AU2020100704A4 - A method of synthesis of ultrathin palladium nanosheet with peroxidase mimetic activity for the colorimetric detection of H2O2 - Google Patents

A method of synthesis of ultrathin palladium nanosheet with peroxidase mimetic activity for the colorimetric detection of H2O2 Download PDF

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AU2020100704A4
AU2020100704A4 AU2020100704A AU2020100704A AU2020100704A4 AU 2020100704 A4 AU2020100704 A4 AU 2020100704A4 AU 2020100704 A AU2020100704 A AU 2020100704A AU 2020100704 A AU2020100704 A AU 2020100704A AU 2020100704 A4 AU2020100704 A4 AU 2020100704A4
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Shumeng Chen
Zhehang Gu
Jiaqi Hu
Zhuoyang Li
Xiaohan Shi
Yipeng Wang
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Chen Shumeng Miss
Li Zhuoyang Miss
Shi Xiaohan Miss
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Li Zhuoyang Miss
Shi Xiaohan Miss
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Abstract

The invention shows a colorimetric method which can detect specific concentration range of hydrogen peroxide (H202) with ultrathin palladium nanosheet. Acetylacetone palladium was ultrasonic mixed with glacial acetic acid, and then carbon monoxide ventilation and centrifugation operation, the ultrathin palladium nanosheet can be obtained. The palladium nanosheet has peroxidase-like activity. pH, temperature, concentration of nanozymes, H20 2 and substrate (TMB) are the facts which influence the intrinsic peroxidase-like activity of palladium nanosheet. The best detection conditions are as following: pH is equal to 4.0, temperature is equal to 60 degrees centigrade, the concentration of nanozymes is 6 pg/mL, the concentration of TMB is 2 mM. Under the best conditions, establish a method to detect hydrogen peroxide (H202).

Description

rq TITLE (N
A method of synthesis of ultrathin palladium nanosheet with peroxidase mimetic activity for the colorimetric detection of H2O2
FIELD OF THE INVENTION
The present invention relates to colorimetric detection of H2O2 by ultrathin palladium nanosheet, which has immense utilization in biosensing, immunoassays, cancer diagnostics and therapy, neuroprotection, stem cell growth, and pollutant removal field.
BACKGROUND OF THE INVENTION
Natural enzymes have high specificity and catalytic efficiency under mild reaction conditions. However, the intrinsic limitations (such as high cost, low stability, difficulty in storage, and sensitive to harsh physiochemical conditions) of natural enzymes have stimulated the emergence and development of various enzyme mimics. [1] Nanomaterials, due to their high catalytic efficiency, stability, economy and large-scale preparation, have successfully demonstrated their ability to mimic natural enzymes, including peroxidases, catalases, oxidases, and nucleases.[2] These nanozymes have already found wide applications in numerous fields, including industrial catalysis, biosensing, immunoassays, cancer diagnostics and therapy, pollutant removal and so on.
2020100704 05 May cq Out of all the nanomaterials, noble metal Nano-enzymes stand out, for their unique
CN optical, electronic and catalytic properties, which are closely related to their size, shape, structure, and composition. Metal Pd, as one of the common precious metals, is widely used in many catalytic reactions. In this context, we chose ultrathin palladium nanosheet as catalyst, which synthesis method is simple, has a large specific surface area, no surfactant, and has a variety of mimic enzyme activities. Compared with other nanomaterials, palladium nanosheet has its unique advantages, so it has a great application prospect in biochemical detection.
Hydrogen peroxide ( H2O2 ) is commonly used in the medical field and industrial field as disinfectants or oxidants. Furthermore, it is also used as a bleaching product in the textile industry. Many countries, including China, have established the standards for the use of hydrogen peroxide in different foods. [3] Excessive intake of hydrogen peroxide is harmful to human body, but China and international organizations have not developed a method for the determination of hydrogen peroxide in solid food. [4] Consequently, an uncomplicated, inexpensive, rapid and sensitive analytical method for H2O2 would be necessary because it could be used for bioassays in food security and environmental applications. Analytical methods for the analysis of this substance have been reported, including fluorescence, cell imaging, electrochemical and colorimetric assays. [5] In this context, the ultrathin palladium nanosheet, due to its ultrahigh peroxide-like activity, was applied to the colorimetric detection of H2O2, which is simple, fast, accurately.
2020100704 05 May
SUMMARY OF THE INVENTION
The object of the invention is to provide a colorimetric method by using nanomatierials to detect H2O2, which is convenient to synthesize. To solve the above problems, this invention provides a new method by using ultrathin palladium nanosheet to detect H2O2. In this invention we produced the nanosheet through a simple method. In order to obtain the best optimum, the specimens were tested by following items: pH value, concentration of substrate, and nanozymes. According to the best optimum, the final results showed that our ultrathin palladium nanosheet achieved sensitive detection of H2O2 with a wider range and high efficiency.
Experiment Instrument
1. Centrifuge
2. Thermostat
3. Seven pH Meters
4. The absorption spectra were collected on a 96-well plate in Molecular Devices Spectramax M5 microplate reader
Experiment Reagents
1. Solid palladium acetylacetone
2. Glacial acetic acid
3. Gas carbon monoxide
2020100704 05 May 2020
4. Deionized water
5. Phosphate buffered solution
6. DMSO solution of TMB
7. Hydrogen peroxide solution
Preparation of Ultrathin Palladium Nanosheet
Weigh some palladium acetylacetone, put it into centrifugal tube, add some glacial acetic acid, ultrasonic 2 min, make it fully dissolved and mixed. Put the CO snorkel into the solution above and continue to ventilate for half an hour. After the snorkel is ventilate, cover the snorkel tightly and seal with a sealing belt. Place the snorkel at room temperature for 24 h. After the reaction, the reaction solution was centrifuged at 10,000 RPM for 5 min, washed with deionized water until neutral, and then dispersed in deionized water.
The Detection of H2O2
The optimal components: pH value, the concentration of substrate (TMB, H2O2) and the nanozymes were determined. Take 0.6 mL optimal pH buffer into the 1.5 mL centrifuge tube, incubating in the optimal temperature for 8 minutes. Add optimal volume of nanozymes and optimal concentration of TMB solution; then add gradient concentration of H2O2. Continue to the constant reaction and observe the color change. 10 minutes later, measure the absorbance of the solution at 652 nm. Repeat this procedure with three different samples. The limit of detection, range and linear
2020100704 05 May rq detection range of H2O2 were determined.
CM
The Detection of H2O2 in Practical Samples
To demonstrate the practical feasibility of the proposed detection assay, three practical samples, were collected and analyzed. All samples were mixed with the optimum volume of pH, temperature, TMB, and nanozymes in 1.5 mL centrifuge tube and had been reacting for 10 minutes. Then the dilute practical H2O2 solution was placed in three samples. Observing the color change, and measuring the absorbance of the mixed solution at 652 nm after 10 minutes. Finally the concentration of H2O2 in practical sample was calculated by the above linear detection curve and compared to the product label to test the accuracy of this method.
DESCRIPTION OF DRAWING
Figure 1. TEM (left) and SEM (right) images of palladium nanosheet.
Figure2. Impacts of pH value on the catalytic activity of palladium nanosheet.
Figure3. Impacts of temperature on the catalytic activity of palladium nanosheet.
Figure4. Impacts of H2O2 substrate concentration on the catalytic activity of palladium nanosheet.
Figure5. Impacts of TMB substrate concentration on the catalytic activity of palladium nanosheet.
Figure6. Impacts of the concentration of nanozymes on the catalytic activity of palladium nanosheet.
Figure?. Schematic representation of the detection of H2O2.
DESCRIPTION OF PREFERRED EMBODIMENTS
The embodiment of the present invention will be explained in details so that the present invention can be more readily understood. The present invention provides a new method to detect H2O2, includes following:
The Preparation of Ultrathin Palladium Nanosheet
2020100704 05 May 2020
1. Weigh 15.2 mg palladium acetylacetone, put it into 50 mL centrifugal tube, add 10 mL glacial acetic acid, ultrasonic 2 min, make it fully dissolved and mixed.
2. Put the CO snorkel into the solution above and continue to ventilate for half an hour. After the snorkel is ventilate, cover the snorkel tightly and seal with a sealing belt. Place the snorkel at room temperature for 24 h.
3. After the reaction, the reaction solution was centrifuged at 10,000 RPM for 5 min, washed with deionized water until neutral, and then dispersed in deionized water.
Impacts of pH value mL of phosphate buffer with different pH value (pH 2.0-8.0) was added into a 1.5 mL centrifuge tube, adding 20 pL sample nanozymes, 10 pL TMB (20 mM) and 10 pL H2O2(50mM). Observing the color change and the absorbance could be determined at 652 nm after 10 minutes, determining the best pH Value buffer. Each group repeated for 3 times. Figure 2 could be deduced that in the buffer solution with pH 4.0, the absorbance achieves the stage; it means that the catalytic activity of nanozyme is the strongest when it is in the environment of pH 4.0.
Impacts of temperature
0.6 mL of phosphate buffer (pH 4.0) was put into a 1.5mL centrifuge tube with different temperature of 0, 10, 20, 30, 40, 50, 60, 70, 80 degrees centigrade, 10 pL 50nM TMB, 10 pL H2O2 (50mM) and 20 pL sample nanozymes were added. After that the absorbance could be determined at 652 nm after 10 minutes. These steps were repeated for 3 times. As shown in Figure 3, when the volume reached 60 degrees centigrade. So, the temperature of 60 degrees centigrade was chosen for nanozymes in the following experiments.
Impacts of TMB Concentration
0.6 mL of phosphate buffer (pH 4.0) was put into a 1.5mL centrifuge tube in 60 degrees centigrade. After that, TMB with different concentration of 0.5, 1, 1.5, 2, 3, 4, 5mM TMB 10 pL H2O2 and 20 pL sample nanozymes were added. After that the absorbance could be determined at 652 nm after 10 minutes. These steps were repeated for 3 times. As shown in Figure 3, when the concentration reach 2nM TMB. So, the concentration of 2 nM was chosen for nanozymes in the following experiments.
Impacts of Palladium nanosheets Concentration
0.6 mL of phosphate buffer (pH 4.0) was put into a 1.5 mL centrifuge tube, then sample of palladium nanosheets with different concentration of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0pg/mL. 10.0 pL TMB and lO.OpL H2O2 were added. The solution was kept reacting at best temperature, then color change could be observed. After 10 minutes,
2020100704 05 May 2020 the solution could be detected by identifying light absorbance at 652 nm. These steps were repeated for 3 times. Figure 5 shows that it achieved stage when the concentration of palladium nanosheets reaches 6.0pg/mL. Therefore, metering of 6.0pg/mL was chosen for palladium nanosheets in the following experiments.
Detection of H2O2
0.6 mL of phosphate buffer (pH 4.0) was added to the 1.5mL centrifuge tube with 6 pL of sample multi-metal cubic nanozymes, and 2nM of TMB. After 8 minutes, H2O2 with different concentrations of 0.5, 1, 2.5, 5, 10, 25, 50mM were added to it. Setting a sample which is without H2O2 for comparison; observing color changes after 8 minutes, solution can be detected by identifying absorbance at 652 nm. Each sample was repeated 3 times. When the concentration achieved 10 mM, the color of solution (blue) faded almost.

Claims (4)

cq WHAT WE CLAIM IS: rd
1. A method of synthesis of ultrathin palladium nanosheet with peroxidase mimetic activity for the colorimetric detection of H2O2, characterized in that: including following steps:
The preparation of Thin layer palladium nanosheet :
Weigh palladium acetylacetone of a certain mass, put it into a centrifugal tube, add glacial acetic acid, ultrasonic for a few minutes, make it fully dissolved and mixed; And then, put the CO snorkeler into the solution above and continue to ventilate for several hours; after the snorkeler is ventilate, cover the snorkeler tightly and seal with a sealing belt, in additional, after the reaction is completed, the reaction liquid is centrifuged, waited for a few minutes at a certain atmospheric pressure, washed with deionized water until neutral, and finally dispersed in deionized water.
2. According to method of claim 1, wherein palladium with a mass of 15.2 mg acetylacetone was weighed; and add 10 mL glacial acetic acid.
3. According to method of claim 1, wherein the best response time is 24 hours.
4. According to method of claim 1, wherein the optimum centrifugal speed is 10000 RPM, the centrifugation time is 5 minutes, and the centrifugation is 3 times.
AU2020100704A 2020-05-05 2020-05-05 A method of synthesis of ultrathin palladium nanosheet with peroxidase mimetic activity for the colorimetric detection of H2O2 Ceased AU2020100704A4 (en)

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CN111965136A (en) * 2020-08-14 2020-11-20 陕西科技大学 Preparation method of peroxidase-like nanoenzyme beta-FeOOH and its application in H2O2Applications in assays
CN112604684A (en) * 2020-12-18 2021-04-06 许昌学院 Gold-carbon composite nanoparticle mimic enzyme and preparation method thereof
CN112643045A (en) * 2020-12-18 2021-04-13 华侨大学 Palladium-ruthenium ultrathin nanosheet, preparation method thereof and application of palladium-ruthenium ultrathin nanosheet as electrocatalyst
CN112844431A (en) * 2020-12-23 2021-05-28 江苏师范大学 Graphite phase single layer C chelated with copper ions3N4Nano enzyme and preparation method thereof
CN112945945A (en) * 2021-01-23 2021-06-11 西安交通大学 Detection of H based on mesoporous CuO nanoenzyme2O2Paper-based sensor and preparation method
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