AU2019101137A4 - A Novel Method Using Flower-like Manganese Oxide Nanozymes for Colorimetric Detection of Ascorbic Acid - Google Patents

A Novel Method Using Flower-like Manganese Oxide Nanozymes for Colorimetric Detection of Ascorbic Acid Download PDF

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AU2019101137A4
AU2019101137A4 AU2019101137A AU2019101137A AU2019101137A4 AU 2019101137 A4 AU2019101137 A4 AU 2019101137A4 AU 2019101137 A AU2019101137 A AU 2019101137A AU 2019101137 A AU2019101137 A AU 2019101137A AU 2019101137 A4 AU2019101137 A4 AU 2019101137A4
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ascorbic acid
nanozyme
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Qimiao Li
Sirui Li
Keyu Pan
Yi Song
Haixin Tan
Yunxiao Zheng
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Li Qimiao Miss
Li Sirui Miss
Pan Keyu Miss
Tan Haixin Miss
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Li Sirui Miss
Pan Keyu Miss
Tan Haixin Miss
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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01C—AMMONIA; CYANOGEN; COMPOUNDS THEREOF
    • C01C3/00—Cyanogen; Compounds thereof
    • C01C3/08—Simple or complex cyanides of metals
    • C01C3/12—Simple or complex iron cyanides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J27/00—Catalysts comprising the elements or compounds of halogens, sulfur, selenium, tellurium, phosphorus or nitrogen; Catalysts comprising carbon compounds
    • B01J27/24—Nitrogen compounds
    • B01J27/26—Cyanides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/80—Complexes comprising metals of Group VIII as the central metal
    • B01J2531/84—Metals of the iron group
    • B01J2531/842—Iron
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    • C01P2004/03—Particle morphology depicted by an image obtained by SEM
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    • C01P2004/01—Particle morphology depicted by an image
    • C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/30—Particle morphology extending in three dimensions
    • C01P2004/38—Particle morphology extending in three dimensions cube-like
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    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/62—Submicrometer sized, i.e. from 0.1-1 micrometer
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7783—Transmission, loss
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

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Abstract

This invention lies in the field of digital signal processing. It is a video recognition system of different contents based on deep learning. The invention comprises the following procedures:First,the videos will be uploaded and the system will extract their key frames. After that ,these frames will be sent to the neural network resnet34.The neural network will output the frames categories and a statistics will be made to decide the content of the videos. The models used by our neural network can be trained by the following processes:The videos will be uploaded at first and their key frames will be extracted. These key frames will be used to generate the data set . Figure 1. Figure 2.

Description

A Novel Method Using Flower-like Manganese Oxide Nanozymes for Colorimetric Detection of Ascorbic Acid
FIELD OF THE INVENTION
This invention belongs to the field about nanocomposite application of colorimetric detection of ascorbic acid, facilitating the utilization of novel nanoparticles in biomedical research and manufacturing food calibration.
BACKGROUND OF THE INVENTION
Natural enzymes with high efficiency have been widely investigated and applied in real practices. However, natural enzymes suffer from the high risk of instability and inactivation due to their high susceptibility to the exterior factors, including temperature, pH value, substrate concentration, etc. A novel natural enzyme mimetic called “nanozyme” has gradually became a supplant of natural enzyme with abundant advantages, owning to its high stability, low cost for preparation and storage. Characteristics including high specific surface area and customizable functions contribute to its reactivity, which, in turn, make nanozymes suitable to be used in biosensing, disease treatment, and immunoassay (D. L. Nelson, M. M. Cox, 2005). Among all the widely used nanozymes, Prussian blue nanoparticles (PBNPs) and its analogue are outstanding due to their ability to enhance the speed of electron transfer reaction to achieve electrochemical catalytic effect. Researchers around the world have
2019101137 11 Oct2019 developed a variety of usage of Prussian blue, including glucose detection and antioxidant. As (Zhang, et al. 2010) reported, Prussian blue and its analogues possess the peroxidase-like activity. However, recent studies mainly placed their focus on the advantage of Prussian blue in colorimetric sensing in the existence of peroxide. (Zhang, 2018) Ascorbic acid (AA), commonly known as vitamin C, abundantly exists in fruits and vegetables. Since AA was utilized to treat scurvy, a vast number of researches have already proved its regulatory role in metabolism process. However, starting from last century, addition studies showed that an overdose of AA will lead to drawbacks like diarrhea or urinary stone. In such a case, quantifying the daily intake of AA to ensure an appropriate amount becomes a topic of global concern. Currently, titrimetry, spectrophotometry and electrochemistry were already used. Consider the advantages in the aspect of cost, colorimetric detection with the help of enzyme catalytic reaction makes a favorable method for the application in quantitative assay of AA. (Zhang, 2013) Materials like MOF808, quinoxaline and AACD were all studied and used a lot. (Zheng et al. 2018)
Dong, et al. (2014) reported that manganese dioxide can act as oxidase, while fitting a wide range of temperature. Herein, an experiment was operated to synthesize manganese dioxide on a framework of Prussian
2019101137 11 Oct2019 blue analogue, creating a flowerlike structure
This invention exposes a convenient method of utilizing novel flower-like manganese oxide enzyme to detect ascorbic acid based on colorimetric method. The flower-like nanozyme with enhancing peroxidase-like catalytic properties could facilitate the reaction activity. On the basis of this patent, flower-like nanozyme is first employed as novel peroxidase mimetics to offer a simple, sensitive and selective colorimetric method for ascorbic acid.
SUMMARY OF THE INVENTION
This invention aims to find a viable, simple way for colorimetric detection of detecting ascorbic acid (AA) by using flower-like manganese oxide nanozyme. Nanozyme is synthesized by using the coprecipitation method. The optimal catalytic parameters, including pH, substrate concentration, 3,3’,5,5’-tetramethylbenzidine (TMB) concentration, and H2O2 concentration, etc., were obtained based on testifying the absorbance which can monitor the catalytic reactivity vividly. It is proved that this novel nanozyme possesses the ability to detect AA with high efficiency and accuracy.
Experiment instruments:
1. Magnetic stirrer
2. Centrifuge
2019101137 11 Oct2019
3. Five pH Meter
4. The absorption spectra were collected on a 96-well plate in Molecular
Devices Spectramax M5 microplate reader
5. Transmission electron microscopy (TEM) images of flower-like MnOx nanozymes were obtained by a transmission electron microscope (FEI Tecnai G2 20 S-TWIN) operating at an accelerating voltage of 200 kV
6. Scanning electron microscopy (SEM) images of flower-like MnOx nanozymes were obtained by a scanning electron microscopy (SEM, Hitachi S-4800)
Experiment reagents:
1. Monosodium phosphate (Sinopharm Chemical Reagent Beijing Co.,
Ltd)
2. Disodium phosphate (Sinopharm Chemical Reagent Beijing Co., Ltd)
3. Ethanol (Beijing Chemical Works)
4. Manganese (II) sulfate (Sigma-Aldrich)
5. poly(vinylpyrrolidone) (Sigma-Aldrich)
6. Potassium ferricyanide (Acros Organics)
7. Hydrogen peroxide (Beijing Chemical Works)
8. 3,3’,5,5’-tetramethylbenzidine (TMB) (Acros Organics)
9. Ammonium fluoride (Acros Organics)
10. Ascorbic acid (Alfa Aesar)
2019101137 11 Oct2019
11. Ascorbic acid effervescent tablets (DAS gesunde PLUS)
The water used throughout all experiments was purified by a Milli-Q system (18 ΜΩ-cm)
Synthesis of Flower-like MnOx Nanozyme
MnSO4-H2O and PVP were dissolved in a mixture of 10 mL deionized water and 10 mL ethanol, denoted as solution 1. K3[Fe(CN)6] was dissolved in 10 mL deionized water, denoted as solution 2. Under magnetic stirring, solution 2 was added dropwise into the solution 1, continuing to magnetic stir for 2 hours. The precipitation was obtained, washed, and dispersed in a mixture of 10 mL ethanol and 10 mL deionized water. NH4F solution was added into the mixture as mentioned earlier while keeping stirring for 20 min. After being centrifuged, washed and dried, nanozyme could be obtained.
The detection of ascorbic acid includes following steps:
The optimum pH value, concentration of H2O2, flower-like nanozyme, and TMB were determined. 0.6 mL of phosphate buffer with optimal pH was placed in a 1.5 mL centrifuge tube, and different volume of H2O2, nanozyme, and TMB with optimal concentration were added subsequently. After 12 minutes, AA with different concentration were added, keeping reacting for 8 minutes. Then, the absorbance of the solution at 652 nm was measured. The whole process repeated for 2 times. The detection range of AA was determined
2019101137 11 Oct2019
DESCRIPTION OF DRAWINGS
Figure 1. Scanning electronic microscope (SEM) image of flower-like nanozyme.
Figure 2. Transmission electronic microscope (TEM) image of flower-like nanozyme.
Figure 3. Influences of pH value on the catalytic activity of flower-like nanozyme.
Figure 4. Effects of H2O2 concentration on the catalytic activity of flower-like nanozyme.
Figure 5. Effects of TMB substrate concentration on the catalytic activity of flower-like nanozyme
Figure 6. The dose-response curves for ascorbic acid detection. (ΔΑ = Ao - A, Ao is the absorbance of standard without adding ascorbic acid, A is absorbance of ascorbic acid in different concentration)
Figure 7. The corresponding linear calibration plots for ascorbic acid. (ΔΑ = Ao- A, Ao is the absorbance of standard without adding ascorbic acid, A is absorbance of ascorbic acid in different concentration)
Figure 8 absorbance is linearly correlated to AA concentration from 2 μΜ to 20 μΜ.
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.
2019101137 11 Oct2019
The present invention provides a new method to detect ascorbic acid, includes following:
Preparation of Flower-like MnOx nanocomposites
0.05g MnSO4-H2O and 0.25g PVP was dissolved in a mixture of 10 mL ethanol and 10 mL deionized water, recorded as solution 1. 0.07g K3[Fe(CN)6] was dissolved in 10 mL deionized water, recorded as solution 2. As the solution 1 was stirred, solution 2 was added dropwise to solution 1 under magnetic stirring for 2 hours. Subsequently, the mixture was washed with a solution mixed with ethanol and water (ratio of volume equals to 1:1) for 3 times by repeated centrifugation with 5000 rpm for 8 min. The precipitation was obtained and dispersed in a mixture of 10 mL of ethanol and 10 mL of deionized water. Ig NH4F was dissolved in 8 mL of water. NH4F solution was quickly added into the mixture as mentioned earlier while keeping stirring. Continuously, the mixture was stirred at room temperature for 20 min and respectively washed with water and ethanol by repeated centrifugation for 2 times. Finally, the mixture was vacuum-dried at 60 °C. The concentration of solution of the nanozyme is 1 mg / mL
Effects of pH value
0.6 mL of phosphate buffer with different pH value (pH 3.0-10.0) was added into a 1.5 mL centrifuge tube. Subsequently, 25 pL TMB, 20 pL flower-like nanozyme sample, and 30 pL H2O2 were added. The solution
2019101137 11 Oct2019 was kept reacting at the room temperature, and the absorbance could be determined at 652 nm after 8 minutes. Overall, the whole process repeated for 2 times. As shown in Figure 3, the optimal pH (3.0) was determined for flower-like nanozyme in the following experiments. Effects of H2O2 Concentration
0.6 mL of phosphate buffer (pH 3.0) was put into a 1.5 mL centrifuge tube. After that, 30 pL H2O2 with different concentration of 83.33, 166.67, 250.00, 333.33, 416.67, 500.00 mM, 30 pL flower-like nanozyme sample, and 25 pL 8.3 mM TMB were added. The solution was kept reacting at the room temperature, and absorbance could be determined at 652 nm after 8 minutes. The whole process repeated for 2 times. As shown in Figure 4. the optimal concentration of 5 mM H2O2 was chosen for flower-like nanozyme in the following experiments.
Effects of TMB Concentration
0.6 mL of phosphate buffer (pH 3.0) was put into a 1.5 mL centrifuge tube. 25 pL TMB with different concentration of 5.0, 10.0, 20.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0 mM, 20 pL flower-like nanozyme sample, and 30 pL H2O2 were added. The solution was kept reacting at the room temperature, and absorbance could be determined at 652 nm after 8 minutes. The whole process repeated for 2 times. As shown in Figure 5, the optimal concentration of 8.3 mM TMB was determined for Flower-like nanozyme in the following experiments.
2019101137 11 Oct2019
Effects of Flower-like MnOx Nanozyme Concentration
0.6 mL of phosphate buffer (pH 3.0) was put into a E5mL centrifuge tube. After that, 10 pL flower-like nanozyme sample with different concentration of 0.033, 0.05, 0.066, 0.083, 0.100 mg / mL, 30 pL 5 mM H2O2 and 25 pL 8.3 mM TMB were added. The solution was kept reacting at the room temperature, and absorbance could be determined at 652 nm after 8 minutes. The whole process repeated for 2 times. As shown in Figure 5, the optimal concentration of 0.1 mg / mL flower-like Nanozyme was chosen in the following experiments.
Detection of Ascorbic Acid
0.6 mL of phosphate buffer (pH 3.0) was put into a 1.5mL centrifuge tube. 30 pL flower-like nanozyme sample, 30 pL 5 mM H2O2, and 25 pL 8.3 mM TMB were added. AA with different concentration 2.0, 4.0, 8.0, 12.0, 16.0, 18.0, 20.0, 22.0, 24.0, 26.0 mM were respectively added after 12 minutes. The color changes of solution were observed after 9 minutes, and the absorbance could be determined at 652 nm (Figure 7.). As Figure 8 illustrates, absorbance is linearly correlated to AA concentration from 2 pM to 20 pM. In order to evaluate the applicability and accuracy of the proposed method, the real sample of AA concentration was measured. The theorical value of concentration of the sample is 5.45 mM, and the value in our measurement is 5.38 mM. Our result has a 0.013% (< 0.05%) margin of error. AA concentration of the real sample based on the
2019101137 11 Oct2019 detection curve was proved to be consistent with the concentration shown in the ingredients. Therefore, it provides a sensitive, convenient method to detect ascorbic acid.

Claims (4)

1. Method of the preparation for flower-like nanozyme, wherein
MnSO4 · H2O and polyvinyl pyrrolidone (PVP) are dissolved in the mixture of 10 mL deionized water and 10 mL ethanol, denoted as solution 1. K3[Fe(CN)6] is dissolved in 10 mL deionized water, denoted as solution 2; as the solution 1 was stirred, solution 2 was added drop wise to solution 1 under magnetic stirring for 2 hours; the mixture was centrifuged, washed with a solution mixed with ethanol and water for 3 times, and dried.
2. According to said method of claim 1, wherein the optimum volume ration of water and ethanol for washing solution is 1:1.
3. According to said method of claim 1, wherein said sample is further dissolved into the mixture of ethanol and deionized water, and NH4F is dissolved into 8 mL water; under stirring, the solution of NH4F is added rapidly into the precipitation sample mixture and stirred for 20 minutes; after stirring, the solution was centrifuged, washed, and dried.
4. According to said method of claim 1, wherein said sample which is
Prussian blue analogue, possess high peroxidase-like activity.
AU2019101137A 2019-09-30 2019-09-30 A Novel Method Using Flower-like Manganese Oxide Nanozymes for Colorimetric Detection of Ascorbic Acid Ceased AU2019101137A4 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113200556A (en) * 2021-05-20 2021-08-03 黑龙江大学 Preparation method and application of high-stability micron-sized cubic cobalt-based Prussian blue analogue
CN117430135A (en) * 2023-12-21 2024-01-23 山东海化集团有限公司 Method for synthesizing ferromanganese-based Prussian blue sodium electric positive electrode material and positive electrode material prepared by method

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113200556A (en) * 2021-05-20 2021-08-03 黑龙江大学 Preparation method and application of high-stability micron-sized cubic cobalt-based Prussian blue analogue
CN117430135A (en) * 2023-12-21 2024-01-23 山东海化集团有限公司 Method for synthesizing ferromanganese-based Prussian blue sodium electric positive electrode material and positive electrode material prepared by method
CN117430135B (en) * 2023-12-21 2024-04-05 山东海化集团有限公司 Method for synthesizing ferromanganese-based Prussian blue sodium electric positive electrode material and positive electrode material prepared by method

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