized in that, For immediate detection of one or more metabolic markers, such as glucose, xanthine, uric acid, and creatinine, in whole blood or plasma samples without separation, step (2) involves first adding a Cu / Co-MOG dispersion with a concentration of 6 mg / mL as a filter medium, and then adding the whole blood or plasma sample.

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