CN111122486A - Rapid hydrogen peroxide detection method of platinum monatomic nanoenzyme and sterilization application - Google Patents
Rapid hydrogen peroxide detection method of platinum monatomic nanoenzyme and sterilization application Download PDFInfo
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- CN111122486A CN111122486A CN202010000772.0A CN202010000772A CN111122486A CN 111122486 A CN111122486 A CN 111122486A CN 202010000772 A CN202010000772 A CN 202010000772A CN 111122486 A CN111122486 A CN 111122486A
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title claims abstract description 56
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 28
- 230000001954 sterilising effect Effects 0.000 title claims abstract description 15
- 238000001514 detection method Methods 0.000 title claims abstract description 13
- 238000004659 sterilization and disinfection Methods 0.000 title claims abstract description 13
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 10
- 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 claims abstract description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 230000031700 light absorption Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 6
- 239000003054 catalyst Substances 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 238000006555 catalytic reaction Methods 0.000 abstract 2
- 230000003115 biocidal effect Effects 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 206010052428 Wound Diseases 0.000 description 12
- 208000027418 Wounds and injury Diseases 0.000 description 12
- 239000002086 nanomaterial Substances 0.000 description 4
- 230000029663 wound healing Effects 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- -1 hydroxyl radicals Chemical class 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 239000010970 precious metal Substances 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- 206010048038 Wound infection Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002951 depilatory effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention provides a method for rapidly detecting hydrogen peroxide by using platinum monatomic nanoenzyme and sterilization application, belonging to the technical fields of nano catalysis, environmental detection, medical antibiosis and the like. The method realizes the non-enzyme chromogenic detection of hydrogen peroxide by using platinum monatomic nanoenzyme as a catalyst and 3,3',5,5' -tetramethyl benzidine as a chromogenic agent. And the platinum monatomic nanoenzyme activity is utilized to realize good in-vitro and living body wound sterilization, so that the H is greatly improved2O2Has high bactericidal activity, reduces H2O2Is used in a concentration such that H is avoided2O2The toxic and side effects of (1). The nano enzyme material is expected to be widely applied to the fields of nano catalysis, environmental detection, biological medicine and the like.
Description
Technical Field
The invention relates to the technical fields of enzyme engineering, environmental monitoring technology and medical antibacterial technology, in particular to a platinum monatomic nanoenzyme-based hydrogen peroxide rapid detection and sterilization application.
Background
Natural enzymes have a wide range of potential applications due to their high specificity and outstanding substrate efficiency. Its inherent instability, limited resources and high cost of the process have prevented its widespread use. The nano enzyme is a nano material with natural enzyme function, has the advantages of low cost, strong biological degradation resistance, large preparation amount and the like compared with natural protease, and is more and more concerned by people when applied to the fields of biosensors and the like. Platinum is a rare precious metal material and has received much attention due to its high temperature oxidation resistance and thermoelectric properties. Research shows that the platinum nanoenzyme has peroxidase activity and can activate hydrogen peroxide (H)2O2) Generating hydroxyl radicals, have been used for H2O2However, most of the existing platinum-based nano materials are particles, and the activity is low. There is an urgent need to develop highly active platinum-based nanomaterials for H2O2And (4) activating. The metal monatomic catalyst becomes a new research frontier by the advantages of active metal centers of metal atom distribution, and the active centers are clear and have high activity. Therefore, the development of the precious metal nano enzyme with the peroxidase activity has important academic significance and economic value.
H2O2As an important Reactive Oxygen Species (ROS), it plays an important role in various biological processes such as wound healing, cell proliferation, and immunity. However, excess of H2O2(166mM-1M) causes adverse effects on normal epidermal tissues. Thus, for environmental and medical H2O2The detection of (2) is also of great significance for the prevention of diseases. At the same time, H2O2As an antibacterial agent, it is widely used in the actual medical treatment of bacterial infections for wound healing, because it decomposes to produce hydroxyl radicals having strong oxidizing properties, thereby destroying the active components of bacteria. Thus, the low concentration H is increased2O2Is a need for wound healing in order to achieve effective bactericidal effects.
Disclosure of Invention
The invention aims to provide a method for quickly detecting hydrogen peroxide of platinum monatomic nanoenzyme and sterilization application.
The technical scheme of the invention is as follows:
a method for rapidly detecting hydrogen peroxide of platinum monatomic nanoenzyme comprises the following steps: adding hydrogen peroxide solution to be detected, 0.1mg/mL monoatomic nanoenzyme and organic color developing agent into acetic acid buffer solution, incubating for 30min at 4-100 ℃, immediately detecting the corresponding light absorption value of the oxide of the organic color developing agent by using a spectrophotometer, and realizing quantitative detection due to the chemical quantitative relationship between the organic color developing agent and the hydrogen peroxide solution.
The organic color developing agent is 3,3',5,5' -tetramethyl benzidine.
A method for sterilizing hydrogen peroxide of platinum monatomic nanoenzyme comprises the following steps:
putting monatomic nanoenzyme, hydrogen peroxide solution and gram-negative bacterium escherichia coli into acetic acid buffer solution, and incubating for more than 6 hours at 0-40 ℃; centrifuging the solution after reaction, coating a flat plate, observing the growth condition of bacteria, wherein the smaller the number of the bacteria on the flat plate, the better the sterilization condition is;
and (3) coating the monatomic nanoenzyme and the hydrogen peroxide solution on the wound, and observing the recovery condition of the wound, wherein the faster the wound is recovered, the better the antibacterial treatment effect is.
The invention has the beneficial effects that:
(1) the method takes platinum monoatomic nanoenzyme as a catalyst and 3,3',5,5' -tetramethyl benzidine (TMB) as a color developing agent to realize H2O2Enzyme-free detection of the solution.
(2) Platinum monatomic nanoenzyme is used as catalyst, and can convert H2O2Efficiently catalyzing the hydroxyl radical with strong oxidizing property, and greatly improving H2O2Sterilization performance of (1), reduction of H2O2Is used in a concentration such that H is avoided2O2The toxic and side effects of the traditional Chinese medicine composition are realized, and a good sterilization effect is realized.
(3) Platinum monatomic nanoenzyme is used as a catalyst, and H is added2O2Effectively catalyzes the hydroxyl free radical with strong oxidizing property, can effectively prevent wound infection and accelerate wound healing.
Drawings
FIG. 1 is a diagram of the sterilization mechanism based on platinum monatomic nanoenzyme according to the present invention.
FIG. 2 is a standard curve for hydrogen peroxide detection based on platinum monatomic nanoenzyme according to the present invention.
Fig. 3 is a graph of the bactericidal effect of the platinum monatomic nanoenzyme-based bactericidal effect of the present invention, wherein a is the bactericidal effect of the nanoenzyme plus hydrogen peroxide treatment, b is the bactericidal effect of the hydrogen peroxide treatment, c is the bactericidal effect of the nanoenzyme treatment, and d is a control.
FIG. 4 is a graph showing the effect of the treatment of contaminated wounds of a living body according to the present invention based on a platinum monatomic nanoenzyme.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the drawings.
Example 1H2O2Detection of (2):
(1) detection method
0.1mg/mL platinum monatomic nanomaterial with 1mM TMB and 1mM H2O2Mix in acetate buffer (pH 4) for 20min at room temperature. The absorbance of each solution was measured in a 652nm excited cuvette using UV-visible absorption spectroscopy.
(2) Drawing a standard working curve:
in the step (1), the H in the sample is followed2O2The oxidation degree of TMB is continuously enhanced, and the absorption intensity of the system at 652nm and H are in the range of 5-500 mu M2O2The concentration has good linear relation and linear correlation coefficient R20.99 (fig. 2).
Example 2
Increasing H by platinum monatomic nanoenzyme2O2The antibacterial performance is investigated:
(1) coli (e.coli) culture
Coli was transferred to LB liquid medium and placed in a shaker at 120rpm for 12h at 37 ℃. Subsequently, the bacteria were collected by centrifugation at 10000rpm for 10min and diluted to 10 with LB medium7CFU mL-1。
(2) Sterilization method
Four groups of experiments were set as control group (e.coli), e.coli + H, respectively2O2Coli + nanoenzyme, E.coli + nanoenzyme + H2O2Group, wherein e.coli is 107CFU, platinum monatomic nanoenzyme 0.1mg/mL and H2O2After incubation at pH 4 for 6 hours at 1mM, the cells were plated on LB medium and cultured at 37 ℃ for 15 hours, and the growth of the plate was observed.
(3) Result analysis of sterilization status
The bactericidal effect is inversely related to the number of colonies on the plate. The smaller the number of colonies on the plate, the better the bactericidal effect (FIG. 3), and it can be seen that the bacterial concentration in E.coli + nanoenzyme + H was higher than that in the other three groups2O2The best bactericidal effect was obtained in the case of the group.
Example 3 an artificial wound model was constructed to examine the therapeutic effect of nanoenzymes:
(1) contamination of wound with bacteria
The depilatory cream was used to depilate the back of the experimental mice. Subsequently, a wound was cut on the back of the dehaired mice with sterilized and disinfected surgical scissors.
(2) Wound management
The mice with wounds on the back are divided into four groups, and the four groups are all coated with 107CFU e.coli, set up four experiments, respectively control, H2O2Nanoenzyme, nanoenzyme + H2O2Group, platinum monatomic nanoenzyme 0.1mg/mL, H2O2Applied to the wound at 1mM in PBS neutral conditions for 6 consecutive days.
(3) Observation of wound recovery
As can be seen from FIG. 4, nanoenzymes + H are present in comparison with the other three groups2O2The group treated wounds had significant scabbing during treatment. More importantly, H used in the experimental procedure2O2The concentration is 1mM, which is much lower than the conventionally used concentration (166mM-1M), and the H is greatly reduced2O2The toxic and side effects of (1).
Claims (4)
1. A method for rapidly detecting hydrogen peroxide of platinum monatomic nanoenzyme is characterized by comprising the following steps: adding hydrogen peroxide solution to be detected, 0.1mg/mL monoatomic nanoenzyme and organic color developing agent into acetic acid buffer solution, incubating for 30min at 4-100 ℃, immediately detecting the corresponding light absorption value of the oxide of the organic color developing agent by using a spectrophotometer, and realizing quantitative detection due to the chemical quantitative relationship between the organic color developing agent and the hydrogen peroxide solution.
2. The method for rapidly detecting hydrogen peroxide by using platinum monatomic nanoenzyme according to claim 1, wherein the organic color-developing agent is 3,3',5,5' -tetramethylbenzidine.
3. A method for sterilizing hydrogen peroxide of platinum monatomic nanoenzyme is characterized by comprising the following steps:
putting monatomic nanoenzyme, hydrogen peroxide solution and gram-negative bacterium escherichia coli into acetic acid buffer solution, and incubating for more than 6 hours at 0-40 ℃; and centrifuging the solution after reaction, coating a flat plate, observing the growth condition of the bacteria, wherein the smaller the number of the bacteria on the flat plate, the better the sterilization condition is.
4. A method for sterilizing hydrogen peroxide of platinum monatomic nanoenzyme is characterized by comprising the following steps:
and (3) coating the monatomic nanoenzyme and the hydrogen peroxide solution on the wound, and observing the recovery condition of the wound, wherein the faster the wound is recovered, the better the antibacterial treatment effect is.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113499773A (en) * | 2021-07-08 | 2021-10-15 | 辽宁大学 | Nano enzyme of nano zinc oxide supported palladium nanoparticles and preparation method and application thereof |
| CN113599506A (en) * | 2021-05-31 | 2021-11-05 | 长沙理工大学 | Platinum nanoenzyme/glucose oxidase @ hyaluronic acid composite antibacterial material and preparation and application thereof |
| CN114011422A (en) * | 2021-11-01 | 2022-02-08 | 广东工业大学 | Monoatomic nanoenzyme and preparation method and application thereof |
| CN114831134A (en) * | 2022-01-05 | 2022-08-02 | 北京化工大学 | Platinum nano particle/short peptide hydrogel and preparation method and antibacterial application thereof |
| CN115569147A (en) * | 2022-09-29 | 2023-01-06 | 浙江瑞邦药业股份有限公司 | Preparation method and application of platinum monatomic supported MXene nanosheet |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113599506A (en) * | 2021-05-31 | 2021-11-05 | 长沙理工大学 | Platinum nanoenzyme/glucose oxidase @ hyaluronic acid composite antibacterial material and preparation and application thereof |
| CN113499773A (en) * | 2021-07-08 | 2021-10-15 | 辽宁大学 | Nano enzyme of nano zinc oxide supported palladium nanoparticles and preparation method and application thereof |
| CN114011422A (en) * | 2021-11-01 | 2022-02-08 | 广东工业大学 | Monoatomic nanoenzyme and preparation method and application thereof |
| CN114831134A (en) * | 2022-01-05 | 2022-08-02 | 北京化工大学 | Platinum nano particle/short peptide hydrogel and preparation method and antibacterial application thereof |
| CN114831134B (en) * | 2022-01-05 | 2022-10-21 | 北京化工大学 | Platinum nano-particle/short peptide hydrogel and preparation method and antibacterial application thereof |
| CN115569147A (en) * | 2022-09-29 | 2023-01-06 | 浙江瑞邦药业股份有限公司 | Preparation method and application of platinum monatomic supported MXene nanosheet |
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