Analytical reagent for quantitative analysis of hydrogen peroxide and preparation and use methods thereof

Abstract

The invention provides an analysis reagent for quantitative analysis of hydrogen peroxide and preparation and use methods thereof, and the analysis reagent for quantitative analysis of hydrogen peroxide comprises a sludge protein-based carbon nanodot dispersion liquid, a BR buffer solution and a TMB solution, the sludge protein-based carbon nanodot dispersion liquid is prepared by the following steps: performing hydrothermal reaction on sludge-based protein extracted from activated sludge at 160-200 DEG C for at least 4 hours, centrifuging, and taking a product of which the molecular weight is not more than 500 Da from supernate. The analysis reagent provided by the invention takes activated sludge as a main raw material, can effectively promote hydrogen peroxide to generate hydroxyl radicals with high oxidation activity, further oxidizes TMB (Tetramethylbenzidine) to generate a blue product, and can directly, accurately and quantitatively analyze the concentration of hydrogen peroxide by utilizing the color change. Meanwhile, the invention also provides a feasible way for high-value utilization of the sludge protein.

Description

Technical Field

[0001] The invention belongs to the fields of analytical chemistry and environmental protection, and particularly relates to an analytical reagent for quantitatively analyzing hydrogen peroxide and a preparation and use method thereof. Background Art

[0002] Hydrogen peroxide (H2O2) is a common strong oxidant widely used in the pharmaceutical, chemical, and food industries. However, high concentrations of hydrogen peroxide are highly corrosive and irritating, easily damaging the skin, eyes, and respiratory tract. In severe cases, they can cause burns or poisoning. In industrial production or laboratory operations, exposure to heat or friction can also cause explosions. Furthermore, in areas such as food, the environment, and pharmaceuticals, excessive hydrogen peroxide residues can pose potential hazards to human health and even cause poisoning. Therefore, accurate detection of hydrogen peroxide levels is crucial for ensuring safe production, product quality, and human health. Establishing highly sensitive and selective detection methods is a key measure for preventing accidents and regulating their use.

[0003] Common methods for detecting H2O2 include electrochemistry, chemiluminescence, chromatography, fluorescence, titration, and colorimetry. Colorimetry allows for direct visual quantification of target analytes and offers advantages such as simplicity, rapidity, reliability, and low cost. In recent decades, nanomaterial-based colorimetry has garnered significant attention due to its high sensitivity and specificity. For example, methods using noble metals, transition metal oxides / sulfides, metal-organic frameworks, carbon nanodots (CNDs), and other nanomaterials as colorimetric probes have been developed for the detection of H2O2. Among these, CNDs have attracted increasing attention in the field of visual analysis due to their small size, high surface area, high enzymatic activity, low toxicity, and abundance of raw materials and ease of preparation.

[0004] Activated sludge is one of the main by-products produced during municipal wastewater treatment. Sludge is typically composed of dissolved organic matter such as nucleic acids, humic acid, protein, and polysaccharides. The organisms in sludge contain a large amount of protein, accounting for approximately 50% of the dry weight of bacterial cells. Protein recovered from sludge can be used as fertilizer, adhesives, or animal feed, and sludge protein is considered a new renewable resource for energy and resource recovery. As the production of activated sludge continues to increase, finding new ways to increase the value of sludge protein remains one of the most pressing issues in the environmental protection field. Summary of the Invention

[0005] To address the above issues, the present invention provides an analytical reagent for quantitative analysis of hydrogen peroxide. This analytical reagent can accurately and quantitatively analyze the concentration of hydrogen peroxide and achieve high-value utilization of sludge protein. The present invention also provides a preparation method and use method of the analytical reagent.

[0006] To achieve the above-mentioned objectives, the first aspect of the present invention provides an analytical reagent for quantitative analysis of hydrogen peroxide, comprising a sludge protein-based carbon nanodot dispersion, a Burritan-Robinson (BR) buffer, and a 3,3',5,5'-tetramethylaniline (TMB) solution, wherein the sludge protein-based carbon nanodot dispersion is extracted from activated sludge, the sludge-based protein is first incubated at 160-200°C for at least 4 hours, and then centrifuged to obtain a product with a molecular weight not exceeding 500 Da in the supernatant.

[0007] This method uses protein extracted from activated sludge as a carbon source and produces carbon nanodots through a simple hydrothermal method. These carbon nanodots exhibit peroxidase-like activity, effectively promoting the production of highly active hydroxyl radicals from hydrogen peroxide. This hydroxyl radical then oxidizes 3,3',5,5'-tetramethylaniline to produce a blue product. This color change can be used to directly quantify the concentration of hydrogen peroxide.

[0008] Further experiments revealed that rationally controlling the ratios of the three components in the analytical reagent can improve the accuracy of hydrogen peroxide detection. Specifically, the volume ratio of the sludge protein-based carbon nanodot dispersion, BR buffer, and TMB solution is 0.4-0.6:1.0-4.0:5-10. The concentration of the sludge protein-based carbon nanodot dispersion is 0.3-1.8 mg / mL, the pH of the BR buffer is 3-6, and the concentration of the TMB solution is 0.015-0.15 mM. In one embodiment, the volume ratio of the sludge protein-based carbon nanodot dispersion, BR buffer, and TMB solution is 0.5:3:6; in another embodiment, the volume ratio of the sludge protein-based carbon nanodot dispersion, BR buffer, and TMB solution is 0.5:1:6.

[0009] Britton-Robinson buffer is a mixture of phosphoric acid, boric acid, and acetic acid. Adding varying amounts of sodium hydroxide to the mixture creates a buffer solution with a wide pH range. Experimental results indicate that the optimal pH value for preparing analytical reagents is between 3 and 4, with pH values ​​of 3, 3.3, 3.6, and 4.0, for improved detection accuracy.

[0010] The concentration of the TMB solution used to prepare the analytical reagent also affects the detection accuracy of hydrogen peroxide. Specifically, as the TMB concentration increases, the detection accuracy first increases and then slightly decreases. Relatively speaking, when the concentration of the TMB solution is in the range of 0.05-0.15 mM, the detection accuracy is higher, and the optimal TMB concentration is 0.1 mM.

[0011] The concentration of the sludge protein-based carbon nanodot dispersion used in the preparation of analytical reagents will also affect the detection accuracy of hydrogen peroxide. Specifically, as the concentration of the sludge protein-based carbon nanodot dispersion used in the preparation of analytical reagents becomes lower, the A 650 The smaller the concentration, the best concentration of sludge protein-based carbon nanodots dispersion is 0.3 mg / mL.

[0012] A second aspect of the present invention provides a method for preparing an analytical reagent for quantitative analysis of hydrogen peroxide, comprising the following steps:

[0013] Sludge-based protein is extracted from activated sludge, and the sludge-based protein is hydrothermally reacted at 160-200°C for at least 4 hours. After cooling, the protein is centrifuged, and the supernatant is collected and purified using a 500 Da dialysis bag to obtain a sludge protein-based carbon nanodot dispersion. The sludge protein-based carbon nanodot dispersion is mixed with BR buffer and TMB solution to obtain an analytical reagent.

[0014] In the above-mentioned analytical reagent, the volume ratio of the sludge protein-based carbon nanodot dispersion, BR buffer and ABTS solution is 0.4-0.6:2.5-4.0:5-10; wherein, the concentration of the sludge protein-based carbon nanodot dispersion is 0.3-1.8 mg / mL, preferably 0.3-1.0 mg / mL; the pH value of the BR buffer is 2-6, preferably 3-4; the concentration of the TMB solution is 0.015-0.15 mM, preferably 0.05-0.15 mM.

[0015] Extracting sludge-based protein from activated sludge may specifically include adjusting the pH of the activated sludge to above 11, followed by ultrasonic cell disruption, and then centrifuging to obtain the supernatant. In a specific implementation, an alkaline substance such as NaOH is added to activated sludge with a moisture content of 90% to adjust the pH to 12. The sludge is then placed in an ultrasonic cell disruptor and ultrasonicated at 600 W for 20 minutes. The sludge is then centrifuged at 8,000 rpm for approximately 10 minutes, and the supernatant is obtained to obtain the sludge-based protein.

[0016] Subsequently, the sludge-based protein is placed in a reactor and placed at 160-200° C. for more than 4 hours. The sludge protein-based carbon nanodots are obtained by high-speed centrifugation, such as at a rotation speed of 10,000 rpm, and dialysis.

[0017] Experiments show that there is no obvious direct or inverse relationship between the temperature of the hydrothermal reaction and the peroxidase-like activity of the sludge protein-based carbon nanodots. In comparison, when the temperature of the hydrothermal synthesis of sludge protein-based carbon nanodots is 200°C, the final analytical reagent responds most obviously to H2O2; there is no obvious direct or inverse relationship between the time of the hydrothermal reaction and the peroxidase-like activity of the sludge protein-based carbon nanodots. Taking into account the time cost, the duration of the hydrothermal reaction can generally be controlled at 4 to 24 hours, preferably 8 hours.

[0018] The third aspect of the present invention provides a method for using the analytical reagent provided or prepared in the above two aspects. Specifically, the detection and quantitative analysis of hydrogen peroxide can be achieved by drawing a standard curve or establishing a standard colorimetric card.

[0019] The method for drawing a standard curve specifically includes: mixing the analytical reagent with a predetermined volume but different concentrations of a hydrogen peroxide standard solution to obtain a mixed system; after the mixed system is allowed to stand at a predetermined temperature for a predetermined time, measuring the absorbance of the mixed system at 650 nm; and drawing a standard curve with the concentration of the hydrogen peroxide standard solution as the horizontal axis and the absorbance as the vertical axis.

[0020] When conducting the test, the analytical reagent is mixed with a predetermined volume of the sample to be tested to obtain a test system; after the test system is also allowed to stand at a predetermined temperature for a predetermined time, the absorbance of the test system at 650 nm is measured, and the concentration of hydrogen peroxide in the test sample can be determined using the standard curve.

[0021] In practice, a regression equation can also be fitted according to the standard curve, and when performing the test, the concentration of hydrogen peroxide in the sample to be tested can be determined according to the regression equation.

[0022] The method for establishing a standard colorimetric card specifically includes: mixing an analytical reagent with a predetermined volume of a standard hydrogen peroxide solution of different concentrations to obtain a mixed system; after the mixed system is allowed to stand at a predetermined temperature for a predetermined time, a standard colorimetric card is drawn according to the color of the mixed system; when performing a test, the analytical reagent is mixed with a predetermined volume of a sample to be tested to obtain a test system; after the test system is allowed to stand at a predetermined temperature for a predetermined time, the test system is compared with the standard color card to determine the concentration of hydrogen peroxide in the test sample.

[0023] Experimental results show that the method for using the analytical reagent provided by the present invention is suitable for H2O2 detection under a wide range of temperature conditions. However, relatively speaking, when the predetermined temperature is 20-100°C, especially 40-60°C, the measurement accuracy is higher.

[0024] This invention prepares sludge protein-based carbon nanodots by directly carbonizing them in a hydrothermal reactor. These carbon nanodots are then used as a visual detection probe for hydrogen peroxide. Combined with BR buffer and ABTS solution, the analytical reagent enables high-precision detection of hydrogen peroxide, enabling quantitative analysis of 0.0045–0.5 mM hydrogen peroxide. A UV-visible spectrophotometer can detect hydrogen peroxide as low as 4.5 μM, and the presence of hydrogen peroxide as low as 0.01 mM can be determined by color. Furthermore, this analytical reagent enables rapid detection of hydrogen peroxide concentrations, offering advantages such as low cost and simple operation. Furthermore, this invention achieves resource utilization of activated sludge and provides new ideas for the high-value utilization of sludge protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 1 is a UV-vis absorption spectrum of the analytical reagent prepared by the sludge protein-based carbon nanodot dispersion prepared at different hydrothermal temperatures after the reaction with H2O2 in Experimental Example 1 of the present invention;

[0026] Figure 2 UV-vis absorption spectra of the analytical reagent prepared from the sludge protein-based carbon nanodot dispersions prepared with different hydrothermal times after reaction with H2O2 in Experimental Example 2 of the present invention;

[0027] Figure 3 In Experimental Example 3 of the present invention, the absorbance graph of the characteristic peak at 650 nm after the analytical reagent prepared with BR buffer solutions of different pH values ​​reacts with H2O2;

[0028] Figure 4 In Experimental Example 4 of the present invention, the absorbance graph of the characteristic peak at 650 nm after the reaction of the analytical reagent with H2O2 at different temperatures;

[0029] Figure 5 In Experimental Example 5 of the present invention, the absorbance graph of the characteristic peak at 650 nm after the analytical reagent prepared with different concentrations of sludge protein-based carbon nanodot dispersion reacted with H2O2;

[0030] Figure 6 In Experimental Example 6 of the present invention, the absorbance graph of the characteristic peak at 650 nm after the analytical reagent prepared with TMB solution of different concentrations reacted with H2O2;

[0031] Figure 7 In Experimental Example 7 of the present invention, the absorbance histogram corresponding to the reaction with H2O2 under the conditions of adding different interfering substances to the analytical reagent;

[0032] Figure 8The UV-vis absorption spectra of the analytical reagent after reacting with H2O2 standard solutions of different concentrations in Application Example 1 of the present invention are shown in FIG.

[0033] Figure 9 This is the working curve drawn in Application Example 1 of the present invention;

[0034] Figure 10 The color of the analytical reagent after reacting with H2O2 standard solutions of different concentrations in Application Example 2 of the present invention;

[0035] Figure 11 This is the standard colorimetric card drawn in Application Example 2 of the present invention. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0037] Example 1 This embodiment provides a method for preparing an analytical reagent, comprising the following steps:

[0038] S1. Add 10 mol / L NaOH aqueous solution to 300 mL of activated sludge with a moisture content of 90%, adjust the pH value of the activated sludge to 12, then place it in an ultrasonic cell disruptor, ultrasonicate it at a power of 2 W / mL for 20 minutes, and then centrifuge it at a speed of 8000 rpm for 10 minutes. Take the supernatant to obtain sludge-based protein.

[0039] S2. Take 10 g of sludge-based protein and add it to the hydrothermal reactor, then put it into the oven, gradually heat it to 160°C, keep it at this temperature for 6 hours, and then naturally cool it to room temperature. Then centrifuge it at 10,000 rpm for 15 minutes to separate the upper brown liquid; take the supernatant and purify it with a 500 Da dialysis bag for 24 hours. During this period, replace the deionized water every 4 hours to obtain sludge protein-based carbon nanotubes.

[0040] S3. A 5.98 mg / mL sludge protein-based carbon nanodot dispersion, a 3.3 pH BR buffer solution, and a 0.5 mM TMB solution were mixed in a volume ratio of 0.5:3:6 to obtain an analytical reagent.

[0041] Example 2 This embodiment provides a method for preparing an analytical reagent, the process of which is basically the same as that of Example 1, with the only difference being that in step S2, the holding temperature is 180°C.

[0042] Example 3 This embodiment provides a method for preparing an analytical reagent, the process of which is substantially the same as that of embodiment 1, with the only difference being that in step S2, the holding temperature is 200°C.

[0043] Experimental Example 1 Take 0.95 mL of each analytical reagent prepared in Examples 1-3, add 2.05 mL of H2O2 standard solution (0.1 mM) to each, react in a 60°C water bath for 10 minutes, and then measure the absorption spectrum of the mixed system in the range of 500-800 nm using a UV-visible spectrophotometer. The results are as follows: Figure 1 As shown, curve 1 represents Example 1, in which the insulation temperature during the preparation of the sludge protein-based carbon nanodot dispersion in the analytical reagent is 160°C. Similarly, curves 2 and 3 represent Example 2 (insulation temperature 180°C) and Example 3 (insulation temperature 200°C), respectively. Figure 1 It can be seen that the analytical reagents prepared in Examples 1-3 all have sufficient response to H2O2 and can be used for the visual detection of H2O2, but the degree of response has no obvious proportional or inverse relationship with the insulation temperature during the preparation process of the sludge protein-based carbon nanodot dispersion. Relatively speaking, the analytical reagent (sludge protein-based carbon nanodots) prepared in Example 3 has the most obvious response to H2O2.

[0044] Example 4 This embodiment provides a method for preparing an analytical reagent, the process of which is basically the same as that of Example 1, with the only difference being that in step S2, the holding temperature is 200° C. and the holding time is 4 hours.

[0045] Example 5 This embodiment provides a method for preparing an analytical reagent, the process of which is basically the same as that of Example 1, with the only difference being that in step S2, the holding temperature is 200° C. and the holding time is 8 hours.

[0046] Example 6 This embodiment provides a method for preparing an analytical reagent, the process of which is substantially the same as that of embodiment 1, with the only difference being that in step S2, the holding temperature is 200°C and the holding time is 12 hours.

[0047] Example 7 This embodiment provides a method for preparing an analytical reagent, and the process is basically the same as that of Example 1, with the only difference being that in step S2, the holding temperature is 200° C. and the holding time is 16 hours.

[0048] Example 8 This embodiment provides a method for preparing an analytical reagent, and the process is basically the same as that of Example 1, with the only difference being that in step S2, the holding temperature is 200° C. and the holding time is 20 hours.

[0049] Example 9 This embodiment provides a method for preparing an analytical reagent, the process of which is substantially the same as that of embodiment 1, with the only difference being that in step S2, the holding temperature is 200°C and the holding time is 24 hours.

[0050] Experimental Example 2 Take 0.95 mL of each analytical reagent prepared in Examples 4-9, add 2.05 mL of H2O2 standard solution (0.1 mM) to each, react in a 60°C water bath for 10 minutes, and then measure the absorption spectrum of the mixed system in the range of 500-800 nm using a UV-visible spectrophotometer. The results are as follows: Figure 2 As shown, curve 1 represents Example 4, in which the insulation time during the preparation of the sludge protein-based carbon nanodot dispersion in the analytical reagent is 4 hours, and so on, curves 2-6 represent Examples 5-9 respectively. Figure 2 It can be seen that the analytical reagents prepared in Examples 4-9 all have sufficient response to H2O2 and can be used for the visual detection of H2O2, but the degree of response has no obvious proportional or inverse relationship with the insulation time during the preparation of the sludge protein-based carbon nanodot dispersion. Relatively speaking, the sludge protein-based carbon nanodots prepared in Example 5 (insulation time of 8 hours) have the most obvious response to H2O2.

[0051] Example 10 This embodiment provides a method for preparing an analytical reagent, and the process is basically the same as that of Example 5, with the only difference being that in step S3, the pH value of the BR buffer solution is 4.0.

[0052] Comparative group This group of comparative examples each provides a method for preparing an analytical reagent, the process of which is basically the same as that of Example 5, with the only difference being that in step S3, the pH value of the BR buffer solution is different.

[0053] Experimental Example 3 Take 0.95 mL of each analytical reagent prepared in Example 5, Example 10 and Comparative Example Group A, add 2.05 mL of H2O2 standard solution (0.1 mM) thereto, react in a 60°C water bath for 10 minutes, and then measure the absorbance using a UV-visible spectrophotometer. The absorbance value of the characteristic peak at 650 nm (A 650 )like Figure 3 shown.

[0054] Depend on Figure 3It can be seen that when the pH value of the BR buffer solution used in the analytical reagent is 3.3 and 4.0, the analytical reagent, more specifically the sludge protein-based carbon nanodots, has a sufficient response to H2O2. However, if the pH value of the BR buffer solution exceeds 6, H2O2 cannot cause a sufficiently obvious signal change. Therefore, the detection method provided by the present invention is only suitable for H2O2 detection under acidic conditions, wherein the pH value of the BR buffer solution is preferably 3-4.

[0055] Experimental Example 4 Take four portions of the analytical reagent prepared in Example 5, each 0.95 mL, add 2.05 mL of H2O2 standard solution (0.1 mM) to each sample, react at different temperatures (20℃, 40℃, 60℃, 80℃) for 10 minutes, and record the absorbance value of the characteristic peak at 650 nm (A 650 ), the results are as follows Figure 4 As shown. Figure 4 It can be seen that at all tested temperatures, significant signal changes can be generated, proving that the detection method provided by the present invention is suitable for H2O2 detection under a wide range of temperature conditions. 650 Relatively higher, the test accuracy is relatively higher.

[0056] Examples 11-15 Examples 11-15 respectively provide a method for preparing an analytical reagent, the process of which is basically the same as that of Example 5, with the only difference being that in step S3, the concentrations of the sludge protein-based carbon nanodot dispersion used to prepare the analytical reagent are 0.3 mg / mL, 0.9 mg / mL, 1.2 mg / mL, 1.5 mg / mL and 1.8 mg / mL, respectively.

[0057] Experimental Example 5 Take 0.95 mL of each analytical reagent of Example 5 and Examples 11-15, add 2.05 mL of H2O2 standard solution (0.1 mM) respectively, react at 60°C for 10 minutes, and record the absorbance value of the characteristic peak at 650 nm (A 650 ),like Figure 5 As shown in the figure, in the sludge protein-based carbon nanodot detection system with different concentrations, relatively obvious signal changes were produced, and as the concentration of the sludge protein-based carbon nanodot dispersion used to prepare the analytical reagent became lower, the A 650 The smaller the concentration, the best concentration of sludge protein-based carbon nanodots dispersion is 0.3 mg / mL.

[0058] Examples 16-23 Examples 16-23 respectively provide a method for preparing an analytical reagent, and the process is basically the same as that of Example 5, with the only difference being that in step S3, the concentration of the TMB solution used to prepare the analytical reagent is different.

[0059] Experimental Example 6 Take 0.95 mL of each analytical reagent of Example 5 and Examples 16-23, add 2.05 mL of H2O2 standard solution (0.1 mM) to each, react at 60°C for 10 minutes, and record the absorbance value of the characteristic peak at 650 nm (A 650 ),like Figure 6 As shown in the figure, when preparing the analytical reagents, different concentrations of TMB solutions (0.015-0.15 mM) were selected, all of which produced relatively obvious signal changes. As the concentration of the TMB solution increased, the degree of signal change showed a trend of first increasing and then slightly decreasing. The optimal TMB concentration was 0.1 mM.

[0060] Example 24 This example provides a method for preparing an analytical reagent. The process is basically the same as that of Example 5, except that in step S3, the volume ratio of the sludge protein-based carbon nanodot dispersion (5.98 mg / mL), BR buffer solution (pH = 3.3), and TMB solution (0.1 mM) is 0.5:1:6.

[0061] Experimental Example 7 Take several 0.75 mL portions of the analytical reagent prepared in Example 24, add 2.05 mL of H2O2 standard solution (0.1 mM) and 2.05 mL of interfering substance (1 mM) respectively, and react at 60°C for 10 minutes. Plot the A values ​​corresponding to different interfering substances. 650nm The results are as follows Figure 7 As shown, the horizontal axis 1 represents the addition of only hydrogen peroxide to the analytical reagent, and the horizontal axes 2-20 represent the simultaneous addition of hydrogen peroxide and the following interfering substances: Cu 2+ , Fe 3+ , Al 3+ , Ca 2+ , Co 2+ ,K + , Mg 2+ , Na + , Fe 2+ , Ba 2+ , Ni 2+ , Zn 2+ , Cl - , SO4 2- , NO3 - , HCO3 -, CO3 2- , ClO4 - , and ClO - The results showed that these possible interfering substances did not affect the determination of H2O2 by analytical reagents.

[0062] Application Example 1 Take several 0.75 mL portions of the analytical reagent prepared in Example 24, add 2.05 mL of H2O2 standard solution of different concentrations to each portion, and react at 60°C for 10 minutes. Record the UV-vis absorption spectrum in the range of 500-800 nm, as shown in Figure 2. Figure 8 As shown. Figure 8 It can be seen that as the concentration of H2O2 standard solution gradually increases, the absorbance at 650 nm gradually increases, indicating that the response of sludge protein-based carbon nanodots to H2O2 gradually increases. 650nm With the concentration of H2O2 standard solution as the vertical axis and the concentration of H2O2 standard solution as the horizontal axis, draw a working curve, such as Figure 9 shown.

[0063] When testing the hydrogen peroxide content of the sample to be tested, 0.75 mL of the analytical reagent prepared in Example 24 can be taken in the above manner, mixed with 2.05 mL of the sample to be tested, and reacted at 60° C. for 10 minutes. The absorbance of the test system at 650 nm is measured, and the concentration of hydrogen peroxide in the sample to be tested can be determined by the above working curve.

[0064] Application Example 2 Take several 0.75 mL portions of the analytical reagent prepared in Example 24, add 2.05 mL of H2O2 standard solution of different concentrations to each portion, and react at 60°C for 10 minutes. Record the color of the reaction system, as shown in Figure 24. Figure 10 As shown, and draw a standard color chart, such as Figure 11 shown.

[0065] When testing the hydrogen peroxide content of the sample to be tested, 0.75 mL of the analytical reagent prepared in Example 24 can be taken in the above manner and mixed with 2.05 mL of the sample to be tested. After reacting at 60° C. for 10 minutes, the test system is compared with the above standard color card to determine the concentration of hydrogen peroxide in the sample to be tested.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An analytical reagent for quantitative analysis of hydrogen peroxide, characterized in that: Including sludge protein-based carbon nanodot dispersion, BR buffer and TMB solution, The sludge protein-based carbon nanodot dispersion is obtained by extracting sludge-based protein from activated sludge, subjecting it to a hydrothermal reaction at 160-200° C. for at least 4 hours, and then centrifuging the supernatant to obtain a product with a molecular weight not exceeding 500 Da.

2. The analytical reagent according to claim 1, characterized in that The volume ratio of the sludge protein-based carbon nanodot dispersion, BR buffer and TMB solution is 0.4-0.6:1.0-4.0:5-10; wherein the concentration of the sludge protein-based carbon nanodot dispersion is 0.3-1.8 mg / mL, preferably 0.3-1.0 mg / mL; the pH value of the BR buffer is 2-6, preferably 3-4; and the concentration of the TMB solution is 0.015-0.15 mM.

3. A method for preparing an analytical reagent for quantitative analysis of hydrogen peroxide, characterized in that: The steps include: extracting sludge-based protein from activated sludge, subjecting the sludge-based protein to a hydrothermal reaction at 160-200° C. for at least 4 hours, cooling and centrifuging, collecting the supernatant and purifying it using a 500 Da dialysis bag to obtain a sludge protein-based carbon nanodot dispersion; The sludge protein-based carbon nanodot dispersion is mixed with BR buffer and TMB solution to obtain the analytical reagent.

4. The preparation method according to claim 3, characterized in that The volume ratio of the sludge protein-based carbon nanodot dispersion, BR buffer and TMB solution is 0.4-0.6:2.5-4.0:5-10; The concentration of the sludge protein-based carbon nanodot dispersion is 0.3-1.8 mg / mL, preferably 0.3-1.0 mg / mL; the pH value of the BR buffer is 2-6, preferably 3-4; and the concentration of the TMB solution is 0.015-0.15 mM.

5. The preparation method according to claim 3 or 4, characterized in that The method of extracting sludge-based protein from activated sludge comprises: The pH of the activated sludge is adjusted to above 11, followed by ultrasonic cell disruption and centrifugation, and the supernatant is collected to obtain the sludge-based protein.

6. The method for using the analytical reagent according to any one of claims 1 to 5, characterized in that: The steps include: Mixing the analytical reagent with a predetermined volume of a hydrogen peroxide standard solution of different concentrations to obtain a mixed system, allowing the mixed system to stand at a predetermined temperature for a predetermined time, measuring the absorbance of the mixed system at 650 nm, and plotting a standard curve with the concentration of the hydrogen peroxide standard solution as the abscissa and the absorbance as the ordinate; The analytical reagent is mixed with a predetermined volume of a sample to be tested to obtain a test system; after the test system is allowed to stand at a predetermined temperature for a predetermined time, the absorbance of the test system at 650 nm is measured, and the concentration of hydrogen peroxide in the test sample is determined using a standard curve.

7. The method of use according to claim 6, characterized in that: The method further includes the steps of fitting a regression equation according to the standard curve, and determining the concentration of hydrogen peroxide in the sample to be tested according to the regression equation.

8. The method for using the analytical reagent according to any one of claims 1 to 5, characterized in that: The steps include: Mixing the analytical reagent with a predetermined volume of a standard hydrogen peroxide solution of different concentrations to obtain a mixed system, and after the mixed system is allowed to stand at a predetermined temperature for a predetermined time, drawing a standard colorimetric card according to the color of the mixed system; The analytical reagent is mixed with a predetermined volume of a sample to be tested to obtain a test system; after the test system is allowed to stand at a predetermined temperature for a predetermined time, the test system is compared with a standard color card to determine the concentration of hydrogen peroxide in the test sample.

9. The method of use according to any one of claims 6 to 8, characterized in that: The predetermined temperature is 20-100°C, preferably 40-60°C.

10. The method of use according to any one of claims 6 to 8, characterized in that: The concentration of hydrogen peroxide in the sample to be tested is not less than 0.0045 mM.

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