Paper chip for simultaneously detecting multiple prostate cancer markers, preparation method and application thereof

Abstract

The present invention relates to the technical field of prostate cancer biomarker analysis and detection, and particularly relates to a paper chip for simultaneously detecting multiple prostate cancer biomarkers, a preparation method thereof, and an application thereof. The paper chip comprises a sample injection layer, an identification layer, and a detection layer arranged from top to bottom, and three hydrophilic regions are respectively arranged on the sample injection layer, the identification layer, and the detection layer, namely a control region A, a detection region B, and a detection region C. The paper chip is mainly used for quantitatively detecting the contents of two prostate cancer biomarkers (Sar and PSA).

Description

Technical Field

[0001] The present invention relates to the technical field of prostate cancer marker analysis and detection, and particularly relates to a paper chip for simultaneously detecting multiple prostate cancer markers, a preparation method thereof, and an application thereof. Background Art

[0002] Recently, prostate cancer has become the most common malignant tumor in the male urinary system, characterized by early positive signs and unclear clinical symptoms, and poor prognosis. The detection methods of traditional cancer serum markers are relatively accurate, but they have the disadvantages of complex detection operation processes, long time consumption, and the need for dedicated detection sites. As an important analytical device, functionalized paper-based sensors have received extensive attention in simplifying the analysis process, improving the timeliness of analysis results, and diversifying analysis methods, and have been applied to the field of medical diagnosis.

[0003] In addition, compared with the detection of single cancer markers, the combined detection of multiple related markers can improve the detection efficiency and diagnostic accuracy. In the detection of prostate tumor serum markers, prostate-specific antigen (PSA) secreted by prostate epithelial cells is generally used as the traditional target for prostate cancer screening. However, there is a "gray zone" (4 - 10 ng / mL) in PSA diagnosis for differentiating benign and malignant prostate diseases in prostate cancer. In addition, the research and statistical results published in N. Engl. J. Med. show that the effectiveness of screening for prostate cancer solely based on PSA levels is not clear. Therefore, the diagnostic results of prostate cancer obtained by detecting the single serum marker PSA are of low accuracy, often resulting in false positive / negative results and misdiagnosis. The occurrence, development, and metastasis of cancer are complex processes involving multiple genes and multiple factors. Therefore, clinically, it is necessary to jointly detect multiple related markers of the same cancer to improve the diagnostic accuracy. Sarcosine (Sar) is involved in the carcinogenesis process of the prostate and is one of the prime culprits of cell carcinogenesis. Some studies even suggest that Sar is more effective than PSA in the diagnosis of prostate cancer. Yan et al. developed a novel electrochemical aptasensor using a MoS2 hierarchical interface and functionalized SiO2 nanoprobes to sensitively detect two biomarkers, PSA and Sar, simultaneously by designing different DNA hybridization modes (Biosens. Bioelectron. 2022, 197, 113797). Currently, there are relatively few reports on the simultaneous detection of multiple prostate cancer markers, and there is no relevant research on paper-based sensors for the simultaneous detection of two cancer markers, Sar and PSA. Summary of the Invention

[0004] Although the detection results of traditional cancer serum markers are relatively accurate, there are disadvantages such as complex detection procedures, long time consumption, and the need for special detection sites. In addition, the diagnostic results of prostate cancer obtained based on the detection of a single serum marker PSA have low accuracy, and false positive / negative results often occur, leading to misdiagnosis. The present invention provides a paper chip for simultaneously detecting multiple prostate cancer markers, a preparation method thereof, and an application. The paper chip designed and prepared in the present invention is mainly used for quantitatively detecting the contents of two prostate cancer markers (Sar and PSA), and has the advantages of high sensitivity, wide linear range, high accuracy, good precision, simple and rapid operation, etc. It realizes the transfer of chemical reactions from solution to paper, marking an important step in the field of rapid diagnosis technology. It can not only provide rapid detection results, but also be used in resource-limited environments, thus achieving the purpose of point-of-care testing and instant testing.

[0005] The present invention is realized through the following technical solutions:

[0006] A paper chip for simultaneously detecting multiple prostate cancer markers, the paper chip includes a sample injection layer, an identification layer, and a detection layer arranged from top to bottom, and three hydrophilic regions are respectively provided on the sample injection layer, the identification layer, and the detection layer, which are control region A, detection region B, and detection region C;

[0007] At the central positions of control region A, detection region B, and detection region C of the sample injection layer, a sample injection region is provided, and a flow channel is provided between the sample injection region and control region A, detection region B, and detection region C;

[0008] The three hydrophilic regions on the identification layer and the detection layer are independent of each other and do not interfere with each other to avoid cross-linking reactions during the detection process;

[0009] The positions of control region A, detection region B, and detection region C of the sample injection layer, the identification layer, and the detection layer correspond to each other;

[0010] Except for the hydrophilic regions, the other regions of the sample injection layer, the identification layer, and the detection layer are hydrophobic;

[0011] Except for the hydrophilic regions, the sample injection layer, the identification layer, and the detection layer are bonded together by waterproof glue between the layers.

[0012] Furthermore, the material of the sample injection layer is Grade589 / 3 Whatman quantitative cellulose paper, which can retain impurities with a particle size of more than 2μm in the liquid, has a moderate thickness, can filter proteins and exogenous pollutants in the actual serum sample, and the target molecules Sar and PSA to be detected can enter the identification layer;

[0013] The materials of the recognition layer and the detection layer are Grade42 Whatman quantitative cellulose paper, which has a uniform pore size distribution and a high reagent loading capacity, can control the slow flow of liquid, and is more suitable for the recognition and detection of analytes than ordinary cellulose filter paper.

[0014] Furthermore, the hydrophilic region of the detection layer is loaded with TMB chromogenic reagent, the hydrophilic region of the sample injection layer is provided with pH = 7 Tris-HCl buffer solution, the control region A of the recognition layer is loaded with HRP@MAF-7 composite material, and the detection regions B and C of the recognition layer are respectively loaded with Apt Sar / HRP@MAF-7 and Apt PSA / HRP@MAF-7 composite material.

[0015] Furthermore, the hydrophilic regions of the recognition layer and the detection layer are separated by a water-soluble double-sided tape to control the flow of the solution during the reaction process, thereby controlling the reaction time of Apt and the target substance. In addition, the water-soluble polyvinyl alcohol dissolved from the water-soluble double-sided tape is dispersed in the detection layer, which can effectively improve the uniformity of the paper fibers and stabilize the TMB chromogenic reagent loaded in the detection region, and improve the TMB chromogenic sensitivity.

[0016] Furthermore, the preparation method of the Apt / HRP@MAF-7 composite material includes the following steps:

[0017] Mix 2 mL of 0.0594 g Zn(NO3)2 solution with 1 mL of Hmtz aqueous solution containing 10 mg of HRP, stir at room temperature for 24 h, stop the reaction, centrifuge at 10000 rpm for 10 min, collect the precipitate, wash it with deionized water for several times, and then freeze-dry to prepare HRP@MAF-7;

[0018] Disperse 10 mg of HRP@MAF-7 in 5 mL of 10 mM Tris-HCl (pH 7.4) buffer solution, add 20 μM Apt, and incubate at 37 °C for 6 h to obtain Apt / HRP@MAF-7 composite material.

[0019] By in-situ encapsulation, horseradish peroxidase (HRP) molecules are embedded in the hydrophilic metal-organic framework (MAF-7) carrier matrix, which can significantly improve the catalytic activity of the enzyme. Then, the negatively charged aptamer (Apt) can pass through π−π stacking and electrostatic interaction are adsorbed on the surface of HRP@MAF-7 to obtain Apt / HRP@MAF-7 probe. At this time, the pores on the surface of HRP@MAF-7 are closed by the Apt shell layer, and the catalytic activity is in an inhibited state.

[0020] The present invention also provides a method for preparing the paper chip for simultaneous detection of multiple prostate cancer markers, comprising the following steps:

[0021] S1. Use Adobe illustrator CC software to design the patterns of the sample injection layer, recognition layer, and detection layer. Use a wax printer to print the designed patterns on papers of different materials. Do not spray wax on the pre-designed hydrophilic areas, respectively forming the paper substrates of the sample injection layer, recognition layer, and detection layer. Heat the wax-sprayed paper substrates in an oven at 180 °C for 2 min to melt the wax printed on the paper surface and penetrate it into the paper to form the hydrophobic areas of each layer;

[0022] S2. Uniformly drop 40 μL of 10 mM Tris-HCl buffer with pH = 7 on the hydrophilic area of the paper substrate of the sample injection layer obtained in step S1; uniformly drop 50 μL of the aqueous dispersion of 10 mg / mL HRP@MAF-7 on the control area A of the hydrophilic area of the paper substrate of the recognition layer, and uniformly drop 50 μL of the aqueous dispersion of 10 mg / mL Apt / HRP@MAF-7 on the detection areas B and C; uniformly drop 20 μL of 15 mM TMB on the hydrophilic area of the detection layer;

[0023] S3. Paste Japanese clover water-soluble double-sided tape 57-899 between the adjacent hydrophilic areas of the recognition layer and the detection layer;

[0024] S4. Bond the hydrophobic areas between adjacent layers with waterproof glue, and finally press tightly with a laminator.

[0025] The present invention also provides the application of the paper chip in the quantitative detection of the contents of Sar and PSA.

[0026] The present invention also provides a method for quantitatively detecting the contents of Sar and PSA by using the paper chip, comprising the following steps:

[0027] Drop the actual sample onto the paper chip. After reacting for 30 min, sequentially add 300 μL of 20 mM H2O2 solution. After continuing to react for 10 min, turn the paper chip over so that the detection layer faces up, and read with a handheld color difference meter device. Calculate the concentrations of Sar and PSA in the sample by using the standard curve between the concentration and the change value of ΔC.

[0028] Compared with the prior art, the beneficial technical effects of the present invention:

[0029] (1) The present invention has break-throughly designed a paper chip that can be portable and simultaneously detect multiple prostate cancer markers in a sample. The paper chip is mainly used for quantitatively detecting the contents of two prostate cancer markers (Sar and PSA), and has the advantages of high sensitivity, wide linear range, high accuracy, good precision, simple and rapid operation, etc. It realizes the transfer of chemical reactions from solution to paper, marking an important step in the field of rapid diagnosis technology. It can not only provide rapid detection results, but also be used in resource-limited environments, thus achieving the purpose of point-of-care testing and instant detection.

[0030] (2) For the paper chip of the present invention, through the setting of introducing the water-soluble double-sided adhesive material, it can prevent the samples modified in the hydrophilic regions of the recognition layer and the detection layer from interfering with each other, and improve the stability of the paper chip. After the sample is added, the reaction time can be regulated by changing the thickness of the double-sided adhesive, so that the sample can fully react and be recognized, enhancing the sensitivity of the detection signal; the water-soluble double-sided adhesive dissolves and is loaded in the detection layer, having good film-forming property and adsorption property, which can enhance the flatness of the paper surface and the fixing effect of the chromogenic agent. Through the dissolution and coating of the soluble double-sided adhesive, the chromogenic agent can be more evenly distributed on the paper surface, reducing diffusion and improving the uniformity and stability of color development.

[0031] (3) The present invention combines the paper chip with a handheld colorimeter for the simultaneous detection of multiple prostate cancer markers. It is expected to establish a sensitive combined detection method for multiple prostate cancer markers, providing a reliable detection basis for the instant and accurate diagnosis of prostate cancer. This detection device is simple, cheap, portable, disposable and easy to use.

[0032] (4) During use, aptamers of different target substances can be introduced to expand the application of this paper chip detection platform.

[0033] (5) The detection result of the prostate cancer marker takes the change of the difference between the detection signals of the detection area and the reference area as the quantitative detection standard, ensuring the accuracy of the detection result. Description of the Drawings

[0034] Figure 1 SEM image of the prepared HRP@MAF-7;

[0035] Figure 2 Synthesis schematic diagram of the Apt / HRP@MAF-7 composite material;

[0036] Figure 3 SEM image of the prepared Apt / HRP@MAF-7;

[0037] Figure 4 Zeta potential diagrams of MAF-7, HRP@MAF-7 and Apt / HRP@MAF-7;

[0038] Figure 5 It is a schematic structural diagram of a paper chip;

[0039] Figure 6 They are schematic diagrams of the sample injection layer, recognition layer, and detection layer;

[0040] Figure 7 It is an SEM image of the hydrophilic region of the recognition layer without the loaded composite material;

[0041] Figure 8 It is an SEM of the hydrophilic region of the recognition layer loaded with Apt / HRP@MAF-7;

[0042] Figure 9 It is an SEM image of the hydrophilic region of the detection layer fixed with TMB chromogenic reagent;

[0043] Figure 10 It is an SEM image of the hydrophilic region of the detection layer fixed with TMB chromogenic reagent after the water-soluble double-sided tape is dissolved;

[0044] Figure 11 It is a schematic diagram of the detection process for detecting Sar and PSA with the paper chip;

[0045] Figure 12 It is for C Sar and the standard curve graph of ΔC;

[0046] Figure 13 It is for C PSA and the standard curve graph of ΔC. Specific implementation mode

[0047] Example 1 Preparation of Apt / HRP@MAF-7 composite material

[0048] S1. Mix 2 mL of 0.0594 g Zn(NO3)2 solution with the Hmtz aqueous solution containing 10 mg of HRP, stir at room temperature for 24 h, stop the reaction, centrifuge at 10000 rpm for 10 min, collect the precipitate, wash it with deionized water multiple times, and then freeze-dry to prepare HRP@MAF-7. The SEM image of the prepared HRP@MAF-7 is shown in Figure 1 .

[0049] S2. Disperse 10 mg of HRP@MAF-7 in 5 mL of 10 mM Tris-HCl (pH 7.4) buffer solution, add 20 μM Apt, and incubate at 37 °C for 6 h to obtain the Apt / HRP@MAF-7 composite material. The synthesis schematic diagram of the Apt / HRP@MAF-7 composite material is shown in Figure 2 , and the SEM image of the obtained Apt / HRP@MAF-7 is shown in Figure 3 .

[0050] Figure 4 Zeta potential diagrams of MAF-7, HRP@MAF-7, and Apt / HRP@MAF-7. After embedding negatively charged HRP into the MAF-7 framework, the ζ potential of HRP@MAF-7 decreased from +22.0 mV of bare MAF-7 to +17.3 mV. This change indicates that HRP was successfully loaded into MAF-7, confirming the successful preparation of the HRP@MAF-7 composite. After introducing Apt, the ζ potential of the composite significantly reversed from +17.3 mV of HRP@MAF-7 to -11.5 mV of Apt / HRP@MAF-7, indicating that Apt was successfully modified onto the surface of HRP@MAF-7.

[0051] Example 2 Structure of the Paper Chip

[0052] A paper chip for simultaneously detecting multiple prostate cancer markers, which includes a sample injection layer, an identification layer, and a detection layer arranged from top to bottom, as shown in Figure 5 , three hydrophilic regions are respectively provided on the sample injection layer, the identification layer, and the detection layer, which are control region A, detection region B, and detection region C respectively. An injection region is provided at the central position of control region A, detection region B, and detection region C of the sample injection layer. A flow channel is provided between the injection region and control region A, detection region B, and detection region C. The three hydrophilic regions on the identification layer and the detection layer are independent of each other and do not interfere with each other to avoid cross-linking reactions during the detection process, as shown in Figure 6 ; the positions of control region A, detection region B, and detection region C on the sample injection layer, the identification layer, and the detection layer correspond to each other; except for the hydrophilic regions, the other regions of the sample injection layer, the identification layer, and the detection layer are hydrophobic; except for the hydrophilic regions, the layers of the sample injection layer, the identification layer, and the detection layer are bonded together by waterproof glue; the hydrophilic regions on the identification layer and the detection layer are separated by a water-soluble double-sided adhesive.

[0053] In this example, as shown in Figure 6 : The flow channels between circular regions are hydrophilic regions, and the rest are hydrophobic regions; the square paper is 5 cm, and the diameter of the circular reaction region is 1 - 1.6 cm; the length of the liquid flow channel is 0.5 cm - 1.1 cm, and the width is 0.3 cm - 0.5 cm.

[0054] The configurations of each layer in Example 1 are shown in Table 1.

[0055] Table 1. Hydrophilic Region Configurations of Each Layer of the Paper Chip

[0056]

[0057] Grade 589 / 3 cellulose filter paper: pore size <2 μm, filtration rate (approximate value) Herzberg (s): 375, bottom thickness 160 μm;

[0058] Grade 42 cellulose filter paper: pore size is about 2.5 μm, filtration rate (approximate value) Herzberg (s): 1870, bottom thickness 200 μm;

[0059] Japanese clover water-soluble double-sided tape 57-899: width 6 mm.

[0060] Example 3 Preparation of paper chip

[0061] S1. Use Adobe illustrator CC software to design the patterns of the sample injection layer, recognition layer, and detection layer. As shown in Figure 5 and Figure 6 shown, use a waxjet printer to print the designed patterns on papers of different materials. Do not spray wax on the pre-designed hydrophilic areas, and respectively form the paper substrates of the sample injection layer, recognition layer, and detection layer. Heat the wax-sprayed paper substrates in an oven at 180 °C for 2 min to melt the wax printed on the paper substrate surface and penetrate it into the paper to form the hydrophobic areas of each layer;

[0062] S2. Uniformly drop 40 μL of 10 mM Tris-HCl buffer with pH = 7 on the hydrophilic area of the paper substrate of the sample injection layer obtained in step S1; uniformly drop 50 μL of the aqueous dispersion of 10 mg / mL HRP@MAF-7 on the control area A of the hydrophilic area of the paper substrate of the recognition layer, and uniformly drop 50 μL of the aqueous dispersion of 10 mg / mL Apt / HRP@MAF-7 on the detection areas B and C; uniformly drop 20 μL of 15 mM TMB on the hydrophilic area of the detection layer;

[0063] S3. Paste Japanese clover water-soluble double-sided tape 57-899 between the adjacent hydrophilic areas of the recognition layer and the detection layer;

[0064] S4. Bond the hydrophobic areas between adjacent layers with waterproof glue, and finally press tightly with a laminator.

[0065] Figure 7 is the SEM image of the hydrophilic area of the recognition layer without the loaded composite material, Figure 8 is the SEM of the hydrophilic area of the recognition layer loaded with Apt / HRP@MAF-7. By comparison, it is found that the Apt / HRP@MAF-7 composite material is uniformly dispersed in the paper fibers of the hydrophilic area of the recognition layer to form a stable covering layer.

[0066] Figure 9 is the SEM image of the hydrophilic area of the detection layer fixing the TMB chromogenic reagent, Figure 10 is the SEM image of the hydrophilic area of the detection layer fixing the TMB chromogenic reagent after the water-soluble double-sided tape is dissolved. It can be seen from the figure that compared with Figure 9, the paper fibers are denser, which can improve the uniformity and sensitivity of color development.

[0067] Example 4 Application of the paper chip in the quantitative detection of Sar and PSA contents

[0068] The detection process of the paper chip for detecting Sar and PSA is as Figure 11 shown. The sample enters through the sampling layer, and the pH buffer salt is redissolved to control the pH of the sample. When the sample reaches the recognition layer and encounters the water-soluble double-sided tape, the liquid flow is slowed down. The prostate cancer markers in the sample will specifically bind to the prostate cancer marker Apt in the recognition layer and form a specific three-dimensional structure, releasing the catalytic active site of HRP@MAF-7 in a concentration-dependent manner. After the water-soluble double-sided tape is completely dissolved, the sample can flow into the detection layer, and in the presence of H2O2, it can effectively catalyze the conversion of TMB in the detection layer into an obvious bright blue oxidation product (oxTMB). The water-soluble double-sided tape is dissolved and loaded in the detection layer, which has good film-forming properties and adsorption properties, and can enhance the flatness of the paper surface and the fixing effect of the color developer. Through the dissolution and coating of the water-soluble double-sided tape, the color developer can be more evenly distributed on the paper surface, reducing diffusion and improving the uniformity and stability of color development.

[0069] The specific detection process is as follows:

[0070] (1) First, the analytical performance was investigated using standard solution samples of different concentrations of Sar and PSA

[0071] Standard solutions containing different concentrations of 300 μL of PSA and Sar, two markers, were added to the paper chip. After reacting for 30 min, 300 μL of H2O2 (20 mM) solution was added successively. After continuing to react for 10 min, the paper chip was flipped so that the detection layer faced up, and readings were taken using a handheld color difference meter device. The color change value (ΔC) was calculated using the Euclidean distance equation:

[0072]

[0073] ΔR, ΔG, and ΔB are the change values of R, G, and B in the detection channel relative to the control region, respectively.

[0074] The experimental results are as Figure 12 and Figure 13 shown. As the concentrations of Sar and PSA increase, there is a good linear relationship between the marker concentration and the change value of ΔC. The linear ranges are 0.01−20 ng / mL and 0.05−150 ng / m, respectively, and the standard curves obtained are y = 5.52* C Sar + 0.61 (R 2 = 0.9960) and y = 1.39*C PSA +0.97(R 2 =0.9995). The calculated detection limits of the corresponding two markers are 0.023 and 0.045 ng / mL, respectively.

[0075] (2) To evaluate the accuracy of the proposed method, the contents of Sar and PSA in human serum samples were investigated and determined by the addition-recovery method. 300 μL of the serum sample solution was dropped onto the paper chip, and the test results are shown in Table 2. It can be seen from Table 2 that the recovery rate of the paper chip is 99.4 - 104.2% (n = 5), and the RSD is 2.0 - 4.1%, which is applicable to the determination of Sar and PSA in actual samples.

[0076] Table 2 Determination of the contents of Sar and PSA in different human serum samples (n = 5). a

[0077]

Claims

1. A paper chip for simultaneously detecting multiple prostate cancer markers, characterized in that: The paper chip includes a sample injection layer, an identification layer, and a detection layer arranged from top to bottom; Three hydrophilic regions are respectively provided on the sample injection layer, the identification layer, and the detection layer, namely a control region A, a detection region B, and a detection region C; At the central positions of the control region A, the detection region B, and the detection region C of the sample injection layer, a sample injection region is provided, and a flow channel is provided between the sample injection region and the control region A, the detection region B, and the detection region C; The three hydrophilic regions on the identification layer and the detection layer are independent of each other and do not interfere with each other; The positions of the control region A, the detection region B, and the detection region C on the sample injection layer, the identification layer, and the detection layer correspond to each other; Except for the hydrophilic regions, the other regions of the sample injection layer, the identification layer, and the detection layer are hydrophobic regions; Except for the hydrophilic regions, the layers of the sample injection layer, the identification layer, and the detection layer are bonded together by a waterproof adhesive; The hydrophilic region of the detection layer is loaded with TMB chromogenic agent, the hydrophilic region of the sample injection layer is provided with a pH = 7 Tris-HCl buffer solution, the control region A of the recognition layer is loaded with HRP@MAF-7 composite material, and the detection regions B and C of the recognition layer are respectively loaded with Apt Sar / HRP@MAF-7 and Apt PSA / HRP@MAF-7 composite material.

2. The paper chip for simultaneously detecting multiple prostate cancer markers according to claim 1, characterized in that: The material of the sample injection layer is Grade589 / 3 Whatman quantitative cellulose paper, and the materials of the identification layer and the detection layer are Grade42 Whatman quantitative cellulose paper.

3. The paper chip for simultaneously detecting multiple prostate cancer markers according to claim 1, wherein The hydrophilic regions of the identification layer and the detection layer are separated by a water-soluble double-sided adhesive.

4. The paper chip for simultaneously detecting multiple prostate cancer markers according to claim 1, characterized in that, The preparation method of the Apt / HRP@MAF-7 composite material includes the following steps: Mix 2 mL of 0.0594 g Zn(NO3)2 solution with 1 mL of Hmtz aqueous solution containing 10 mg of HRP, stir at room temperature for 24 h, stop the reaction, centrifuge at 10000 rpm for 10 min, collect the precipitate, wash it with deionized water multiple times, and then freeze-dry to prepare HRP@MAF-7; Disperse 10 mg of HRP@MAF-7 in 5 mL of 10 mM Tris-HCl pH 7.4 buffer solution, add 20 μM Apt, and incubate at 37°C for 6 h to obtain the Apt / HRP@MAF-7 composite material.

5. A method for preparing a paper chip for simultaneously detecting multiple prostate cancer markers as described in any one of claims 1-4, characterized in that: Include the following steps: S1. Use Adobe illustrator CC software to design the patterns of the sample injection layer, the identification layer, and the detection layer, use a wax jet printer to print the designed patterns on papers of different materials, and do not spray wax on the pre-designed hydrophilic regions, respectively forming the paper bases of the sample injection layer, the identification layer, and the detection layer. Heat the wax-sprayed paper bases in an oven at 180°C for 2 min to melt the wax printed on the paper base surface and penetrate it into the paper to form the hydrophobic regions of each layer; S2. Uniformly drop 40 μL of 10 mM pH = 7 Tris-HCl buffer solution on the hydrophilic regions of the paper base of the sample injection layer obtained in step S1; uniformly drop 50 μL of the aqueous dispersion of 10 mg / mL HRP@MAF-7 on the control region A of the hydrophilic region of the paper base of the identification layer, and uniformly drop 50 μL of the aqueous dispersion of 10 mg / mL Apt / HRP@MAF-7 on the detection region B and the detection region C; uniformly drop 20 μL of 15 mM TMB on the hydrophilic regions of the detection layer; S3. Paste the Japanese clover water-soluble double-sided adhesive 57-899 between the adjacent hydrophilic regions of the identification layer and the detection layer; S4. Bond the hydrophobic regions between adjacent layers with a waterproof adhesive, and finally press them tightly with a laminator.

6. Use of a paper chip for simultaneously detecting multiple prostate cancer markers as described in any one of claims 1-4 in the quantitative detection of the contents of Sar and PSA, said use being for non-disease treatment purposes and disease diagnosis purposes.

7. A method for quantitatively detecting the contents of Sar and PSA by using a paper chip for simultaneously detecting multiple prostate cancer markers as described in any one of claims 1-4, characterized in that: Comprising the following steps: Drop an actual sample onto the paper chip. After reacting for 30 min, sequentially add 300 μL of 20 mM H2O2 solution. After continuing to react for 10 min, turn the paper chip over so that the detection layer faces upward, and take readings using a handheld color difference meter device. Calculate the concentrations of Sar and PSA in the sample using the standard curve between the concentration and the change value of ΔC.

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