A protein detection sensor array based on nanozymes and protein detection method

Abstract

The present invention discloses a protein detection sensor array based on nanozymes and a protein detection method, which belong to the field of biosensors. The sensor array of the present invention includes three sensing units, each unit containing a nanozyme, namely: PPy@MoS2@Au, PPy@MoS2@Ag and PPy@MoS2@Pd, and the three nanozymes have different enzyme activity change responses to different proteins. The present invention utilizes the constructed protein sensor array to convert the qualitative determination of the target protein into a qualitative and / or quantitative determination of a colorimetric indicator containing hydrogen peroxide, detects the ultraviolet-visible absorbance value by an enzyme marker, and draws a PCA-2D or PCA-3D map of the target protein. On the map, the protein is corresponding to the 95% confidence ellipse, and the qualitative and / or quantitative determination of the target protein is achieved simply and quickly, which can provide a new idea for quickly distinguishing proteins and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biosensors, and in particular to a nanozyme-based protein detection sensor array and a protein detection method. Background Art

[0002] Proteins are essential components of living organisms and are closely linked to many diseases. Protein content and conformational changes are crucial for diagnosing diseases and their progression. For example, elevated transferrin concentrations can lead to acute viral hepatitis and hepatocyte necrosis. Conversely, low transferrin levels can trigger a range of infectious diseases, including primary liver cancer and kidney disease. Traditional sensors often employ a lock-and-key approach, employing antibodies or designed nucleic acid capture probes to achieve high specificity. However, this approach is subject to numerous environmental influences, such as pH and temperature, making it difficult to consider these factors during sensor design, significantly limiting their application. Sensor arrays, on the other hand, utilize a combination of multiple sensing elements that lack specific binding to proteins. When bound to the analyte, these elements do not require a specific, selective response to the protein. Similar to the nose and tongue of mammals, differential detection of analytes is based on the combined responses of different sensing elements. This enables sensor arrays to detect complex mixtures, such as a mixture. The colorimetric sensor array is constructed using three nanozymes. It has the advantages of high sensitivity and fast color development speed, and has gradually become the development focus of sensor arrays. Summary of the Invention

[0003] The inventors of the present invention discovered in their research that proteins interact with three nanozymes (PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd) through electrostatic and hydrophilic interactions, thereby causing changes in the activity of the nanozymes. The color response of the sensor array is essentially a pattern recognition process. Pattern recognition using the PCA algorithm enables differential detection. Three sensing units are used to achieve differential detection of single proteins and mixed proteins of different types and concentrations. Therefore, the present invention is proposed.

[0004] Specifically, the present invention provides a sensor array method for detecting proteins, characterized in that it includes three sensor units, each unit containing a nanozyme, namely: PPy@MoS2@Au, PPy@MoS2@Ag and PPy@MoS2@Pd, and the three nanozymes have different enzymatic activity changes in response to different proteins.

[0005] Furthermore, the contents of PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd in the detection system are 5 to 100 μg / mL, respectively.

[0006] Furthermore, the content of the nanozyme is 20 μg / mL.

[0007] Furthermore, the three nanozymes PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd have obvious absorption values at a wavelength of 652nm.

[0008] Furthermore, the pH value corresponding to the maximum absorbance of the PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd is pH 4.0.

[0009] The present invention also provides a protein detection method based on a nanozyme sensor array, which is characterized by comprising the following steps:

[0010] S1: Identify the target protein;

[0011] S2: constructing a sensor array, wherein the sensor array includes three sensing units corresponding to three nanozymes: PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd. The three nanozymes have different enzymatic activity changes in response to the target protein;

[0012] S3: preparing solutions P1-Pn of a single target protein, and optionally, preparing mixed solutions S1-Sn of all target proteins, wherein the highest target protein concentration in each mixed solution is the same as the concentration of the single protein;

[0013] S4: fully combining the sensor array with the solutions P1-Pn and S1-Sn respectively, adding a color developing reagent, performing ultraviolet detection, and obtaining detection data;

[0014] S5: Processing the detection data using a PCA algorithm and an HCA algorithm to obtain PCA maps and HCA maps of a single target protein solution and a mixed protein solution, respectively;

[0015] S6: adding the analyte to be tested to the sensor array, and identifying the protein according to the position of the analyte in the map.

[0016] Furthermore, the preparation method of the three nanozymes in step S2 is as follows:

[0017] S11: ultrasonically dissolving ammonium molybdate and nitric acid, wherein the concentration of ammonium molybdate in the reaction system is 0.18-0.25 mol / L and the concentration of nitric acid is 1.5-2 mol / L; the mixture is hydrothermally reacted at 175-185°C for 18-24 hours; after the reaction, the product MoO3 is washed and dried;

[0018] S12: MoO3 is dispersed in a mixture of ethanol and water and uniformly dispersed by ultrasonication; pyrrole is added, and the amount ratio of pyrrole to MoO3 is 0.8-1.2 mL / g, preferably 1 mL / g; after stirring, a (NH4)2S2O8 solution with a concentration of 0.25-0.4 mol / L is slowly added, and the molar ratio of MoO3 to (NH4)2S2O8 is 1:2-3; the reaction is stirred at room temperature for 3-6 hours. After the reaction is completed, the mixture is filtered, washed and dried to obtain MoO3@PPy; in the mixture of ethanol and water, the volume ratio of ethanol to water is 1:7-9, preferably 1:8; preferably, the reaction time is 4 hours;

[0019] S13: MoO3@PPy is added to the CN2H4S solution and ultrasonically dispersed. The product is then hydrothermally reacted at 180-220°C for 18-28 hours. After the reaction, the product is washed and dried to obtain the product PPy@MoS2. The ratio of thiourea to MoO3@PPy is 0.08-0.1 mol / g, preferably 0.09 mol / g. The concentration of thiourea in the reaction system is 0.1-0.2 mol / L, preferably 0.15 mol / L.

[0020] S14: PPy@MoS2 is dispersed in water and ultrasonically dispersed uniformly to a concentration of 0.4-0.5 mg / mL; HAuCl4, AgNO3, or PdCl2 are added respectively; the reaction is ultrasonically reacted for 0.5-2 h. After the reaction is completed, the products are washed and dried to obtain PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd, respectively. The ratio of HAuCl4, AgNO3, or PdCl2 to PPy@MoS2 is 0.005-0.015 mol / g, preferably 0.01 mol / g.

[0021] Furthermore, the detection steps in step S4 are as follows:

[0022] S41: pH 4.0 citric acid-sodium citrate buffer was mixed with PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd, and the protein solution was added, mixed thoroughly, and incubated at room temperature; the concentration of the citric acid-sodium citrate buffer was 5-20 mM, preferably 10 mM, and the incubation was for 0.5-2 h, preferably 1 h;

[0023] S42: H2O2 and TMB are added to the mixture and reacted at 37°C for 10 min; the H2O2 content in the mixture is 0.2-15 mmol / L, and the TMB content is 0.5-1.5 mmol / L; preferably, the H2O2 content is 10 mmol / L, and the TMB content is 1 mmol / L;

[0024] S43: Measure the absorbance value at a wavelength of 652 nm using an enzyme marker to obtain detection data.

[0025] Furthermore, in step S5, the PCA map is a PCA-2D map and / or a PCA-3D map; the HCA map is an HCA-2D map and / or an HCA-3D map; and the protein distribution range is determined based on a 95% confidence ellipse in the map.

[0026] Furthermore, the detection method in step S6 is:

[0027] The mixture of protein-containing nanozymes was evenly mixed with a color indicator, and reacted at pH 4.0 and room temperature for 10 minutes to obtain the detection data. The protein distribution range of the atlas was compared to determine the protein to which it belonged.

[0028] The beneficial technical effects of the present invention are:

[0029] 1. The protein sensor array constructed with three three-dimensional nanorods (PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd) provided by the present invention has catalytic activity similar to that of peroxidase, and has good stability, high surface area, and low toxicity.

[0030] 2. This invention utilizes a protein sensor array constructed with three types of three-dimensional nanorods (PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd) to convert the qualitative determination of a target protein into qualitative and / or quantitative determination using a chromogenic indicator containing hydrogen peroxide. UV-visible absorbance is measured using a microplate reader, and PCA-2D or PCA-3D maps of the target protein are generated. Proteins are mapped using 95% confidence ellipses on the maps, enabling simple and rapid qualitative and / or quantitative determination of the target protein. This provides a new approach for rapid protein differentiation and holds broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SEM images of PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods.

[0032] Figure 2 These are the UV absorption spectra of TMB substrate catalyzed by PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods.

[0033] Figure 3 The peroxidase activity trends of PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods with changes in pH.

[0034] Figure 4The protein sensor array constructed by PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods responds to the enzyme activity of different proteins.

[0035] Figure 5 PCA maps of protein sensing arrays constructed with PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods for distinguishing single proteins.

[0036] Figure 6 PCA and LDA maps of protein sensing arrays constructed for PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods for distinguishing different single proteins.

[0037] Figure 7 PCA and LDA maps of protein sensing arrays constructed for PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods for distinguishing different mixed proteins.

[0038] Figure 8 Schematic diagram of the process of protein discrimination using protein sensing arrays constructed with PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods.

[0039] Figure 9 PCA maps of the protein sensing array composed of three-dimensional nanozymes PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd for distinguishing different concentrations of CytC.

[0040] Figure 10 The detection limit of the protein sensor array composed of three-dimensional nanozymes PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd.

[0041] Figure 11 A protein sensing array composed of three-dimensional nanozymes PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd was used to detect and differentiate proteins with different mixing ratios.

[0042] Figure 12 A protein sensing array composed of three-dimensional nanoenzymes PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd is used to distinguish proteins in serum. DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific implementation examples.

[0044] Example 1: Synthesis of PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd nanorods.

[0045] According to the preparation method of the literature (Fabrication of noble metal nanoparticles decorated on one dimensional hierarchical polypyrrole@MoS2 microtubes, The Royal Society of Chemistry, 2020, 8, 7801-7811.), three-dimensional nanorods PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd were prepared as follows:

[0046] (1) Preparation of MoO3

[0047] 1 g of ammonium molybdate was dissolved in 20 mL of water and 5 mL of 8 mol / L nitric acid by ultrasonication. The mixture was then transferred to a 50 mL reactor and incubated at 180 °C for 20 h. After the reaction, the mixture was filtered and washed several times with water and ethanol. The product MoO3 was dried in an oven at 60 °C overnight.

[0048] (2) Preparation of MoO3@PPy

[0049] a. 100 mg MoO3 was dispersed in 5 mL ethanol and 40 mL water and dispersed evenly by ultrasonication;

[0050] b. Add 0.1 mL of pyrrole to step a and stir for 0.5 h;

[0051] c. Dissolve 0.329g (NH4)2S2O8 in 5mL of water and add slowly. Stir the reaction at room temperature for 4h. After the reaction is completed, filter, wash with ethanol and dry.

[0052] (3) Preparation of PPy@MoS2

[0053] a. 0.3425 g of thiourea (CN2H4S) was dispersed in 30 mL of water and evenly dispersed by ultrasonication;

[0054] b. Add a into 50mg MoO3@PPy and disperse evenly by ultrasonication;

[0055] c. Then transfer it into a 50mL reactor at 200℃ for 24h. After the reaction, filter and wash it several times with water and ethanol, and dry it to obtain the product PPy@MoS2.

[0056] (4) Preparation of ppy@MoS2@Au / Ag / Pd

[0057] a. 10 mg PPy@MoS2 was dispersed in 20 mL water and dispersed evenly by ultrasonication;

[0058] b. Add 0.1 mmol HAuCl4 / AgNO3 / PdCl2 respectively;

[0059] c. Ultrasonic reaction was performed for 1 h. After the reaction, the product PPy@MoS2@Au / Ag / Pd was obtained by centrifugation and washing with ethanol several times and drying.

[0060] Example 2: Peroxidase-like activity of PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd.

[0061] 2.1 Peroxidase-like activity

[0062] (1) Take 277.5 μL, 279 μL, and 271.5 μL of 10 mM pH 4.0 citric acid-sodium citrate buffer solution into each centrifuge tube, respectively. Add 0 μL, 6 μL, and 6 μL of PPy@MoS2@Au / Ag / Pd (1 mg / mL each of the three enzymes), 7.5 μL, 0 μL, and 7.5 μL of hydrogen peroxide solution (0.01 M), and 15 μL, 15 μL, and 15 μL of 3,3',5,5'-tetramethylbenzidine (TMB, 20 mM) into each centrifuge tube, respectively. Mix the above solutions evenly.

[0063] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0064] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0065] The results are as follows Figure 2 As shown in the figure, all three nanozymes exhibit a distinct absorption peak at a wavelength of 652 nm. As can be seen from the figure, under the same conditions, the three nanozymes have distinct absorption values at a wavelength of 652 nm, with the order from high to low being PPy@MoS2@Pd > PPy@MoS2@Au > PPy@MoS2@Ag. The signal-to-noise ratios of the catalytic activity of the three nanozymes, PPy@MoS2@Pd, PPy@MoS2@Au, and PPy@MoS2@Au, are 19, 4, and 9 times, respectively. This indicates that PPy@MoS2@Pd has the highest catalytic activity, followed by PPy@MoS2@Au, and PPy@MoS2@Ag has the lowest.

[0066] 2.2 Optimum pH value of PPy@MoS2@Au / Ag / Pd

[0067] (1) 271.5 μL of 10 mM pH 2.0, 10 mM pH 3.0, 10 mM pH 4.0, 10 mM pH 5.0, 10 mM pH 6.0, 10 mM pH 7.0, 10 mM pH 8.0, 10 mM pH 9.0, and 10 mM pH 10.0 citric acid-sodium citrate buffer solutions were placed in separate centrifuge tubes. 6 μL of PPy@MoS2@Au / Ag / Pd (1 mg / mL), 7.5 μL of aqueous hydrogen peroxide (0.01 M), and 15 μL of 3,3',5,5'-tetramethylbenzidine (TMB, 20 mM) were added to each centrifuge tube and the solutions were mixed evenly.

[0068] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0069] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0070] The results are as follows Figure 3 As shown in the figure, the absorbance at 652 nm first increases and then decreases with the increase of pH. In order to make PPy@MoS2@Au / Ag / Pd work under the best conditions, pH 4.0 corresponding to the maximum absorbance was selected as the optimal pH for the reaction.

[0071] Example 3:

[0072] 3.1 H2O2 experiment

[0073] [TMB] = 0.2 mM

[0074] (1) Take an appropriate amount of 10 mM pH = 4.00 citric acid-sodium citrate buffer solution in each centrifuge tube, add 6 μL ppy@MoS2@Au / Ag / Pd (1 mg / mL), 6 μL 3,3',5,5'-tetramethylbenzidine (TMB, 10 mM), and different concentrations of hydrogen peroxide aqueous solution (final concentrations are close to 100, 200, 500, and 1000 μM) to each centrifuge tube in sequence, make up the volume to 300 μL, and mix the above solutions evenly;

[0075] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0076] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0077] [TMB] = 0.1 mM

[0078] (1) Take an appropriate amount of 10 mM pH = 4.00 citric acid-sodium citrate buffer solution in each centrifuge tube, add 6 μL PPy@MoS2@Au / Ag / Pd (1 mg / mL), 3 μL 3,3',5,5'-tetramethylbenzidine (TMB, 10 mM), and different concentrations of hydrogen peroxide aqueous solution (final concentrations are close to 100, 200, 500, and 1000 μM) to each centrifuge tube in sequence, make up the volume to 300 μL, and mix the above solutions evenly;

[0079] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0080] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0081] [TMB] = 0.1 mM

[0082] (1) Take an appropriate amount of 10 mM pH = 4.00 citric acid-sodium citrate buffer solution in each centrifuge tube, and add 6 μL ppy@MoS2@Au / Ag / Pd (1 mg / mL), 1.5 μL 3,3',5,5'-tetramethylbenzidine (TMB, 20 mM), and different concentrations of hydrogen peroxide aqueous solution (final concentrations are close to 100, 200, 500, and 1000 μM, respectively) to each centrifuge tube in sequence, and mix the above solutions evenly;

[0083] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0084] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0085] The trend of the H2O2 redox reaction rate catalyzed by the nanomimetic enzyme PPy@MoS2@Au / Ag / Pd with different TMB concentrations (0.20mM, 0.10mM, 0.05mM) is as follows Figure 3 As shown, after calculation, the Michaelis constant Km of the nanomimetic enzyme PPy@MoS2@Au for the substrate H2O2 is 0.04mM, and the maximum reaction rate Vm is 0.2; the Michaelis constant Km of PPy@MoS2@Ag for the substrate H2O2 is 60, and the maximum reaction rate Vm is 909; the Michaelis constant Km of PPy@MoS2@Pd for the substrate H2O2 is 0.30, and the maximum reaction rate Vm is 1.66.

[0086] 3.2 TMB experiment

[0087] [H2O2]=0.1mM

[0088] (1) Take an appropriate amount of 10 mM pH 4.0 citric acid-sodium citrate buffer solution in each centrifuge tube, add 6 μL ppy@MoS2@Au / Ag / Pd (1 mg / mL), 3 μL hydrogen peroxide aqueous solution (0.01 M), and 1.5 μL 3,3',5,5'-tetramethylbenzidine of different concentrations (final concentrations of approximately 200, 400, 600, and 800 μM) to each centrifuge tube in sequence, make up the volume to 300 μL, and mix the above solutions evenly;

[0089] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0090] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0091] [H2O2]=0.075mM

[0092] (1) Take an appropriate amount of 10 mM pH = 4.0 citric acid-sodium citrate buffer solution in each centrifuge tube, add 6 μL ppy@MoS2@Au / Ag / Pd (1 mg / mL), 2.25 μL hydrogen peroxide aqueous solution (0.01 M), and 1.5 μL 3,3',5,5'-tetramethylbenzidine of different concentrations (final concentrations of approximately 200, 400, 600, and 800 μM) to each centrifuge tube in sequence, make up the volume to 300 μL, and mix the above solutions evenly;

[0093] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0094] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0095] [H2O2]=0.05mM

[0096] (1) Take an appropriate amount of 10 mM pH = 4.0 citric acid-sodium citrate buffer solution in each centrifuge tube, add 6 μL ppy@MoS2@Au / Ag / Pd (1 mg / mL), 1.5 μL hydrogen peroxide aqueous solution (0.01 M), and 1.5 μL 3,3',5,5'-tetramethylbenzidine of different concentrations (final concentrations of approximately 200, 400, 600, and 800 μM) to each centrifuge tube in sequence, make up the volume to 300 μL, and mix the above solutions evenly;

[0097] (2) reacting the mixture obtained in step (1) at room temperature for 10 min;

[0098] (3) The ultraviolet absorption spectrum of the mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0099] At different H2O2 concentrations (0.10mM, 0.075mM, 0.05mM), the rate of H2O2 redox reaction catalyzed by nanomimetic enzyme PPy@MoS2@Au / Ag / Pd changes with H2O2 concentration. Figure 3 As shown, after calculation, the Michaelis constant Km of the nanomimetic enzyme PPy@MoS2@Au for the substrate TMB is 1.53, and the maximum reaction rate Vm is 4.6; the Michaelis constant Km of PPy@MoS2@Ag for the substrate H2O2 is 0.03, and the maximum reaction rate Vm is 2.98; the Michaelis constant Km of PPy@MoS2@Pd for the substrate H2O2 is 0.17, and the maximum reaction rate Vm is 4.46.

[0100] Example 4: Protein sensor array constructed from three types of three-dimensional nanorods (PPy@MoS2@Au, PPy@MoS2@Ag, PPy@MoS2@Pd) to distinguish proteins, the steps are:

[0101] S1. Identify multiple target proteins; select 11 proteins: hemoglobin (Hem, hemoglobin), concanavalin A (ConA, concanavalin), Cytochrome C (CytC, cytochrome C), Chymotrypsinogen A (ChyA, chymosinogen A), bovine hemoglobin from blood (BHB, bovine hemoglobin), papain (Pap, papain), lysosome (Lys, lysosome), Streptavidin (SA, streptavidin), histone (His, histone), fibrinogen (Fib, fibrinogen), casein (Cas, casein).

[0102] S2. Establish a sensor array, which includes three nanorods (PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd) as three sensing units. Add 261 μL of 10 mM pH 4.0 citric acid-sodium citrate buffer, 6 μL (PPy@MoS2@Au, PPy@MoS2@Ag, PPy@MoS2@Pd), and 6 μL of protein solution (250 nM) to a 96-well plate, mix thoroughly, and incubate at room temperature for 1 h.

[0103] S3. Add 7.5 μL of H2O2 (final concentration 10 mmol / L) and 15 μL of TMB (final concentration 1 mmol / L) to the S2 mixture, and make up the volume to 300 μL with buffer. Incubate at 37°C for 10 min, and measure the absorbance at 652 nm using a microplate reader to obtain the test data.

[0104] S4. Process the detection data using the PCA algorithm and the HCA algorithm to obtain PCA maps and HCA maps of the single target protein solution and the mixed protein solution, respectively.

[0105] Test results such as Figure 4 As shown, the protein interacts with three nanozymes (PPy@MoS2@Au, PPy@MoS2@Ag, PPy@MoS2@Pd) through electrostatic and hydrophilic interactions, resulting in different changes in the activity of the nanozymes after each protein binds to different nanozymes.

[0106] The detection data were processed using the PCA algorithm and the HCA algorithm to obtain PCA maps and HCA maps of the single target protein solution and the mixed protein solution, respectively.

[0107] Test results such as Figure 5 、 Figure 6 、 Figure 7 and Figure 8 As shown in the PCA map, the coverage area of each protein's 95% confidence ellipse shows that the protein sensor array can distinguish 11 single protein arrays. In the HCA map, the levels of the phylogenetic tree show that each protein belongs to a different level and has different similarities, further demonstrating that the sensor array can distinguish 11 single protein arrays.

[0108] Test results such as Figure 9 and 10 As shown in FIG, the coverage area of the 95% confidence ellipse of different concentrations of CytC protein in the PCA map shows that the protein sensor array can distinguish single proteins of different concentrations, and its detection limit can reach 17 nM.

[0109] Test results such as Figure 11 As shown in FIG, the coverage area of the 95% confidence ellipse of proteins with different mixing ratios in the PCA map shows that the protein sensor array can distinguish mixed proteins with different mixing ratios.

[0110] Test results such as Figure 12 As shown in FIG, the PCA map shows that the protein sensing array can distinguish five single proteins in serum through the coverage area of the 95% confidence ellipse of the detected proteins in serum.

[0111] The present invention is not limited to the above-mentioned embodiments. Any improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A protein detection sensor array based on nanozymes, characterized by: The system includes three sensing units, each containing a nanozyme: PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd. The three nanozymes have different enzymatic activity changes in response to different proteins. The content of PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd in the detection system is 5-100 μg / mL; The pH value corresponding to the maximum absorbance of the PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd is pH 4.

0.

2. The nanozyme-based protein detection sensor array according to claim 1, characterized in that: The content of the nanozyme is 20 μg / mL.

3. The nanozyme-based protein detection sensor array according to claim 1, characterized in that: The three nanozymes PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd have obvious absorption values at a wavelength of 652nm.

4. A protein detection method based on a nanozyme sensor array, characterized in that: The following steps are involved: S1: Identify the target protein; S2: Constructing a sensor array, wherein the sensor array includes three sensing units corresponding to three nanozymes: PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd. The three nanozymes have different enzymatic activity changes in response to the target protein; S3: preparing solutions P1-Pn of a single target protein, and optionally, preparing mixed solutions S1-Sn of all target proteins, wherein the highest target protein concentration in each mixed solution is the same as the concentration of the single protein; S4: fully combining the sensor array with the solutions P1-Pn and S1-Sn, adding H2O2 and TMB, performing UV detection, and obtaining detection data; S5: Processing the detection data using a PCA algorithm and an HCA algorithm to obtain PCA maps and HCA maps of a single target protein solution and a mixed protein solution, respectively; S6: Add the analyte to be tested to the sensor array, and identify the protein according to the position of the analyte in the spectrum; the detection method is: The mixture of nanozymes containing protein was evenly mixed with H2O2 and TMB, and reacted at pH 4.0 and room temperature for 10 minutes to obtain the detection data. The protein distribution range of the atlas was compared to determine the protein to which it belonged.

5. The protein detection method based on nanozyme sensor array according to claim 4, characterized in that: The preparation method of the three nanozymes described in step S2 is as follows: S11: ultrasonically dissolving ammonium molybdate and nitric acid, wherein the concentration of ammonium molybdate in the reaction system is 0.18-0.25 mol / L and the concentration of nitric acid is 1.5-2 mol / L; the mixture is hydrothermally reacted at 175-185°C for 18-24 hours; after the reaction, the product MoO3 is washed and dried; S12: MoO3 is dispersed in a mixture of ethanol and water and uniformly dispersed by ultrasonication; pyrrole is added at a ratio of 0.8-1.2 mL / g of pyrrole to MoO3, and after stirring, a 0.25-0.4 mol / L (NH4)2S2O8 solution is slowly added, with a molar ratio of MoO3 to (NH4)2S2O8 of 1:2-3; the reaction is stirred at room temperature for 3-6 h. After the reaction is completed, the MoO3@PPy is filtered, washed, and dried to obtain; S13: MoO3@PPy was added to the thiourea CN2H4S solution and ultrasonically dispersed. The product PPy@MoS2 was then hydrothermally reacted at 180-210°C for 18-28 hours. After the reaction, the product was washed and dried to obtain the product PPy@MoS2. The ratio of thiourea to MoO3@PPy was 0.08-0.1 mol / g, and the concentration of thiourea in the reaction system was 0.1-0.2 mol / L. S14: PPy@MoS2 was uniformly dispersed in water by ultrasonication; HAuCl4, AgNO3, or PdCl2 were added at a concentration of 0.4-0.5 mg / mL; the ratio of HAuCl4, AgNO3, or PdCl2 to PPy@MoS2 was 0.005-0.015 mol / g; the reaction was ultrasonically reacted for 0.5-2 h. After the reaction, the products were washed several times by centrifugation with ethanol and dried to obtain the products PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd.

6. The protein detection method based on nanozyme sensor array according to claim 4, characterized in that: The detection steps in step S4 are as follows: S41: pH 4.0 citric acid-sodium citrate buffer was mixed with PPy@MoS2@Au, PPy@MoS2@Ag, and PPy@MoS2@Pd, and the protein solution was added, mixed thoroughly, and incubated at room temperature; S42: H2O2 and TMB were added to the mixture and reacted at 37°C for 10 min; the H2O2 concentration in the mixture was 0.2-15 mmol / L, and the TMB concentration was 0.5-1.5 mmol / L; S43: Measure the absorbance value at a wavelength of 652 nm using an enzyme marker to obtain detection data.

7. The protein detection method based on nanozyme sensor array according to claim 4, characterized in that: In step S5, the PCA map is a PCA-2D map and / or a PCA-3D map; the HCA map is an HCA-2D map and / or an HCA-3D map; and the protein distribution range is determined based on a 95% confidence ellipse in the map.

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