AU2019101136A4 - Synthesis of multi-metal cubic nanozymes with peroxidase mimetic activity for the colorimetric detection of ascorbic acid - Google Patents
Synthesis of multi-metal cubic nanozymes with peroxidase mimetic activity for the colorimetric detection of ascorbic acid Download PDFInfo
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- 239000002184 metal Substances 0.000 title claims abstract description 30
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 9
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 8
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 title abstract description 112
- 235000010323 ascorbic acid Nutrition 0.000 title abstract description 55
- 239000011668 ascorbic acid Substances 0.000 title abstract description 55
- 229960005070 ascorbic acid Drugs 0.000 title abstract description 55
- 238000001514 detection method Methods 0.000 title abstract description 20
- 230000000694 effects Effects 0.000 title abstract description 6
- 102000003992 Peroxidases Human genes 0.000 title description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 17
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 41
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000008367 deionised water Substances 0.000 claims description 13
- 229910021641 deionized water Inorganic materials 0.000 claims description 13
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- -1 Poly(vinylpyrrolidone) Polymers 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- 101710134784 Agnoprotein Proteins 0.000 claims description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims 2
- 230000035484 reaction time Effects 0.000 claims 1
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical class [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 abstract description 8
- 229960003351 prussian blue Drugs 0.000 abstract description 7
- 239000013225 prussian blue Substances 0.000 abstract description 7
- 239000000758 substrate Substances 0.000 abstract description 3
- 238000004737 colorimetric analysis Methods 0.000 abstract description 2
- 238000000975 co-precipitation Methods 0.000 abstract 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 16
- 239000000523 sample Substances 0.000 description 15
- 238000002835 absorbance Methods 0.000 description 11
- 230000003197 catalytic effect Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 5
- 239000012621 metal-organic framework Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PBAYDYUZOSNJGU-UHFFFAOYSA-N chelidonic acid Natural products OC(=O)C1=CC(=O)C=C(C(O)=O)O1 PBAYDYUZOSNJGU-UHFFFAOYSA-N 0.000 description 3
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
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- 239000007938 effervescent tablet Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000555745 Sciuridae Species 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 206010047623 Vitamin C deficiency Diseases 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 235000020774 essential nutrients Nutrition 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002082 metal nanoparticle Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
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- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 208000010233 scurvy Diseases 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
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- 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
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
- B01J31/1691—Coordination polymers, e.g. metal-organic frameworks [MOF]
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- 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
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
- B01J31/22—Organic complexes
- B01J31/2204—Organic complexes the ligands containing oxygen or sulfur as complexing atoms
- B01J31/2208—Oxygen, e.g. acetylacetonates
- B01J31/2226—Anionic ligands, i.e. the overall ligand carries at least one formal negative charge
- B01J31/223—At least two oxygen atoms present in one at least bidentate or bridging ligand
- B01J31/2239—Bridging ligands, e.g. OAc in Cr2(OAc)4, Pt4(OAc)8 or dicarboxylate ligands
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- 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
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- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
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- C07F15/02—Iron compounds
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- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/06—Cobalt compounds
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- 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
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Abstract
The invention shows a colorimetric method which can detect specific concentration range of ascorbic acid (AA) with multi-metal cubic nanozymes (a kind of Prussian blue analogue). Through co-precipitation synthesis of Cu(CH 3 COO) 2 , CoCl2 -6H20, C 6H 5Na 3 07-2H 2 0and K3 [Fe(CN)] solution, the multi-metal cubic nanozymes (a kind of Prussian blue analogue) can be obtained. The multi-metal cubic nanozymes (a kind of Prussian blue analogue) has peroxidase-like activity. pH, concentration ofnanozymesH 2 02 and substrate (TMB) are the facts which influence the intrinsic peroxidase-like activity of multi-metal cubic nanozymes (a kind of Prussian blue analogue). The best detection conditions are as following: pH is equal to 4.0, the metering of nanozymes is 12 pL, the metering of TMB (20 mM) is 20 pL and the metering of H 2 0 2 (200 mM) is 35 pL. Under the best conditions, establish a method to detect ascorbic acid (AA), so the linearity range of ascorbic acid (AA) is from 0 mM to 60 mM and the detection range of ascorbic acid (AA) is from 10 mM to 120 mM. Utilizing this method to detect the ascorbic acid (AA) is accurate.
Description
TITLE
Synthesis of multi-metal cubic nanozymes with peroxidase mimetic activity for the colorimetric detection of ascorbic acid
FIELD OF THE INVENTION
The present invention relates to colorimetric detection of ascorbic acid (AA) by multi-metal cubic nanozymes, which have immense utilization in medical assays and biosensors field.
BACKGROUND OF THE INVENTION
Enzyme-mimicking catalytic nanoparticles, known as Nanozymes, are a type of enzyme-mimics with both distinctive features of nanomaterials and efficient catalytic function that is similar to natural enzymes [1], The main motivation of researching in nanozymes is to resolve the shortages of natural enzymes, such as high cost, complicated preparation process and low stability [2], In order to make up for the deficiencies of natural ones, nanomaterials can be an outstanding choice due to the merits of high productivity of catalysis, great stability in a wide range of temperatures and pH and low cost of preparations, nanozymes have been widely applied and researched in analytes of clinical, chemical, food, agricultural and environmental significance.
Ascorbic acid (AA), normally known as Vitamin C, is an essential nutrient for human body to maintain the normal physiologic function. It widely participates in types of complex metabolic process such as body
2019101136 30 Sep 2019 oxidation, deoxidation, which is able to improve growth and the formation of antibody, strengthening the immunocompetence as well [3], AA can react with the reaction product of antioxidants, reviving the oxidants, so AA is a significant kind of synergist of antioxidants. It is necessary for human to obtain AA from other nutrients when AA is insufficient in a body, or it will easily cause scurvy [4], Although most mammals can synthesis AA in liver, human, primates and marmots cannot synthesis it spontaneously. As a result, human have to intake AA through food and medicine.
To detect ascorbic acid in samples, some methods could be applied. For electrochemical method, the procedures of preparing electrodes are complicated. Some errors might exist when titrimetric analysis is applied, while it is complex to manipulate when fluorescence analysis method is applied [4],
In this invention we produced a method of preparing multi-metal cubic nanozymes, a kind of metal organic frameworks (MOFs) with inherent advantages, like rich metal active sites, high porosity, diverse structures and tunable chemical composition [5], Additionally, the redox active MOFs could be an electrochemical sensing platform, which were used to detect hydrogen peroxide [6], AA [7], 2,4-dichlorophenol [8] and L-cysteine [9], Functional metal nanoparticles can be attached within MOFs’ cages/channels, because of the high porosity and flexibility of
2019101136 30 Sep 2019
MOFs [10], The anchorage Ag+ on the surface of nanozymes were utilized to enhance the normalized activity of nanozymes. Prussian blue nanoparticles are found to have the merit of low electronic position for transfer of H2O2 electrons [11], as a Prussian blue analogue, this invention also has the characteristic, so it has superior affinity for H2O2 that to indirectly reach the detection of AA. The synthesized nanozymes using this method displayed peroxidase-mimic activity, respectively, the detection sensitivity of AA was enhanced. Compared with other AA detection methods (as mentioned in the previous paragraph), this method is more convenient and simpler, wider in linear range, and has more precise selectivity and higher degree in sensitivity. The nanozymes synthesized by this procedure have immense potential in the application of medical diagnostics and bio-nanotechnology and provide a new method and thinking for detection of ascorbic acid.
SUMMATY OF THE INVENTION
The object of the invention is to provide a colorimetric method by using n anomatierials to detect AA, which is convenient to synthesize. To solve the above problems, this invention provides a new method by using multi-metal cubic nanozy mes (a kind of Prussian blue analogue) to detect AA. In this invention we produced the nanozymes through a simple method. In order to obtain the
2019101136 30 Sep 2019 best optimum, the specimens were tested by following items: pH value, concentration of substrate, H2O2 and nanozymes. According to the best optimum, the final results showed that our multi-metal cubic nanozymes achieved sensitive detection of AA with a wider range and high efficiency.
Experiment instrument
1) .Magnetic stirrer
2) . Centrifuge
4) . Five pH Meter
5) . The absorption spectra were collected on a 96-well plate in Molecular Devices Spectramax M5 microplate reader
6) . Transmission electron microscopy (TEM) images of PtRunanozymes were obtained by a transmission electron microscope (FEITecnai G2 20 S-TWIN) operating at an accelerating voltage of 200 kV
Experiment reagents
1) . Cu(CH3COO)2 Sinopharm Chemical Reagent Beijing Co., Ltd
2) . Poly(vinylpyrrolidone) (PVP) Sigma-Aldrich
3) . C6H5Na3O7 -2H2O Sinopharm Chemical Reagent Beijing Co., Ltd
4) . CoC12-6H2O Xilong Scientific Co., Ltd.
5) . K3[Fe(CN)6] Acros Organics
6) . Ethanol solution Beijing Chemical Works
7) . Ethylene glycol Beijing Chemical Works
2019101136 30 Sep 2019
8) . AgNO 3 Alfa Aesar
9) .KOH Sinopharm Chemical Reagent Beijing Co., Ltd
10) .KH2PO4 Sinopharm Chemical Reagent Beijing Co., Ltd
11) . 3,3’,5,5’-tetramethylbenzidine (TMB) Acros Organics
12) .DMSO solution Acros Organics
13) . H2O2 Beijing Chemical Works
14) . Deionized water Milli-Q system (18 ΜΩ-cm) 15). Ascorbic
Acid (AA) Alfa Aesar
16) .Domestic effervescent tablets KANGBAIXIN
17) .Imported effervescent tablets DAS gesunde PLUS
The water used throughout all experiments was purified by a Milli-Q system (18 ΜΩ · cm).
Preparation of Prussion blue analogue nanozymes:
Cu(CH3COO)2, PVP and C6H5Na307-2H20 were dissolved in 10 mL deionized water, denoted as solution 1.
CoCl2 · 6H2O, PVP and C6H5Na307-2H20 were dissolved in 10 mL deionized water, denoted as solution 2. K3[Ee(CN)6] was dissolved in 20 mL deionized water, denoted as solution 3. Mixing solution 1 and 2 uniformly, then solution 3 was added dropwise to the above solution, continuing to stir for 0.5 hours. After being centrifuged, washed and dried, purple powder could be harvested.
2019101136 30 Sep 2019
The detection of AA:
The optimal components: pH value, the concentration of substrate (TMB, H2O2) and the nanozymes were determined. Take the optimal pH 4 buffer into the 1.5 mL centrifuge tube, added optimal volume of nanozymes sample, TMB and H2O2 respectively. Reacting in room temperature for 10 minutes, then added gradient concentration of ascorbic acid(A0: without ascorbic acid). Continue to the constant reaction, observe the color change and measure the absorbance of the solution at 650 nm after 10 minutes. Repeat this procedure with two different samples. The limit of detection, range and linear detection range of AA were determined (ΔΑ = Ao - A).
The detection of AA in the 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, TMB, H2O2 and nanozymes in
1.5 mL centrifuge tube and had been reacting for 10 minutes. Then the dilute practical AA solution was placed in three samples. Observing the color change, and measuring the absorbance of the mixed solution at 650 nm after 10 minutes. Finally the concentration of AA in practical sample was calculated by the above linear detection curve and compared to the product label to test the accuracy of this method.
Accuracy and precision of ascorbic-acid sample in Multi-Metal Cubic
2019101136 30 Sep 2019
Nanozymes based sensing platform.
| Sample | Standard | Detected | Recovery( |
| concentration | concentration | %) | |
| (mM) | (mM) | ||
| KANGBAIXIN | 5.45 | 5.30 | 97 |
| DAS gesunde | 9.54 | 9.75 | 102 |
| PLUS |
Table 1
DESCRIPTION OF DRAWINGS
Figure 1. TEM images of multi-metal cubic nanozymes (a) at 200 nm (b) at 100 nm.
Figure 2. Impacts of pH value on the catalytic activity of multi-metal cubic nanozymes.
Figure 3. Impacts of TMB substrate concentration on the catalytic activity of multi-metal cubic nanozymes.
Figure 4. Impacts of H2O2 concentration on the catalytic activity of multi-metal cubic nanozymes.
Figure 5. Impacts of the concentration of nanozymes on the catalytic activity of multi-metal cubic nanozymes.
Figure 6. Detection limit and detection range of AA (ΔΑ=Α0-Α) (A0:
2019101136 30 Sep 2019 without ascorbic acid, A: concentration of ascorbic acid).
Figure 7. A linear fit of AA (ΔΑ=Α0-Α) (AO: without ascorbic acid, A: concentration of ascorbic acid).
Figure 8 Schematic representation of the detection of AA..
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 AA, includes following:
The Preparation of multi-metal cubic nanozymes (a kind of Prussian blue analogue) > 33.6 mg Cu(CH3COO)2, 150 mg poly(vinylpyrrolidone) (PVP) and
200 mg C6H5Na3O7 -2H2O was added into 10 mL deionized water, marked as solution 1.
2> 40 mg CoC12-6H2O, 150 mg PVP and 219 mg C6H5Na307-2H20 was added into 10 mL deionized water, marked as solution 2.
> 130 mg K3[Fe(CN)6] was added into 20 mL deionized water, marked as solution 3.
4> Mix Solution 1 and Solution 2 evenly, then Solution 3 was dripped into the mixed solution slowly This stirring last for 0.5 hours after Solution 3 was dripped. Then stand at room temperature for 20 hours to observe the color change.
> After the reaction was completed, the reaction was centrifuged at 8000
2019101136 30 Sep 2019 rpm for 6 minutes. Water/ethanol mixture (volume ratio 1:1) was used for washing twice, and pure ethanol solution was used for washing twice. 6> The samples obtained by centrifugation were dissolved in 10 mL ethylene glycol, and AgNO 3 solution (concentration: 2 mmol/L) was dripped to it slowly, and stirred at room temperature for reaction for 3 hours. It was centrifuged at 6000 rpm for 6 minutes, washed with water for 2 times, washed with ethanol for 2 times, dried and weighed. The concentration of the nanozymes is 3.6 mg/mL.
Impacts of pH value mL of phosphate buffer with different pH value (pH 3.0-9.0) was added into a 1.5 mL centrifuge tube, adding 10 pL sample nanozymes (3.6 mg/mL), 10 pL TMB (20 mM) and 20 pL H2O2 (200mM). Observing the color change and the absorbance could be determined at 650 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 TMB Concentration
0.6 mL of phosphate buffer (pH 4.0) was put into a 1.5mL centrifuge tube. After that, TMB (20 mM) with different volume of 3, 5, 10, 15, 20, 25 pL, 20 pL H2O2 and 10 pL sample nanozymes were added. After that the absorbance could be determined at 650 nm after 10 minutes. These steps
2019101136 30 Sep 2019 were repeated for 3 times. As shown in Figure 3, when the volume reached 20 to 25 pL, absorbance was similar. So, the volume of 20 pL was chosen for nanozymes in the following experiments.
Impacts of H2 O2 Concentration
0.6 mL of phosphate buffer (pH 4.0) was put into a 1.5 mL centrifuge tube. After that, H2O2 with different concentrations of 1.48, 2.96, 4.44, 5.93, 7.41, 8.89, 10.37 mM, 20.0 pL TMB and 10.0 pL sample multi-metal cubic nanozymes were added. The absorbance could be determined at 650 nm after 10 minutes. These steps were repeated for 3 times. As shown in Figure 4, the upward tendency of absorbance slowed down obviously when the concentration reached 8.89 to 10.37 mM. Thus, the concentration of 10.37 mM was chosen for multi-metal cubic nanozymes in the following experiments.
Impacts of multi-metal cubic nanozymes Concentration
0.6 mL of phosphate buffer (pH 4.0) was put into a 1.5 mL centrifuge tube, then sample of multi-metal cubic nanozymes (3.6 mg/mL) with different volumes of 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0 pL. 20.0 pLTMB and 35.0 pL H2O2 were added. The solution was kept reacting at room temperature, then color change could be observed. After 10 minutes, the solution could be detected by identifying light absorbance at 650 nm. These steps were repeated for 3 times. Figure 5 shows that it achieved stage when the volume of multi-metal cubic nanozymes reaches 64.5
2019101136 30 Sep 2019 pg/mL. Therefore, metering of 64.5 pg/mL was chosen for multi-metal cubic nanozymes in the following experiments.
Detection of AA
0.6 mL of phosphate buffer (pH 4.0) was added to the 1.5mL centrifuge tube with 12 pL of sample multi-metal cubic nanozymes, 20 pL of TMB and 35 pL of H2O2. After 10 minutes, AA with different concentrations of 9.8, 14.7, 19.6,39.2,58.8, 78.4,98.0, 117.6, 137.3 pMwere added to 500 pL solution. Setting a sample which is without AA for comparison; observing color changes after 10 minutes, solution can be detected by identifying absorbance at 650 nm. Each sample was repeated 2 times. When the concentration achieved 137.3 pM, the color of solution (blue) faded almost. As shown in Eigure 6, Eigure 7, the detected range of AA is from 10 to 120 pM, the linear range is from 10 to 60 pM.
Detection of AA among real-life sample
0.6 mL of phosphate buffer (pH 4.0) was added to the 1.5 mL centrifuge tube with 12 pL multi-metal cubic nanozymes, 20 pL TMB and 35 of H2O2. After 10 minutes, 10 pL diluted sample was added to 500 pL solution. Setting a sample which is without AA for comparison; observing color changes after 10 minutes, solution can be detected by identifying absorbance at 650 nm. Each sample was repeated 2 times. Calculate the concentration of the sample according to the curve above and compare the result with the label of the commodity to test the accuracy of the
2019101136 30 Sep 2019 invention. Table 1 shows that our invention can measure the concentration of AA accurately with a recovery of 97% and 102% separately.
2019101136 30 Sep 2019
Editorial Note
There are two pages of claims only
Claims (5)
1. The preparation of multi-metal cubic nanozymes’ metal organic frame, wherein said preparation including: Cu(CH3COO)2, Poly(vinylpyrrolidone)(PVP) and C6H5Na307-2H20 are dissolved in deionized water, denoted as solution 1 CoCl2 -6H2O, PVP and C6H5Na307 -2H2O is dissolved in deionized water, denoted as solution 2, K3[Fe(CN)6] is dissolved in deionized water, denoted as solution 3;Mix Solution 1 and Solution 2 evenly, then dropwise add Solution 3 into the mixed solution slowly, then stir the mixed solution after Solution 3 is completely dropped; next, stands the solution at room temperature and observe the color change; after the reaction is completed, centrifuge the solution; using Water/ethanol mixture and pure ethanol to wash the sediment; the samples obtained by centrifugation are dissolved in ethylene glycol, and dropwise add AgNO 3 (aq) to the solution slowly, and stirring at room temperature in order to let the components react completely; the mixture was centrifuged, washed with water and ethanol, dried and weighed subsequently; after the synthesis process is completed, centrifuge the solution; using water/ethanol mixture and pure ethanol to wash the sediment.
2. According to claim 1, wherein said the optimal time for stirring in the synthesis process of metal organic frame is 30 minutes.
3. According to claim 1, wherein said the optimal reaction time for the
2019101136 30 Sep 2019 synthesis of metal organic frame is 20 hours.
4. According to claim 1, wherein said the optimal volumes of reagents used in each solutions are:
Solution 1: 33.6 mg Cu(CH3COO)2, 150 mg PVP, 219 mg C6H5Na307-2H20 and 10 mL deionized water; Solution 2: 40 mg CoC12-6H2O, 150 mg PVP, 219 mg C6H5Na307-2H20 and lOmL deionized water; Solution 3: 130 mg K3[Fe(CN)6] and 20 mL deionized water.
5. According to claim 1, wherein said the optimal ratio of the washing reagent (water/ ethanol) is 1:1.
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| CN110801866A (en) * | 2019-11-15 | 2020-02-18 | 中国地质大学（北京） | Method for preparing monatomic copper artificial simulation peroxidase |
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| CN110801866A (en) * | 2019-11-15 | 2020-02-18 | 中国地质大学（北京） | Method for preparing monatomic copper artificial simulation peroxidase |
| CN110927378A (en) * | 2019-11-18 | 2020-03-27 | 广东工业大学 | Nano enzyme-linked immunosorbent assay method for detecting glycocholic acid |
| CN112630179A (en) * | 2020-12-09 | 2021-04-09 | 安徽师范大学 | Prussian blue quantum dot with oxide mimic enzyme property, preparation method thereof and method for detecting L-cysteine |
| CN112759514A (en) * | 2021-02-04 | 2021-05-07 | 福州大学 | Synthetic method of columnar copper fumarate with double-enzyme activity |
| CN113686936A (en) * | 2021-08-18 | 2021-11-23 | 南京工业大学 | Preparation method of nano sensing slurry for printing sucrose detection chip |
| CN114414514A (en) * | 2022-01-20 | 2022-04-29 | 中山大学 | Preparation method of manganese Prussian blue nano-enzyme and application of manganese Prussian blue nano-enzyme in alcohol concentration detection |
| CN114414514B (en) * | 2022-01-20 | 2023-12-22 | 中山大学 | Preparation method of manganese Prussian blue nano enzyme and application of manganese Prussian blue nano enzyme in alcohol concentration detection |
| CN114594062A (en) * | 2022-03-15 | 2022-06-07 | 济南大学 | AuRu nano enzyme and application thereof in visual colorimetric detection of ascorbic acid |
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