CN113295682B - Phenolic compound analysis method based on polyphenol oxidase activity nanoenzyme - Google Patents
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 3
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Abstract
The invention belongs to the technical field of phenol detection, and particularly relates to a phenol compound analysis method based on polyphenol oxidase activity nanoenzyme, which comprises the following steps: preparing an array sensor; (2) performing discriminant analysis on different phenolic pollutants; (3) performing discriminant analysis on different concentrations of the same phenolic substance; (4) carrying out discriminant analysis on the mixed two phenolic substances; and (5) distinguishing and analyzing phenolic substances in the actual sample. The method for analyzing the phenolic compounds based on the polyphenol oxidase activity nanoenzyme constructs the array sensor based on the nanoenzyme system, has high detection sensitivity and strong expandability, realizes the differentiation and analysis of different phenolic pollutants, has strong specificity and high accuracy, combines the colorimetric array with the chemometrics algorithm, and constructs the sensor by utilizing the characteristic wavelength, so that the establishment of the sensor has more theoretical basis.
Description
Technical Field
The invention relates to the technical field of phenol detection, in particular to a phenol compound analysis method based on polyphenol oxidase activity nanoenzyme.
Background
The phenolic compounds are basic raw materials in industrial and agricultural production, have stronger toxicity and difficult degradability, and the solubility of most phenolic pollutants in water is higher, so that the migration capability of the phenolic compounds in the water is improved, and the phenolic compounds gradually become one of main pollutants in the water, thereby having vital significance for the detection of the phenolic pollutants in the water. At present, methods for detecting phenolic substances in water mainly comprise chromatography, spectrophotometry, fluorescence analysis, electrochemical analysis and the like. However, the method has the disadvantages of huge apparatus and equipment, high detection cost and long time consumption, needs complex pretreatment processes such as extraction and separation of samples, needs professional operation, needs a large amount of manpower and material resources, has long detection time and expensive apparatus, needs professional and technical personnel to maintain, has high detection cost, is not suitable for field detection, and cannot meet the detection requirements of various agricultural products. The colorimetric method can well fill the defects of complex sample pretreatment, expensive instruments, complex operation and the like of other methods, is very suitable for on-site rapid analysis and detection, has the accuracy and the sensitivity of other methods in the detection process, and gradually becomes a current research hotspot.
The nano enzyme is a nano material with enzyme-like catalytic activity and nano characteristics, overcomes a plurality of defects of natural enzyme compared with natural enzyme, and has the advantages of high stability, low cost, easy large-scale production, long-term storage and the like. The nano enzyme, the colorimetric sensor and the chemometrics algorithm are combined to form an array, so that high-sensitivity detection of the phenolic pollutants can be realized, rapid distinguishing of the phenolic pollutants with similar structures can be realized, and a new idea is provided for detection of the phenolic pollutants.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
Therefore, the invention provides a low-cost and high-sensitivity detection method for different phenolic pollutants, a nano-enzyme-based array sensor detection system is constructed, a new method is provided for detecting different types of phenolic substances, a sensor array is one of multi-dimensional sensing technologies and consists of a plurality of sensing units, and the sensor array is the same as a chip constructed by a plurality of functional components, wherein the response degree of a single sensing unit to different substances is different, and different sensing units also have different degrees of response to the same substance, so that the specific distinction of samples can be realized through characteristic patterns generated by the response of different sensing units to target substances and the difference among the characteristic patterns.
In order to solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions:
a method for analyzing phenolic compounds based on polyphenol oxidase activity nanoenzymes, which comprises the following steps: the method utilizes the catalytic characteristic difference of the nanoenzyme with polyphenol oxidase activity on different phenolic compounds, combines the characteristic wavelength selection and the discriminant analysis technology, and carries out the recognition analysis of the phenolic compounds, and comprises the following steps:
(1) Preparing an array sensor: taking the characteristic spectra at different times and under different times as each sensing unit of the array sensor to prepare a sensor array with multiple sensing units;
(2) Discriminant analysis of different phenolic pollutants: adding a water solution of phenolic substances into the array sensor array in the step (1), mixing the water solution with the detection solution to generate a color development reaction, reading an ultraviolet absorption value of the detection solution through an enzyme-labeling instrument, carrying out digital processing on color development results of different phenolic substances in a system, introducing obtained data into Matlab software, and operating a chemometrics algorithm to carry out discriminant analysis to obtain a scoring graph of the discriminant results of the different phenolic substances;
(3) Discrimination analysis of different concentrations of the same phenolic substance: preparing four phenol solutions with different concentrations, adding the phenol solutions into the array sensor array in the step (1), mixing the phenol solutions with the detection solution to generate a color reaction, reading an ultraviolet absorption value of the detection solution through an instrument, carrying out digital processing on color development results of different phenolic substances in a system, and introducing obtained data into Matlab software for discriminant analysis to obtain a score chart of the discrimination results of the different phenolic substances;
(4) And (3) performing discriminant analysis after mixing of two phenolic substances: mixing two phenolic substances in the same proportion, adding the mixed solution into the array sensor array in the step (1), mixing the mixed solution with the detection solution in the array sensor array to generate a color development reaction, reading an ultraviolet absorption value of the detection solution through an instrument, carrying out digital processing on color development results of different phenolic substances in a system, and introducing obtained data into Matlab software for discriminant analysis to obtain a scoring graph of the discrimination results of different phenolic substances;
(5) And (3) distinguishing and analyzing phenolic substances in the actual sample: adding a standard solution of phenolic substances into sewage to obtain a system of an actual sample, adding the solution into the array sensor array in the step (1), mixing the solution with the detection solution in the array sensor array to generate a color reaction, reading an ultraviolet absorption value of the detection solution through an instrument, carrying out digital processing on color development results of different phenolic substances in the system, and introducing the obtained data into Matlab software for discriminant analysis to obtain a score map of the discrimination results of different phenolic substances.
As a preferable embodiment of the method for analyzing a phenolic compound based on a polyphenol oxidase activity nanoenzyme of the present invention, wherein: the polyphenol oxidase was synthesized by mixing 10mM of disodium 5' -guanylate solution, 50mM of copper chloride solution, 100mM of Tris-HCl buffer solution, pH =8.5 and ultrapure water in a ratio of 2,000rpm, and centrifuging 5min with a centrifuge at 10,000rpm; the resulting precipitate was washed 3 times with ultrapure water. Finally, uniformly dispersing the precipitate in ultrapure water to obtain the synthesized nano enzyme.
As a preferable embodiment of the method for analyzing a phenolic compound based on a polyphenol oxidase activity nanoenzyme of the present invention, wherein: preparing an array sensor: forming a system by using a nano enzyme solution, a MES buffer solution 100mM, a pH =7, and 10mg/ml of 4-aminopyridine, ultrapure water and a phenolic substance solution; for hydroquinone, catechol, resorcinol, phenol, phloroglucinol and parachlorophenol, the addition amount of each solution in a 1ml system is 100 mul of nano enzyme, 100 mul of MES buffer solution, 100 mul of 4-aminopyridine, 100 mul of ultrapure water and 100 mul of phenol solution respectively; in 1ml of the system, 2,4-dichlorophenol was added in an amount of 100. Mu.L of nanoenzyme, 100. Mu.L of MES buffer, 100. Mu.L of 4-aminopyridine, 200. Mu.L of ultrapure water and 500. Mu.L of phenol solution.
As a preferable embodiment of the method for analyzing a phenolic compound based on a polyphenol oxidase activity nanoenzyme of the present invention, wherein: in the step (2), the phenolic substances are hydroquinone, catechol, resorcinol, phenol, phloroglucinol and p-chlorophenol respectively, the concentration is 1mg/ml, and the concentration of 2, 4-dichlorophenol is 0.2mg/ml; the concentrations of the phenol in the step (3) in the reaction system are respectively 1, 10, 100 and 1000 mu g/mL; and (4) mixing the two phenolic substances in the step (4) in the same proportion respectively to obtain phenol +2,4-DP, phloroglucinol + p-chlorophenol, hydroquinone + phloroglucinol, catechol + phenol, hydroquinone +2,4-DP and resorcinol + p-chlorophenol.
Compared with the prior art: the invention constructs the array sensor based on the nano enzyme system, has high detection sensitivity and strong expandability, realizes the differentiation and analysis of different phenolic pollutants, has strong specificity and high accuracy, combines the colorimetric array with a chemometrics algorithm, and constructs the sensor by utilizing characteristic wavelength, so that the establishment of the sensor has more theoretical basis.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail with reference to the accompanying drawings and detailed embodiments, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise. Wherein:
FIG. 1 is a result graph of characteristic wavelength calculation by a genetic algorithm, and finally characteristic wave points of the top five ranked in each time period are obtained;
FIG. 2 is a partial least squares linear discriminant analysis plot of the present invention for the same concentration of different types of classified contaminants and the response of an array sensor;
FIG. 3 is a plot of partial least squares linear discriminant analysis of phenol and array sensor responses at different concentrations in accordance with the present invention;
FIG. 4 is a partial least squares linear discriminant analysis plot of the present invention for equal ratio mixing of two phenolics and array sensor response.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and it will be apparent to those of ordinary skill in the art that the present invention may be practiced without departing from the spirit and scope of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
A phenol pollutant detection and distinguishing method based on a nano enzyme colorimetric sensor comprises the following specific steps:
(1) Preparing an array sensor: the colorimetric array is constructed by combining the laccase property of the nano enzyme by the coordination of the nucleotide and the copper, and each sensing unit consists of reaction time and optimal wavelength. And 5 optimal reaction wave points of the reaction system under the full wave band are obtained through calculation of a chemometrics algorithm-genetic algorithm, and the optimal reaction wave points and the reaction time form each sensing unit of the array sensor with the wavelength multiplied by the time (5 multiplied by 3), so that the sensor array with multiple sensing units is prepared.
(2) Discriminant analysis of different phenolic pollutants: adding a phenolic substance aqueous solution into the array sensor array in the step (1), mixing the phenolic substance aqueous solution with a detection solution (an enzyme substrate for generating a colorimetric signal) in the array sensor array to generate a color reaction, reading an ultraviolet absorption value of the detection solution through an instrument, carrying out digital processing on color development results of different phenolic substances in a system, and introducing obtained data into Matlab software for discriminant analysis to obtain a scoring chart of the discrimination results of the different phenolic substances.
(3) Discrimination analysis of different concentrations of the same phenolic substance: preparing four phenol solutions with different concentrations, adding the phenol solutions into the array sensor array in the step (1), mixing the phenol solutions with detection liquid (enzyme substrates for generating colorimetric signals) in the array sensor array to generate a color reaction, reading the ultraviolet absorption value of the detection liquid through an instrument, carrying out digital processing on color development results of different phenolic substances in a system, and introducing obtained data into Matlab software for discriminant analysis to obtain a score map of the discrimination results of the different phenolic substances.
(4) And (3) performing discriminant analysis after mixing of two phenolic substances: mixing two phenolic substances in the same proportion, adding the mixed solution into the array sensor array in the step (1), mixing the mixed solution with detection liquid (enzyme substrate for generating colorimetric signals) in the array sensor array to generate a color reaction, reading the ultraviolet absorption value of the detection liquid through an instrument, carrying out digital processing on the color development results of different phenolic substances in a system, and introducing the obtained data into Matlab software for discriminant analysis to obtain a scoring chart of the discrimination results of different phenolic substances.
(5) And (3) distinguishing and analyzing phenolic substances in the actual sample: adding a standard solution of phenolic substances into sewage to obtain a system of an actual sample, adding the solution into the array sensor array in the step (1), mixing the solution with a detection solution (an enzyme substrate for generating a colorimetric signal) in the array sensor array to generate a color reaction, reading an ultraviolet absorption value of the detection solution through an instrument, performing digital processing on color development results of different phenolic substances in the system, and introducing obtained data into Matlab software for discriminant analysis to obtain a scoring chart of the discrimination results of the different phenolic substances.
The nanoenzyme solution described in step (1) was prepared by mixing a 5' -guanylate disodium solution (10 mM), a copper chloride solution (50 mM), a Tris-HCL buffer solution (100mm, ph = 8.5), and ultrapure water at a ratio of 2,000rpm, and centrifuging the mixture for 5min with a centrifuge. The resulting precipitate was washed 3 times with ultrapure water. And finally, uniformly dispersing the precipitate in ultrapure water to obtain the synthesized nano enzyme (GMP-Cu).
The reaction time in the step (1) is respectively 30min,60min and 120min, the absorbance values under three times respectively form a matrix, MATLAB is utilized to run a genetic algorithm to obtain characteristic spectrum values under three times, and five spectrum points with the largest number of times are selected to form an array, which is respectively 30min: 383. 394, 398, 414, 465nm; and (5) 60min:360nm, 366, 390, 495 and 498nm;120min: 377. 381, 407, 421 and 509nm.
The phenolic substances in the step (2) are hydroquinone, catechol, resorcinol, phenol, phloroglucinol and parachlorophenol with the concentration of 1mg/ml and 2, 4-dichlorophenol with the concentration of 0.2mg/ml respectively.
The concentrations of the phenol in the step (3) in the reaction system are respectively 1, 10, 100 and 1000 mu g/mL.
Further, the two phenolic substances in the step (4) are respectively phenol +2,4-DP, phloroglucinol + p-chlorophenol, hydroquinone + phloroglucinol, catechol + phenol, hydroquinone +2,4-DP resorcinol + p-chlorophenol in the same proportion.
In embodiments, the sensing unit comprises a 96-well plate, and the 96-well plate is a fully transparent microplate, but is not limited thereto.
The principle of the invention is as follows: the metal organic framework formed by the coordination of the nucleotide and the copper has laccase property, can oxidize phenolic substances to generate quinone, and generates obvious color change after the addition of a color developing agent. Based on the properties, the nanoenzyme-based array sensor is constructed.
Example 1 identification and differentiation of various phenolic contaminants
A reaction system was prepared in accordance with embodiment (1), and the reaction was carried out for 30min,60min, and 120min.
The sensing unit 1: and the characteristic spectra are 383 nm, 394 nm, 398 nm, 414 nm and 465nm respectively when the reaction is carried out for 30 min.
The sensing unit 2: the characteristic spectra at 60min of the reaction were 360nm, 366 nm, 390 nm, 495 nm and 498nm, respectively.
The sensing unit 3: the characteristic spectra at 120min of reaction were 377, 381, 407, 421, 509nm, respectively.
The reaction system configured in the embodiment (1) is placed in a 96-well plate in a certain volume, the absorbance value under the sensing unit of the array sensor for detecting the phenolic substances is read by a microplate reader, the experiment is performed for 10 times in parallel, the obtained data is introduced into MATLAB software for partial least squares discriminant analysis, the score maps of the main components of different phenolic substances are obtained, and the different phenolic substances can be correctly identified and distinguished.
While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the disclosed embodiments of this invention can be used in any combination as long as there is no structural conflict, and the combination is not exhaustively described in this specification merely for the sake of brevity and resource savings. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (4)
1. A phenol compound analysis method based on polyphenol oxidase activity nanoenzyme is characterized in that the method utilizes the catalytic characteristic difference of the nanoenzyme with polyphenol oxidase activity on different phenol compounds, combines characteristic wavelength selection and discriminant analysis technology, and carries out the recognition analysis of the phenol compounds, and comprises the following specific steps:
(1) Preparing an array sensor: taking characteristic spectra at different time and different time as each sensing unit of the array sensor to prepare a sensor array with multiple sensing units, constructing a colorimetric array by using the laccase property of nucleotide and copper coordination combined nano-enzyme, wherein each sensing unit consists of reaction time and optimal wavelength, and the sensing unit 1: the characteristic spectra are 383 nm, 394 nm, 398 nm, 414 nm and 465nm respectively when the reaction is carried out for 30 min; the sensing unit 2: reacting for 60min, wherein the characteristic spectra are respectively 360nm, 366 nm, 390 nm, 495 nm and 498nm; the sensing unit 3: characteristic spectra of the reaction at 120min are 377 nm, 381 nm, 407 nm, 421 nm and 509nm respectively;
(2) Discriminant analysis of different phenolic pollutants: adding a water solution of phenolic substances into the array sensor array in the step (1), mixing the water solution with the detection liquid in the array sensor array to generate a color development reaction, reading an ultraviolet absorption value of the detection liquid through an enzyme-labeling instrument, performing digital processing on color development results of different phenolic substances in a system, introducing obtained data into Matlab software, and operating a chemometrics algorithm to perform discriminant analysis to obtain a scoring graph of the discriminant results of different phenolic substances;
(3) Discrimination analysis of different concentrations of the same phenolic substance: preparing four phenol solutions with different concentrations, adding the four phenol solutions into the array sensor array in the step (1), mixing the four phenol solutions with the detection liquid in the array sensor array to generate a color development reaction, reading an ultraviolet absorption value of the detection liquid through an instrument, carrying out digital processing on color development results of different phenols in a system, and introducing obtained data into Matlab software for discriminant analysis to obtain a score chart of the discriminant results of the different phenols;
(4) And (3) performing discriminant analysis after mixing of two phenolic substances: mixing two phenolic substances in the same proportion, adding the mixed solution into the array sensor array in the step (1), mixing the mixed solution with the detection solution in the array sensor array to generate a color development reaction, reading an ultraviolet absorption value of the detection solution through an instrument, carrying out digital processing on color development results of different phenolic substances in a system, and introducing obtained data into Matlab software for discriminant analysis to obtain a scoring graph of the discrimination results of different phenolic substances;
(5) And (3) distinguishing and analyzing phenolic substances in the actual sample: adding a standard solution of phenolic substances into sewage to obtain a system of an actual sample, adding the solution into the array sensor array in the step (1), mixing the solution with the detection liquid in the array sensor array to generate a color reaction, reading the ultraviolet absorption value of the detection liquid through an instrument, carrying out digital processing on the color development results of different phenolic substances in the system, and introducing the obtained data into Matlab software for discriminant analysis to obtain a score map of the discriminant results of different phenolic substances.
2. The method for analyzing a phenolic compound based on polyphenol oxidase activity nanoenzyme according to claim 1, wherein polyphenol oxidase is synthesized by mixing a mixture of 10mM of 5' -disodium guanylate solution, 50mM of copper chloride solution, 100mm of Tris-HCL buffer solution, ph =8.5 and ultrapure water at a ratio of 2,000 rpm in a centrifuge for 5 min; washing the generated precipitate with ultrapure water for 3 times, and finally uniformly dispersing the precipitate in the ultrapure water to obtain the synthesized nano enzyme.
3. The method for analyzing phenolic compounds based on polyphenol oxidase activity nanoenzymes of claim 1, wherein the preparation of the array sensor comprises: forming a system by using a nano enzyme solution, a MES buffer solution 100mM, a pH =7, and 10mg/ml of 4-aminopyridine, ultrapure water and a phenolic substance solution; for hydroquinone, catechol, resorcinol, phenol, phloroglucinol and parachlorophenol, the addition amount of each solution in a 1ml system is 100 mul of nano enzyme, 100 mul of MES buffer solution, 100 mul of 4-aminopyridine, 100 mul of ultrapure water and 100 mul of phenol solution respectively; in 1ml of the system, 2,4-dichlorophenol was added in an amount of 100. Mu.L of nanoenzyme, 100. Mu.L of MES buffer, 100. Mu.L of 4-aminopyridine, 200. Mu.L of ultrapure water and 500. Mu.L of phenol solution.
4. The method for analyzing phenolic compounds based on polyphenol oxidase activity nanoenzyme as claimed in claim 1, wherein the phenolic substances in step (2) are hydroquinone, catechol, resorcinol, phenol, phloroglucinol, p-chlorophenol with concentration of 1mg/ml, and 2, 4-dichlorophenol with concentration of 0.2mg/ml; the concentrations of the phenol in the step (3) in the reaction system are respectively 1, 10, 100 and 1000 mu g/mL; and (4) mixing the two phenolic substances in the step (4) in the same proportion respectively to obtain phenol +2,4-DP, phloroglucinol + p-chlorophenol, hydroquinone + phloroglucinol, catechol + phenol, hydroquinone +2,4-DP and resorcinol + p-chlorophenol.
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