CN113281289B - Ratio sensing detection method for activity of CeO2 mimic organophosphorus hydrolase and oxidase - Google Patents
Ratio sensing detection method for activity of CeO2 mimic organophosphorus hydrolase and oxidase Download PDFInfo
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- CN113281289B CN113281289B CN202110554047.2A CN202110554047A CN113281289B CN 113281289 B CN113281289 B CN 113281289B CN 202110554047 A CN202110554047 A CN 202110554047A CN 113281289 B CN113281289 B CN 113281289B
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- CETPSERCERDGAM-UHFFFAOYSA-N ceric oxide Chemical compound O=[Ce]=O CETPSERCERDGAM-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 229910000422 cerium(IV) oxide Inorganic materials 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 28
- 230000000694 effects Effects 0.000 title claims abstract description 24
- 102000004316 Oxidoreductases Human genes 0.000 title claims abstract description 18
- 108090000854 Oxidoreductases Proteins 0.000 title claims abstract description 18
- 102000006996 Aryldialkylphosphatase Human genes 0.000 title claims abstract description 17
- 108010008184 Aryldialkylphosphatase Proteins 0.000 title claims abstract description 17
- 230000003278 mimic effect Effects 0.000 title description 8
- 238000010521 absorption reaction Methods 0.000 claims abstract description 36
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical group OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims abstract description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 24
- WYMSBXTXOHUIGT-UHFFFAOYSA-N paraoxon Chemical compound CCOP(=O)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 WYMSBXTXOHUIGT-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229960004623 paraoxon Drugs 0.000 claims abstract description 23
- 230000003647 oxidation Effects 0.000 claims abstract description 19
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 19
- 239000002244 precipitate Substances 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- VYLVYHXQOHJDJL-UHFFFAOYSA-K cerium trichloride Chemical compound Cl[Ce](Cl)Cl VYLVYHXQOHJDJL-UHFFFAOYSA-K 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000010276 construction Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 3
- 102000004157 Hydrolases Human genes 0.000 abstract description 2
- 108090000604 Hydrolases Proteins 0.000 abstract description 2
- 229910052684 Cerium Inorganic materials 0.000 description 3
- 230000003592 biomimetic effect Effects 0.000 description 2
- -1 cerium ion Chemical class 0.000 description 2
- 239000000447 pesticide residue Substances 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 235000018645 Allium odorum Nutrition 0.000 description 1
- 240000008654 Allium ramosum Species 0.000 description 1
- 235000005338 Allium tuberosum Nutrition 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- GWXLDORMOJMVQZ-UHFFFAOYSA-N cerium Chemical compound [Ce] GWXLDORMOJMVQZ-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
Abstract
The invention relates to a ratio sensing detection method based on the activities of CeO2 simulated organophosphorus hydrolase and simulated oxidase, which is characterized in that the ratio sensing detection method is constructed by the following specific construction methods: the method comprises the following steps: incubating paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain a paranitrophenol signal A400; step two: and (3) dissolving the precipitate obtained after centrifugation in the first step by using an acetic acid buffer solution, and then reacting the solution with the prepared TMB to measure the ultraviolet-visible absorption so as to obtain an absorption peak A653 of the oxidation state TMB. Finally, the ratio of the signal of p-nitrophenol to the signal of oxidation state TMB is determined. The sensing method has the advantages of low experimental cost, good accuracy, high selectivity and high detection speed in the detection process, so that the ratio sensing detection method based on the activities of the CeO2 organophosphorus-imitating hydrolase and the oxidases can quickly and sensitively detect paraoxon.
Description
Technical Field
The invention relates to the technical field of agricultural chemistry, in particular to a ratio sensing detection method for activity of CeO2 organophosphorus-imitated hydrolase and oxidase-imitated enzyme.
Background
The pesticide residue exceeding the standard can cause harm to the health of eaters, and the immunity of people is reduced and even poisoned and died after the vegetables or other agricultural products with pesticide residue exceeding the standard are eaten for a long time. Organophosphorus pesticides are neurotoxins and cause nerve dysfunction. The existing analysis method commonly used in laboratories has the problems of complex sample pretreatment, high cost, dependence on trained workers, time consumption and the like, and limits the practical application of the method. The biosensing method has the advantages of simplicity, rapidness, high accuracy, good portability, low cost benefit, easy field detection and the like, reduces the time and cost required by sample preparation and signal output, and has simple and convenient operation and high sensitivity.
The mimic enzyme is a catalyst which is synthesized by an artificial method and has enzyme property, has simpler structure, more stable chemical property, enzyme property, and the like, and also has the advantages of high efficiency, high selectivity, low price, easy obtainment and the like. Ceria is an oxide of cerium in which the cerium ion has two valences, and the valences of the cerium ion can be changed over with each other by oxidation or reduction. Thus, ceria has a variety of properties including optical, magnetic, electrochemical, biomimetic oxidase and biomimetic phosphatase activities.
The invention aims at the problem and provides a ratio sensing detection method based on the activities of CeO2 organophosphorus hydrolase and oxidase.
Disclosure of Invention
The invention aims to provide a ratio sensing detection method based on the activities of CeO2 simulated organophosphorus hydrolase and simulated oxidase to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a ratio sensing detection method based on the activities of CeO2 mimic organophosphorus hydrolase and mimic oxidase is constructed by the following specific construction methods:
the method comprises the following steps: incubating paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain a paranitrophenol signal A400;
step two: and (3) dissolving the precipitate obtained after centrifugation in the first step by using an acetic acid buffer solution, and then reacting the solution with the prepared TMB to measure the ultraviolet-visible absorption so as to obtain an absorption peak A653 of the oxidation state TMB. Finally, the ratio of the signal of p-nitrophenol to the signal of oxidation state TMB is determined.
Preferably, the preparation method of the cerium dioxide nanoenzyme comprises the following steps: dissolving cerium chloride in 75mL of ultrapure water, adding ammonia water and hydrogen peroxide, heating at 100 ℃ for 1h while stirring to obtain a light yellow precipitate, centrifuging the yellow precipitate, washing with ultrapure water until the pH value is neutral, and drying the obtained precipitate in an oven for later use.
Preferably, the construction of the ratio sensing detection method specifically comprises the following steps: reacting paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme with the concentration of 1-10mg/mL for 0.5-1h at 36-90 ℃, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain a paranitrophenol signal A400; dissolving the precipitate obtained in the step to 1-5mg/mL by using an acetic acid buffer solution with pH being 4, then reacting the solution with prepared TMB to measure ultraviolet visible absorption to obtain an absorption peak A653 of the oxidation state TMB, and finally taking a ratio of a signal of p-nitrophenol to a signal of the oxidation state TMB.
Compared with the prior art, the invention has the beneficial effects that:
the invention constructs a ratio sensing detection method based on the activities of CeO2 mimic organophosphorus hydrolase and mimic oxidase, and realizes the sensitive detection of paraoxon in Chinese chives. CeO2 imitating the activity of organophosphorus hydrolase hydrolyzes paraoxon to generate yellow paranitrophenol, and the yellow paranitrophenol has obvious ultraviolet visible absorption peak at 400 nm. Meanwhile, CeO2 has the activity of imitating oxidase, and oxidizes colorless TMB into a blue oxidation state TMB, and the TMB has an obvious ultraviolet visible absorption peak at 653 nm. When a target object paraoxon exists, the CeO2 nano enzyme hydrolyzes paraoxon to generate paranitrophenol, a higher ultraviolet visible absorption value is obtained at 400nm, the paraoxon can inhibit the activity of the oxidases-like enzyme of CeO2 nano enzyme, and the absorption peak at 653nm is obviously reduced; when no target substance exists, no p-nitrophenol molecule exists in the system, no ultraviolet visible absorption peak exists at 400nm, and an obvious absorption peak exists at 653 nm.
The invention provides a ratio sensing detection method based on the activities of CeO2 organophosphorus hydrolase and oxidase, which is used for simply, conveniently, quickly, sensitively and efficiently detecting paraoxon;
the ratio sensing detection method based on the activities of the CeO2 simulated organophosphorus hydrolase and the simulated oxidase adopts a sensing method designed according to the ratio sensing principle, has the characteristics of simple operation and high detection speed, and is applied to the detection of antibiotic paraoxon. The main design is as follows:
(a) incubating paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain paranitrophenol signal A400
(b) And (b) dissolving the precipitate obtained after centrifugation in the step (a) by using an acetic acid buffer solution, and then reacting the solution with the prepared TMB to measure the ultraviolet-visible absorption so as to obtain an absorption peak A653 of the oxidation state TMB. Finally, the ratio of the signal of p-nitrophenol to the signal of oxidation state TMB is determined.
In the whole detection process, the operation flow is simple, the consumed time is less, the price of instruments and equipment is low, the use is convenient, and the adaptability to temperature and environment is strong. The design of the invention has the advantages of low cost, easy operation and strong practicability, and is favorable for conveniently and efficiently detecting paraoxon.
The CeO2 nano enzyme imitating the activities of the organophosphorus hydrolase and the oxidase has high stability and good repeatability, and avoids the defect that biological enzyme is easy to survive in extreme environments.
According to the ratio sensing detection method based on the activities of the CeO2 organophosphorus hydrolase and the oxidases, sample separation and complex cleaning procedures are not needed in the detection process, the use amount of the sample is small, the response time is short, the detection speed is high, the measurement range is wide, the test result can be directly read by an instrument, the field real-time detection can be realized, and the rapid and sensitive detection of the paraoxonium is facilitated.
Drawings
FIG. 1 is a schematic diagram of the principle of the ratio sensing detection method based on the activities of CeO2 mimic organophosphorus hydrolase and peroxidase.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The ratio sensing detection method based on the activities of the CeO2 simulated organophosphorus hydrolase and the simulated oxidase of the embodiment is constructed by the following specific construction method:
the method comprises the following steps: incubating paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain a paranitrophenol signal A400;
step two: and (3) dissolving the precipitate obtained after centrifugation in the first step by using an acetic acid buffer solution, and then reacting the solution with the prepared TMB to measure the ultraviolet-visible absorption so as to obtain an absorption peak A653 of the oxidation state TMB. Finally, the ratio of the signal of p-nitrophenol to the signal of oxidation state TMB is determined.
The preparation method of the cerium dioxide nanoenzyme of the embodiment comprises the following steps: dissolving cerium chloride in 75mL of ultrapure water, adding ammonia water and hydrogen peroxide, heating at 100 ℃ for 1h while stirring to obtain a light yellow precipitate, centrifuging the yellow precipitate, washing with ultrapure water until the pH value is neutral, and drying the obtained precipitate in an oven for later use.
The specific construction method of the ratio sensing detection method in this embodiment is as follows: reacting paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme with the concentration of 1-10mg/mL for 0.5-1h at 36-90 ℃, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain a paranitrophenol signal A400; dissolving the precipitate obtained in the above steps to 1-5mg/mL by using acetic acid buffer solution with pH being 4, then taking the solution to react with prepared TMB to measure ultraviolet visible absorption, obtaining absorption peak A653 of oxidation state TMB, and finally taking the ratio of the signal of p-nitrophenol to the signal of oxidation state TMB.
CeO2 imitating the activity of the organophosphorus hydrolase hydrolyzes paraoxon to generate yellow paranitrophenol, and the yellow paranitrophenol has an obvious ultraviolet visible absorption peak at 400 nm. Meanwhile, CeO2 has the activity of imitating oxidase, and oxidizes colorless TMB into a blue oxidation state TMB, and the TMB has an obvious ultraviolet visible absorption peak at 653 nm. When a target object paraoxon exists, the CeO2 nano enzyme hydrolyzes paraoxon to generate paranitrophenol, a higher ultraviolet visible absorption value is obtained at 400nm, the paraoxon can inhibit the activity of the oxidases-like enzyme of CeO2 nano enzyme, and the absorption peak at 653nm is obviously reduced; when no target substance exists, no p-nitrophenol molecule exists in the system, no ultraviolet visible absorption peak exists at 400nm, and an obvious absorption peak exists at 653 nm. As the concentration of paraoxon is increased, A400/A653 is gradually increased, thereby realizing ultraviolet ratio detection of paraoxon.
The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (1)
1. The ratio sensing detection method based on the activities of the CeO2 simulated organophosphorus hydrolase and the simulated oxidase is characterized by comprising the following specific steps:
the method comprises the following steps: incubating paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain a paranitrophenol signal A400;
step two: dissolving the precipitate obtained after centrifugation in the first step by using an acetic acid buffer solution, and then reacting the solution with the prepared TMB to measure the ultraviolet-visible absorption to obtain an absorption peak A653 of the oxidation state TMB; finally, the ratio of the signal of the p-nitrophenol to the signal of the oxidation state TMB is made;
the preparation method of the cerium dioxide nano enzyme comprises the following steps: dissolving cerium chloride in 75mL of ultrapure water, then adding ammonia water and hydrogen peroxide, heating for 1h at 100 ℃ and continuously stirring to obtain a light yellow precipitate, centrifuging the yellow precipitate, washing with ultrapure water until the pH value is neutral, and finally drying the obtained precipitate in an oven for later use;
the ratio sensing detection method based on the activities of CeO2 simulated organophosphorus hydrolase and simulated oxidase specifically comprises the following steps: reacting paraoxon standard samples with different concentrations with cerium dioxide nanoenzyme with the concentration of 1-10mg/mL for 0.5-1h at 36-90 ℃, centrifuging, taking out supernate, and measuring ultraviolet visible absorption to obtain a paranitrophenol signal A400; dissolving the precipitate obtained in the above steps to 1-5mg/mL by using acetic acid buffer solution with pH being 4, then taking the solution to react with prepared TMB to measure ultraviolet visible absorption, obtaining absorption peak A653 of oxidation state TMB, and finally taking the ratio of the signal of p-nitrophenol to the signal of oxidation state TMB.
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| WO2009055128A2 (en) * | 2007-08-17 | 2009-04-30 | Massachusetts Institute Of Technology | Compositions for chemical and biological defense |
| CN102101691B (en) * | 2009-12-18 | 2012-07-18 | 中国石油天然气股份有限公司 | Preparation method of cerium oxide nanoparticles |
| FR3008700B1 (en) * | 2013-07-18 | 2015-08-14 | Univ Nice Sophia Antipolis | ACTIVE OR BIOACTIVE NETWORK POLYMERIZED CERAMIC NANOPARTICLES, TOPIC PROJECTORS, PROCESSES FOR THEIR PREPARATION AND USES THEREOF |
| CN103983777A (en) * | 2014-05-07 | 2014-08-13 | 大连理工大学 | Nanometer cerium dioxide bionic oxidase-based microcystic toxin colorimetric detection method |
| CN107328930B (en) * | 2017-05-09 | 2019-03-29 | 济南大学 | A kind of preparation and application based on dual signal response ratio type screen printing electrode immunosensor |
| CN111334556B (en) * | 2020-03-25 | 2023-11-10 | 南京医科大学 | Colorimetric detection method for acid phosphatase or organophosphorus pesticide based on manganese dioxide simulated biological simulated oxidase activity |
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