A colorimetric system based on precious metals and their alloys and its application

Abstract

The present invention discloses a colorimetric system based on precious metals and their alloys, and application of the system in blood glucose detection. Compared with biochemical, electrochemical and currently available colorimetric methods, the colorimetric system based on precious metals and their alloys described in the present invention has the advantages of low cost, fast reaction speed, simple components, low sample consumption, high sensitivity, good selectivity, convenience and speed, and can realize accurate detection of glucose in complex actual body fluid samples, and has excellent clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to a colorimetric system based on precious metals and their alloys, and application of the system in blood sugar detection. Background Art

[0002] Current blood sugar detection technologies mainly include biochemical, electrochemical and colorimetric methods. For biochemical methods, there are relatively mature fully automatic biochemical analyzers on the market. Due to its fast measurement speed, high accuracy and small reagent consumption, it has been widely used in inspection agencies at all levels, but it is more expensive than electrochemical and colorimetric methods. Electrochemical methods are fast, sensitive, accurate, simple in equipment, low in price and can be automated, but the disadvantage is that most electrochemical methods have poor selectivity and are usually used in combination with other analytical methods such as high performance liquid chromatography and capillary electrophoresis in practical applications.

[0003] Colorimetry is a method for determining the content of a component by comparing and measuring the color depth of a colored substance solution. Compared with traditional fluorescence, electrochemical and electro-biosensors, colorimetric sensors have the advantages of being observable by the naked eye, low cost, fast reaction speed, simple components, high sensitivity, good selectivity, and convenience. However, the existing glucose colorimetric systems are mostly low in sensitivity or cannot be detected in complex samples (such as serum, plasma, etc.).

[0004] With the emergence and development of nanotechnology, a series of nanoparticles have been found to have enzymatic activity. Compared with traditional biological enzymes with high preparation costs, complex separation and purification processes, strict storage conditions, and short storage time, nanozymes with high stability, low cost, long storage time, and simple preparation process have been widely used in various fields due to their unique advantages. Among them, the colorimetric platform of peroxidase nanozymes replacing the traditional biological enzyme HRP has been widely studied. Glucose is a risk marker for many diseases, and the construction of a rapid and convenient quantitative analysis platform is of great significance in disease screening, and the colorimetric platform based on nanozymes provides an excellent idea for the construction of a glucose quantitative analysis platform. At present, the principle of building the relevant platform is mainly that glucose generates gluconic acid and hydrogen peroxide under the action of glucose oxidase, and hydrogen peroxide generates hydroxyl radicals under the action of peroxidase nanozymes, and hydroxyl radicals oxidize the color developer to cause color changes. In addition, the significant color changes of the colorimetric method can be converted into RGB pixel values ​​by collecting digital images, and with the emergence of various small and easy-to-carry detection and intelligent analysis equipment, the colorimetric method has shown great potential for point-of-care testing. Summary of the invention

[0005] In order to solve the problem of low sensitivity of the existing glucose colorimetric system, the inventors have designed a colorimetric system based on precious metals and their alloys. The system is constructed using the peroxidase properties of precious metals and their alloys, and can achieve accurate measurement of human blood glucose content, thereby completing the present invention.

[0006] In one aspect, the present invention provides a colorimetric system based on precious metals and their alloys, which comprises precious metal and their alloy nanoparticles with plasmon effect as peroxidase nanozymes, glucose oxidase, NaAc-HAc buffer, and TMB color developer.

[0007] In a preferred embodiment, the precious metal and alloy nanoparticles having plasmon effect are selected from the group consisting of: single metal nanoparticles Pd, Pt, Au, Ag, Cu; bimetallic alloy nanoparticles PdPt, PtAu, PtAg, PtCu, PdAu, PdAg, PdCu; and trimetallic alloy nanoparticles PdPtAu, PdPtAg, PdPtCu.

[0008] Due to the synergistic effect and electronic effect, multi-metal nanostructures exhibit better catalytic performance, and Au>Ag and Cu, bimetallic alloy nanoparticles are preferred, and trimetallic alloy nanoparticles are more preferred.

[0009] In terms of stability, Ag is easily oxidized and causes partial surface passivation, Cu has high reactivity and is easily oxidized. In addition, considering the shorter time consumption and storage stability when used as peroxidase nanozymes, PdPtAu tri-metal alloy nanoparticles are most preferred, as they have the strongest enzyme activity and are the most stable.

[0010] In a preferred embodiment, the noble metal and alloy nanoparticles having plasmon effect are prepared by the following method:

[0011] Step 1, uniformly mixing a first metal precursor, HCl and Pluronic F127 to obtain a mixed solution, wherein the first metal precursor is selected from a Na2PdCl4 aqueous solution and a H2PtCl6·6H2O aqueous solution;

[0012] Step 2, adding ascorbic acid aqueous solution to the above mixed solution, and continuously ultrasonically treating in a water bath for 3-5 hours;

[0013] Step 3, adding a second metal precursor to the above micelle solution, and continuing water bath ultrasound for 1 hour, wherein the second metal precursor is selected from HAuCl4·4H2O, AgNO3 aqueous solution, CuCl2 aqueous solution, and Cu(NO3)2 aqueous solution;

[0014] Step 4: After standing at room temperature for 30 minutes, the mixture was washed with ultrapure water and ethanol three times respectively to remove the Pluronic F127 template, and then the product was collected by centrifugation at 10,000 rpm for 10 minutes. The collected particles were dried in an oven to obtain the corresponding noble metal or alloy nanoparticles.

[0015] In a preferred embodiment, in step 1, the first metal precursor, water, HCl and Pluronic F127 are ultrasonically treated at 40 kHz for 10 minutes to be uniformly mixed.

[0016] In a preferred embodiment, the pH of the NaAc-HAc buffer is 2.0-5.0, such as 2.0, 2.5, 3.0, 3.6, 4.0, 4.5, 5.0, preferably pH 3.

[0017] In another aspect, the present invention provides a method for detecting blood sugar using the above-mentioned colorimetric system based on precious metals and their alloys, the method comprising:

[0018] A. The detection of standard glucose solution comprises the following steps:

[0019] Glucose solutions of different concentrations (0-1.11×10 -2 M) incubated with glucose oxidase at 37°C for 1 h;

[0020] NaAc-HAc buffer and TMB solution were added to the mixed solution, and then the peroxidase nanozyme suspension (1 mg / mL,) obtained by dispersing precious metal and alloy nanoparticles in deionized water was added and mixed, and the homogenized solution was incubated for 15 min;

[0021] After the incubation, the absorbance of the mixed solutions with different glucose concentrations at 652 nm was measured with an enzyme-labeled instrument; or a mobile phone camera was used to take photos of the mixed solutions and extract the RGB pixel values ​​of the mixed solutions with different glucose concentrations;

[0022] Linearly fit the glucose concentration with the absorbance or RGB pixel value of the corresponding mixed solution to obtain a standard curve;

[0023] B. Detection of glucose in a sample, comprising the following steps;

[0024] The diluted sample was incubated with glucose oxidase at 37°C for 1 h to obtain a culture solution;

[0025] NaAc-HAc buffer and TMB solution are added to the culture solution, and then added to the peroxidase nanozyme suspension obtained by dispersing precious metal and alloy nanoparticles in deionized water, and mixed evenly, and the homogenized solution is incubated at 20°C-50°C for 15 minutes to obtain a mixed solution;

[0026] After the incubation, the absorbance of the mixture at 652 nm was measured using an ELISA reader; or a mobile phone camera was used to take a photo of the mixture and extract the RGB pixel values;

[0027] Compare the absorbance and RGB pixel values ​​obtained in the previous step with the standard curve to obtain the measured concentration value.

[0028] In a preferred embodiment, the pH of the NaAc-HAc buffer is 2.0-5.0, such as 2.0, 2.5, 3.0, 3.6, 4.0, 4.5, 5.0, preferably 3.0.

[0029] In a preferred embodiment, the precious metal and alloy nanoparticles are selected from the group consisting of Pd, Pt, Au, Ag, Cu, PdPt, PtAu, PtAg, PtCu, PdAu, PdAg, PdCu, PdPtAu, PdPtAg, and PdPtCu nanoparticles.

[0030] In a preferred embodiment, the homogenous solution is incubated at 50°C.

[0031] In a preferred embodiment, the sample is a human serum sample.

[0032] Beneficial Effects

[0033] Compared with biochemical, electrochemical and currently available colorimetric methods, the colorimetric system based on precious metals and their alloys described in the present invention has the advantages of low cost, fast reaction speed, simple components, low sample consumption, high sensitivity, good selectivity, convenience and speed, and can achieve accurate detection of glucose in complex actual body fluid samples. The system is based on the glucose oxidase-precious metal and its alloy peroxidase nanozyme-TMB reaction system, with a material cost as low as 0.3 yuan / sample; good selectivity for glucose; good detection performance in complex samples (blood samples) and high sensitivity. The existing patent CN105572065A technical solution requires 40μL human serum sample for measurement; Patent CN10 The 3760161B technical solution requires 30 μL human serum sample for measurement; the patent CN111239125A technical solution requires 50 μL human serum sample for measurement; the patent CN104374769B technical solution requires 20 mL human serum sample for measurement; the patent CN105424690A technical solution requires 10 μL human serum sample for measurement, while the colorimetric system of the present invention only consumes 4 μL blood sample to achieve accurate quantification of blood glucose; it can be screened in hospitals or laboratories through an ELISA instrument for high-throughput screening, and it also has the application potential of extracting and analyzing the RGB pixel value of the color development system through a mobile phone APP scan to enable patients to monitor their blood glucose by themselves. The colorimetric system is convenient, fast, and highly accurate, and has excellent clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a characterization picture of the PdPtAu nanoparticles prepared in Preparation Example 7 of the present invention. Figure 1 a is the SEM characterization picture, Figure 1 b is TEM characterization picture;

[0035] Figure 2 The standard curve is obtained by detecting the standard glucose solution using the colorimetric system containing PdPtAu nanoparticles when the microplate reader is used as the analysis platform in Experimental Example 2;

[0036] Figure 3 The standard curve obtained by detecting the standard glucose solution using the colorimetric system containing PdPtAu nanoparticles when the smartphone is used as the analysis platform in Experimental Example 3;

[0037] Figure 4 This is a graph showing the results of testing human serum samples in Experimental Example 4. Figure 4 a shows the blood glucose values ​​of 24 people measured by the colorimetric system containing PdPtAu nanoparticles. Figure 4 b shows the linear relationship between the quantitative results of the colorimetric system containing PdPtAu nanoparticles and the quantitative results of the commercially available D-glucose assay kit;

[0038] Figure 5 is the relative peroxidase activity of PdPtAu nanoparticles under different pH conditions of NaAc-HAc buffer in Experimental Example 5;

[0039] Figure 6 The relative peroxidase activity of PdPtAu nanoparticles was measured when the solution in Experimental Example 6 was incubated at different temperatures. DETAILED DESCRIPTION

[0040] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0041] Preparation Example 1: Preparation of Pd Nanoparticles

[0042] 0.6 mL of Na2PdCl4 aqueous solution (20 mM), 3.0 mL of H2O, 30 μL of HCl (6.0 M) and 60 mg of Pluronic F127 were ultrasonically mixed. Subsequently, 3.0 mL of 0.1 M ascorbic acid aqueous solution was added to the mixture, and the mixture was ultrasonically treated for 4 h in a 45 ° C water bath. After standing at room temperature for 30 min, the mixture was washed with ultrapure water and ethanol for 3 times to remove the Pluronic F127 template to obtain Pd nanoparticles. The product was collected by centrifugation at 10000 rpm for 10 min, and the collected particles were dried in an oven at 50 ° C.

[0043] Preparation Example 2: Preparation of Pt nanoparticles.

[0044] 0.6mL H2O, 3.0mL H2PtCl6·6H2O aqueous solution (20mM), 30μL HCl (6.0M) and 60mg Pluronic F127 were ultrasonically mixed. Subsequently, 3.0mL 0.1M ascorbic acid aqueous solution was added to the above mixture, and ultrasonication was continued for 4h in a 45℃ water bath. After standing at room temperature for 30min, the mixture was washed with ultrapure water and ethanol three times respectively to remove the Pluronic F127 template to obtain Pt nanoparticles. The product was collected by centrifugation at 10000rpm for 10min, and the collected particles were dried in an oven at 50℃.

[0045] Preparation Example 3: Preparation of Au, Ag, and Cu nanoparticles.

[0046] 3.6mL H2O, 30μL HCl (6.0M) and 60mg Pluronic F127 were ultrasonically mixed. Subsequently, 3.0mL 0.1M ascorbic acid aqueous solution and 1.2mL metal precursor (HAuCl4·4H2O, AgNO3, CuCl2, Cu(NO3)2) aqueous solution (30mM) were added to the above mixture, and ultrasonication was continued for 4h in a 45℃ water bath. After standing at room temperature for 30min, the mixture was washed with ultrapure water and ethanol three times to remove the Pluronic F127 template to obtain Au, Ag, and Cu nanoparticles, respectively. The product was collected by centrifugation at 10000rpm for 10min, and the collected particles were dried in an oven at 50℃.

[0047] Preparation Example 4: Preparation of PtAu, PtAg, PtCu bimetallic alloys.

[0048] 0.6mL H2O, 3.0mL H2PtCl6·6H2O aqueous solution (20mM), 30μL HCl (6.0M) and 60mg Pluronic F127 were ultrasonically mixed. Subsequently, 3.0mL 0.1M ascorbic acid aqueous solution was added to the above mixture, and ultrasonication was continued for 3h in a water bath at 45℃. After that, 1.2mL metal precursor (HAuCl4·4H2O, AgNO3, CuCl2, Cu(NO3)2) aqueous solution (30mM) was added to the above solution, and water bath ultrasonication was continued at 45℃ for 1h. After standing at room temperature for 30min, it was washed with ultrapure water and ethanol three times respectively to remove the Pluronic F127 template to obtain PtAu, PtAg, and PtCu bimetallic alloy nanoparticles respectively. The product was collected by centrifugation at 10000rpm for 10min, and the collected particles were dried in an oven at 50℃;

[0049] Preparation Example 5: Preparation of PdAu, PdAg, PdCu bimetallic alloys.

[0050] First, 0.6 mL of Na2PdCl4 aqueous solution (20 mM), 3.0 mL of H2O, 30 μL of HCl (6.0 M) and 60 mg of Pluronic F127 were ultrasonically mixed. Subsequently, 3.0 mL of 0.1 M ascorbic acid aqueous solution was added to the above mixture, and the mixture was ultrasonically treated for 3 hours in a water bath at 45 ° C. After that, 1.2 mL of metal precursor (HAuCl4·4H2O, AgNO3, CuCl2, Cu(NO3)2) aqueous solution (30 mM) was added to the above solution, and the water bath ultrasonication was continued at 45 ° C for 1 hour. After standing at room temperature for 30 minutes, the mixture was washed with water and ethanol three times to remove the Pluronic F127 template to obtain PdAu, PdAg, and PdCu bimetallic alloy nanoparticles. The product was collected by centrifugation at 10000 rpm for 10 minutes, and the collected particles were dried in an oven at 50 ° C.

[0051] Preparation Example 6: Preparation of PdPt bimetallic alloy.

[0052] First, 0.6 mL of Na2PdCl4 aqueous solution (20 mM), 3.0 mL of H2PtCl6·6H2O aqueous solution (20 mM), 30 μL of HCl (6.0 M) and 60 mg of Pluronic F127 were ultrasonically mixed. Subsequently, 3.0 mL of 0.1 M ascorbic acid aqueous solution was added to the above mixture, and the mixture was ultrasonically mixed for 3 hours in a 45°C water bath to obtain PdPt micelles. After standing at room temperature for 30 minutes, the mixture was washed with water and ethanol three times respectively to remove the Pluronic F127 template. The product was collected by centrifugation at 10,000 rpm for 10 minutes, and the collected particles were dried in an oven at 50°C.

[0053] Preparation Example 7: Preparation of PdPtAu, PdPtAg, and PdPtCu multi-element alloys.

[0054] 0.6 mL Na2PdCl4 aqueous solution (20 mM), 3.0 mL H2PtCl6·6H2O aqueous solution (20 mM), 30 μL HCl (6.0 M) and 60 mg Pluronic F127 were ultrasonically mixed. Subsequently, 3.0 mL 0.1 M ascorbic acid aqueous solution was added to the above mixture, and the mixture was ultrasonicated continuously for 3 h in a water bath at 45 ° C to obtain PdPt micelles. After that, 1.2 mL metal precursor (HAuCl4·4H2O, AgNO3, CuCl2, Cu(NO3)2) aqueous solution (30 mM) was added to the above PdPt micelle solution, and the water bath ultrasonication was continued at 45 ° C for 1 h. After standing at room temperature for 30 min, the mixture was washed with ultrapure water and ethanol for 3 times to remove the Pluronic F127 template to obtain PdPtAu, PdPtAg, and PdPtCu alloy nanoparticles, respectively. The product was collected by centrifugation at 10,000 rpm for 10 min, and the collected particles were dried in an oven at 50°C.

[0055] The precious metal and alloy nanoparticles prepared in Preparation Examples 1-7 all have plasmon effects.

[0056] The scanning electron microscopy results of the PdPtAu triple alloy nanoparticles prepared in Preparation Example 7 were obtained using Hitachi S-4800. Figure 1 As shown in a, it can be seen that the synthesized PdPtAu triple alloy nanoparticles are porous spherical with a particle size of about 203 nm and uniform particle size.

[0057] The transmission electron microscopy results of the PdPtAu triple alloy nanoparticles prepared in Preparation Example 7 were obtained using JEOL JEM-2100F. Figure 1As shown in b, it also shows that the prepared PdPtAu triple alloy nanoparticles are uniform porous spherical, which is consistent with the scanning electron microscopy results.

[0058] Examples 1-15

[0059] The noble metal and alloy nanoparticles obtained in Preparation Examples 1-7 were used as peroxidase nanozymes, respectively, to form a colorimetric system together with glucose oxidase, NaAc-HAc buffer and TMB color developer.

[0060] When the colorimetric system was used to test the glucose content of the sample, it was found that the enzymatic catalytic activity of the bimetallic alloy nanoparticles was better than that of the single metal nanoparticles, and the catalytic activity of the trimetallic alloy nanoparticles was better than that of the bimetallic alloy nanoparticles.

[0061] In terms of stability, tri-metal alloy nanoparticles have the best stability and can maintain stable nanozyme catalytic activity during long-term storage.

[0062] Overall, PdPtAu triple alloy nanoparticles have the best enzyme activity and stability.

[0063] Experimental Example 1: Detection of Glucose Content in Samples

[0064] A. The detection of standard glucose solution comprises the following steps:

[0065] Glucose solutions of different concentrations (0-1.11×10 -2 M, 20 μL) and glucose oxidase (GOx, 1 mg / mL, 100 μL) were incubated at 37 °C for 1 h;

[0066] 400 μL of NaAc-HAc buffer (0.2 M, pH 3.0) and 100 μL of TMB solution (10 mg / mL, solvent: DMSO) were added to the mixed solution of step 3.1, and then 5 μL of peroxidase nanozyme suspension (1 mg / mL, obtained by dispersing Pd, Pt, Au, Ag, Cu, PdPt, PtAu, PtAg, PtCu, PdAu, PdAg, PdCu, PdPtAu, PdPtAg, PdPtCu nanoparticles in deionized water) was added and mixed, and the homogenous solution was incubated at 50 °C for 15 min;

[0067] After the incubation, the absorbance of the mixed solutions with different glucose concentrations at 652 nm was measured with an enzyme-labeled instrument; or a mobile phone camera was used to take photos of the mixed solutions and extract the RGB pixel values ​​of the mixed solutions with different glucose concentrations;

[0068] Linear fitting is performed between the glucose concentration and the absorbance or RGB pixel value of the corresponding mixed solution;

[0069] B. Detection of glucose in a sample, comprising the following steps;

[0070] The samples were diluted to within the linear range of the glucose colorimetric platform;

[0071] First, the diluted sample was incubated with glucose oxidase (GOx, 1 mg / mL, 100 μL) at 37 °C for 1 h. Subsequently, 400 μL NaAc-HAc buffer (0.2 M, pH 3.0) and 100 μL TMB solution (10 mg / mL, solvent is DMSO) were added to the culture medium, and then 5 μL peroxidase nanozyme suspension (1 mg / mL, Pd, Pt, Au, Ag, Cu, PdPt, PtAu, PtAg, PtCu, PdAu, PdAg, PdCu, PdPtAu, PdPtAg, PdPtCu nanoparticles dispersed in deionized water) was added and mixed, and the homogenized solution was incubated at 50 °C for 15 min. After the incubation, the absorbance of the mixed solution at 652 nm was measured by an ELISA instrument and compared with the standard curve to obtain the glucose concentration value.

[0072] Experimental Example 2: Detection of standard glucose solution based on colorimetric platform of ELISA

[0073] 20 μL of glucose solution of different concentrations (0 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 1 mg / mL) was incubated with 100 μL of glucose oxidase (GOx, 1 mg / mL) at 37 °C for 1 h;

[0074] 400 μL NaAc-HAc buffer (0.2 M, pH 3.0) and 100 μL TMB solution (10 mg / mL, solvent: DMSO) were added to the above mixed solution, and then 5 μL PdPtAu suspension (1 mg / mL) was added and mixed, and the homogenized solution was incubated at 50 °C for 15 min;

[0075] After the incubation, the absorbance of the mixed solutions with different glucose concentrations at 652 nm was measured using an enzyme-labeled instrument;

[0076] The glucose concentration was linearly fitted with the absorbance of the corresponding mixed solution, such as Figure 2 As shown. Detection of standard glucose solution by the colorimetric system containing PdPtAu nanoparticles (linear range 0.1-1.0 mg / mL, R2 = 0.992)

[0077] Experimental Example 3: Detection of standard glucose solution based on colorimetric platform for mobile phone recognition

[0078] 20 μL of glucose solution of different concentrations (0 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 1 mg / mL) was incubated with 100 μL of glucose oxidase (GOx, 1 mg / mL) at 37 °C for 1 h;

[0079] 400 μL NaAc-HAc buffer (0.2 M, pH 3.0) and 100 μL TMB solution (10 mg / mL, solvent: DMSO) were added to the above mixed solution, and then 5 μL PdPtAu suspension (1 mg / mL) was added and mixed, and the homogenized solution was incubated at 50 °C for 15 min;

[0080] After the incubation, the mixed solution was photographed with a mobile phone camera, and the RGB pixel values ​​of the mixed solutions with different glucose concentrations were extracted;

[0081] The glucose concentration is linearly fitted with the RGB pixel value of the corresponding mixed solution, such as Figure 3 Detection of standard glucose solution by the colorimetric system containing PdPtAu nanoparticles (Luminance = 26.50145 + 0.0041R - 0.08092G - 0.05677B, linear range 0.05-2mg / mL, R2 = 0.990)

[0082] Experimental Example 4: Detection of human blood sugar

[0083] Blood glucose levels were measured in serum samples of 12 healthy controls and 12 patients with pancreatitis and pancreatic cancer with abnormal blood glucose. The volume of each sample was 4 μL.

[0084] The original human serum sample was diluted 5 times;

[0085] 20 μL of diluted serum sample was incubated with 100 μL of glucose oxidase (GOx, 1 mg / mL) at 37°C for 1 h. At the same time, different concentrations of glucose standard solutions (0 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 1 mg / mL) were also treated under the same conditions as standard controls;

[0086] 400 μL NaAc-HAc buffer (0.2 M, pH 3.0) and 100 μL TMB solution (10 mg / mL, solvent: DMSO) were added to the above mixed solution, and then 5 μL PdPtAu suspension (1 mg / mL) was added and mixed, and the homogenized solution was incubated at 50 °C for 15 min;

[0087] After the incubation, the absorbance of the mixture at 652 nm was measured using an enzyme-labeled instrument;

[0088] The data is processed to obtain the human serum blood glucose value, such as Figure 4 As shown in a.

[0089] The samples were measured using a commercially available D-glucose assay kit, and the results were linearly correlated with the quantitative results of the colorimetric system containing PdPtAu nanoparticles. Figure 4 As shown in b, the two are linearly correlated (R 2 =0.980). The specific blood glucose values ​​of human serum measured by the present invention and the commercially available D-glucose determination kit are shown in Table 1.

[0090] Table 1

[0091]

[0092] Experimental Example 5: pH optimization experiment (testing the peroxidase activity of PdPtAu nanoparticles at different pH)

[0093] Different amounts of PdPtAu multi-alloy nanoparticles (0.4, 0.3, 0.25, 0.2 and 0.1 μg) were mixed with 1 ml of pH buffer (glycine-hydrochloric acid buffer (0.05 M) with pH values ​​of 2.0 and 2.5, and NaAc-HAc buffer (0.2 M) with pH values ​​of 3, 3.6, 4, 4.5, 5 and 5.5), 50 μL of TMB solution (10 mg / mL, solvent: DMSO) and 117.98 μL of H2O2 aqueous solution (to make the final concentration 1 M), and the absorbance change at 652 nm was immediately measured every 10 seconds for a total of 6 minutes. At the same time, a reaction system without adding H2O2 aqueous solution (117.98 μL of water) was prepared as a control. The formula for calculating the specific activity of peroxidase is as follows:

[0094] b nanozyme =V / (ε×l)×(△A / △t)

[0095] SA=b nanozyme / m

[0096] Where bnanozyme is the catalytic activity of the nanozyme; V is the total volume of the reaction solution; ε is the molar absorption coefficient of TMB at 652 nm, i.e., 39000 M-1 cm -1 ; l is the path length of light propagation in the test tube; △A / △t is the initial change rate of absorbance per min; SA is the activity unit per mg of nanozyme; m represents the weight of nanozyme;

[0097] The data were processed to obtain the peroxidase specific activity of PdPtAu multi-element alloy at different pH values. Figure 5According to the results, the peroxidase specific activity of PdPtAu multi-element alloy reaches the highest value and remains stable when the pH is 3.0-4.0. Based on the fact that glucose oxidase is dissolved in PBS buffer with a pH of 7.4 in the colorimetric system, it will affect the pH of the mixed system. Therefore, pH 3.0 is the best choice.

[0098] Experimental Example 6: Temperature Optimization Experiment

[0099] 1mL NaAc-HAc buffer (0.2M, pH 3.6), 0.01μg PdPtAu alloy and 50μL TMB solution (10mg / mL, solvent is DMSO) were mixed in a tube. Then 117.98μL H2O2 aqueous solution was added to the above solution (to make the final concentration 1M), and at the same time, the same volume of water was added to another solution as background. Incubate at different temperatures (20℃, 25℃, 30℃, 37℃, 40℃, 45℃, 50℃, 55℃, 60℃) for 15min. Then detect the absorbance of the solution at 652nm.

[0100] The experimental results are as follows Figure 6 It can be seen that when other conditions are the same, the absorbance of the mixed system at 652nm is the highest after incubation at 50℃, indicating that the optimal incubation temperature is 50℃.

Claims

1. A colorimetric system based on precious metals and their alloys, the colorimetric system comprising precious metal and their alloy nanoparticles with plasmon effect as peroxidase nanozyme, glucose oxidase, NaAc-HAc buffer, TMB color developer, the precious metal and their alloy nanoparticles with plasmon effect are prepared by the following method: Step 1, uniformly mixing a first metal precursor, HCl and Pluronic F127 to obtain a mixed solution, wherein the first metal precursor is selected from a Na2PdCl4 aqueous solution and a H2PtCl6·6H2O aqueous solution; Step 2, adding ascorbic acid aqueous solution to the above mixed solution, and continuously ultrasonically treating in a water bath for 3-5 hours; Step 3, adding a second metal precursor to the solution obtained in step 2, and continuing water bath ultrasound for 1 hour, wherein the second metal precursor is selected from HAuCl4·4H2O, AgNO3 aqueous solution, CuCl2 aqueous solution, and Cu(NO3)2 aqueous solution; Step 4: After standing at room temperature for 30 minutes, the mixture was washed with ultrapure water and ethanol three times respectively to remove the Pluronic F127 template, and then the product was collected by centrifugation at 10,000 rpm for 10 minutes. The collected particles were dried in an oven to obtain the corresponding noble metal or alloy nanoparticles.

2. The colorimetric system according to claim 1, wherein: The precious metal and alloy nanoparticles having plasmon effect are selected from the group consisting of: single metal nanoparticles Pd, Pt, Au, Ag, Cu; bimetallic alloy nanoparticles PdPt, PtAu, PtAg, PtCu, PdAu, PdAg, PdCu; and trimetallic alloy nanoparticles PdPtAu, PdPtAg, PdPtCu.

3. The colorimetric system according to claim 2, wherein: The precious metal and alloy nanoparticles having plasmon effect are bimetallic alloy nanoparticles PdPt, PtAu, PtAg, PtCu, PdAu, PdAg, PdCu; or trimetallic alloy nanoparticles PdPtAu, PdPtAg, PdPtCu.

4. The colorimetric system according to claim 2, wherein: The noble metal and alloy nanoparticles having plasmon effect are PdPtAu nanoparticles.

5. The colorimetric system according to claim 1, wherein: In step 1, the first metal precursor, water, HCl and Pluronic F127 were ultrasonically treated at 40 kHz for 10 minutes to be mixed uniformly.

6. The colorimetric system according to claim 1, wherein: The pH of the NaAc-HAc buffer is 2.0 to 5.0, including 2.0, 2.5, 3.0, 3.6, 4.0, 4.5, and 5.

0.

7. The colorimetric system according to claim 6, wherein: The pH of the NaAc-HAc buffer was 3.

8. A method for detecting blood sugar using a colorimetric system based on precious metals and their alloys as claimed in any one of claims 1 to 7, the method comprising: A. Detection of standard glucose solution, The steps include: 0-1.11×10 -2 M Glucose solutions with different concentrations were incubated with glucose oxidase at 37°C for 1 h; NaAc-HAc buffer and TMB solution were added to the mixed solution, and then 1 mg / mL peroxidase nanozyme suspension obtained by dispersing precious metal and alloy nanoparticles in deionized water was added and mixed, and the homogenous solution was incubated for 15 min; After the incubation, the absorbance of the mixed solutions with different glucose concentrations at 652 nm was measured with an enzyme-labeled instrument; or a mobile phone camera was used to take photos of the mixed solutions and extract the RGB pixel values ​​of the mixed solutions with different glucose concentrations; Linearly fit the glucose concentration with the absorbance or RGB pixel value of the corresponding mixed solution to obtain a standard curve; B. Detection of glucose in a sample, comprising the following steps; The diluted sample was incubated with glucose oxidase at 37°C for 1 h to obtain a culture solution; NaAc-HAc buffer and TMB solution are added to the culture solution, and then added to the peroxidase nanozyme suspension obtained by dispersing precious metal and alloy nanoparticles in deionized water, and mixed evenly, and the homogenized solution is incubated at 20°C-50°C for 15 minutes to obtain a mixed solution; After the incubation, the absorbance of the mixture at 652 nm was measured using an ELISA reader; or a mobile phone camera was used to take a photo of the mixture and extract the RGB pixel values; Compare the absorbance and RGB pixel values ​​obtained in the previous step with the standard curve to obtain the measured concentration value.

9. The method according to claim 8, wherein: The homogenized solution was incubated at 50 °C.

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