CN110887829A - Nanolase-surface enhanced Raman substrate, fluorine ion detection kit and application thereof - Google Patents
Nanolase-surface enhanced Raman substrate, fluorine ion detection kit and application thereof Download PDFInfo
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- 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
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention discloses a nanoenzyme-surface enhanced Raman substrate, a fluorine ion detection kit and application thereof. The nanoenzyme-surface enhanced Raman substrate comprises R-MnCo2O4@ Au complex. The preparation method of the nanoenzyme-surface enhanced Raman substrate comprises the following steps: to MnCo2O4The nano tube is subjected to chemical reduction treatment to obtain MnCo with oxygen vacancy-rich surface2O4Nanotubes, then on MnCo2O4Growing Au nano particles in situ at oxygen vacancies of the nano tube to form R-MnCo2O4@ Au complex to obtain the nano enzyme-surface enhanced Raman substrate. The invention also discloses a fluorine ion detection kit and a fluorine ion detection method. The nanoenzyme-surface enhanced Raman substrate has SERS activity,the detection kit has the catalytic activity of similar oxidase and similar peroxidase, and the detection kit for the fluorine ions has extremely high sensitivity to the detection of the fluorine ions.
Description
Technical Field
The invention relates to a nano enzyme-surface enhanced Raman substrate and a preparation method thereof, a corresponding kit for rapidly detecting fluorine ions by using a nano enzyme-surface enhanced Raman system, and application of the kit in detecting fluorine ions, and belongs to the technical field of Raman spectrum detection.
Background
Fluorine is the smallest anion and is one of the trace elements necessary for human body, and has important influence on human body functions. It is also an essential component in the processes of formation, metabolism and the like of human teeth and bones, and exists in organs, cells and even cell sap of a human body. To ensure that the human body is sufficiently enriched with fluoride ions, fluorine is often used as an important additive in toothpaste, medicines and drinking water. However, if the fluorine content in the body is excessive, a series of diseases such as dental fluorosis and fluorosis can be caused. Fluorine is widely present in natural water, the concentration of fluoride ions in human bodies is mainly determined by the external environment, the drinking water contains 2.4-5mg/L of fluorine bone disease can occur, high-concentration sodium fluoride can interfere normal cell metabolism, and 6-12 g of sodium fluoride can kill the disease. Various diseases caused by fluorine, such as chronic poisoning of teeth and bones, calculus, acute stomach diseases, renal dysfunction and the like, are increasing, and the fluorine poses a serious threat to the health of human beings.
Therefore, the development of a kit capable of rapidly detecting the fluoride ions and the content thereof has extremely important significance for the prevention and diagnosis of diseases caused by the fluoride.
Disclosure of Invention
The invention mainly aims to provide a nano enzyme-surface enhanced Raman substrate, a preparation method and application thereof, so as to overcome the defects of the prior art.
The invention also aims to provide a fluorine ion detection kit based on the nanoenzyme-surface enhanced Raman substrate.
Another object of the present invention is to provide a method for detecting fluorine ions.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a nanoenzyme-surface enhanced Raman substrate which comprises R-MnCo2O4@ Au complex.
In some embodiments, the R-MnCo2O4The @ Au complex is in MnCo2O4R-MnCo generated by chemically reducing nano tube2O4And Au nano particles grow in situ at the oxygen vacancy.
The embodiment of the invention also provides a preparation method of the nano enzyme-surface enhanced Raman substrate, which comprises the following steps:
to MnCo2O4The nano tube is subjected to chemical reduction treatment to obtain MnCo with oxygen vacancy-rich surface2O4A nanotube;
making the MnCo2O4Mixing and reacting the nanotubes and the gold source to form a mixture in MnCo2O4Growing Au nano particles in situ at oxygen vacancies of the nano tube to form R-MnCo2O4And @ Au compound, namely obtaining the nano enzyme-surface enhanced Raman substrate.
The embodiment of the invention also provides application of the nanoenzyme-surface enhanced Raman substrate in the field of fluorine ion detection.
The embodiment of the invention also provides a fluorine ion detection reagent, which comprises the nano enzyme-surface enhanced Raman substrate and 3,3',5,5' -tetramethylbenzidine color development liquid.
The embodiment of the invention also provides a fluorine ion detection kit, which comprises the nano enzyme-surface enhanced Raman substrate or the fluorine ion detection reagent.
Accordingly, the embodiment of the present invention further provides a method for detecting fluoride ions, which is implemented by the kit described above, and the method includes:
and in the presence of hydrogen peroxide and 3,3',5,5' -tetramethylbenzidine color developing solution, applying the solution to be detected possibly containing fluorine ions to the nano enzyme-surface enhanced Raman substrate for reaction, detecting by using a Raman spectrometer, recording the detection result, and realizing the detection of the solution to be detected.
Compared with the prior art, the invention has the advantages that:
1) the MnCo in the nanoenzyme-surface enhanced Raman substrate provided by the invention2O4The preparation of the nano tube adopts an electrostatic spinning technology and a roasting method, the reaction is simple and convenient, the time and the efficiency are saved, the problems of high reaction temperature and long reaction time of a high-temperature solid phase method and a sol-gel method are solved, sodium borohydride is used to ensure that the surface of the nano tube is rich in oxygen vacancies, and reduced MnCo is generated2O4Nanotube (R-MnCo)2O4) Abundant electrons generated by oxygen vacancies are beneficial to reducing metal ions into metal nano particles, and the difficulty that the cobalt-manganese composite oxide synthesized by the traditional method is difficult to be further compounded with other substances is overcome;
2) the nano enzyme-surface enhanced Raman substrate provided by the invention is a nano enzyme, has catalytic activity, does not need to be additionally compounded with other enzymes to enable the nano enzyme to be functionalized, and simultaneously has high-activity surface enhanced Raman performance;
3) the nanoenzyme-surface enhanced Raman substrate provided by the invention has SERS activity, and also has oxidase-like enzyme and peroxidase-like enzyme catalytic activity, the catalytic activity can catalyze the charge transfer reaction of TMB, the SERS activity can greatly enhance the Raman signal of TMB oxidation products, can catalyze the color reaction, and realize trace detection of fluorine ions;
4) the fluorine ion detection kit provided by the invention has in-situ non-destructive property, can be recycled for other tests, has small dosage, short reaction time and low cost, and can monitor the reaction process in situ in real time;
5) the minimum detection concentration of the fluorine ion detection kit provided by the invention is 0.1nmol/L, and the fluorine ion concentration in the range of 100nmol/L-0.1nmol/L can be rapidly detected, so that the kit is proved to have extremely high sensitivity for fluorine ion detection, can realize qualitative and quantitative analysis of the fluorine ion concentration, and has a good linear relation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1a and 1b are SERS spectra obtained by time monitoring using SERS according to example 1 of the present invention.
FIGS. 2a and 2b are SERS spectra after adding different concentrations of fluoride ion according to example 2 of the present invention.
Detailed Description
The nano enzyme is a functional nano material with enzyme-like catalytic activity, and compared with natural enzyme and other artificial simulated enzymes, the nano enzyme has the advantages of high stability, large-area utilization and modification of biomolecules, multiple functions and the like. The Surface Enhanced Raman Spectroscopy (SERS) effect is very widely applied, has the advantages of good selectivity, high sensitivity, in-situ nondestructive detection and the like, can quickly obtain an analysis result, and can monitor the reaction process in real time. Compared with many traditional fluorine ion detection methods, the electrode method has the advantages of more interference factors and poor repeatability, the chromatography method and the fluorescence method have longer time, and the nanoenzyme-surface enhanced Raman system has unique advantages due to quick reaction, high sensitivity and good selectivity. Therefore, the preparation of the nanoenzyme-surface enhanced Raman substrate has very important significance for rapidly detecting the fluorine ions by applying the surface enhanced Raman effect.
In view of the defects of the prior art, the inventor of the present invention has made extensive studies and extensive practices to provide a kit for rapidly detecting fluoride ions by using a nanoenzyme-surface enhanced raman system, wherein the kit relates to detection of fluoride ions by using the nanoenzyme-surface enhanced raman system, and specifically, the prepared high-activity surface enhanced raman R-MnCo is used for detection of fluoride ions2O4The reagent kit of the substrate solution of the @ Au (per) oxide enzyme compound inhibits TMB (Tetramethylbenzidine) color reaction through fluorine ions, and rapidly detects SERS (surface enhanced Raman spectroscopy) spectrum signals of the fluorine ions by utilizing a nanoenzyme-surface enhanced Raman system.
The technical solution, its implementation and principles, etc. will be further explained as follows.
The technical solution of the present invention will be explained in more detail below. It is to be understood, however, that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with one another to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
An aspect of an embodiment of the present invention provides a nanoenzyme-surface enhanced raman substrate including R-MnCo2O4The @ Au class (per) oxide enzyme complex.
In some embodiments, the R-MnCo2O4The @ Au (per) oxide enzyme complex is formed in MnCo2O4R-MnCo generated by chemically reducing nano tube2O4And Au nano particles grow in situ at the oxygen vacancy.
Further, the nano enzyme-surface enhanced Raman substrate is prepared by adopting an electrostatic spinning technology to prepare MnCo2O4The surface of the nanotube is enriched with oxygen vacancy by a chemical reduction method, and the MnCo is enriched with oxygen vacancy2O4The nanotubes are called Reduced-MnCo2O4(as: R-MnCo)2O4) Finally growing Au nano particles in situ at oxygen vacancy to form R-MnCo2O4The @ Au class (per) oxide enzyme complex.
Furthermore, the diameter of the Au nano particles is 10-30 nm.
The nano enzyme-surface enhanced Raman substrate provided by the invention is a nano enzyme, has catalytic activity, does not need to be additionally compounded with other enzymes to be functionalized, and also has high-activity surface enhanced Raman performance.
The nanoenzyme-surface enhanced Raman substrate provided by the invention has SERS activity, and also has catalytic activity of an oxidase-like enzyme and a peroxidase-like enzyme, the catalytic activity can catalyze charge transfer reaction of TMB, the SERS activity can greatly enhance Raman signals of TMB oxidation products, and can catalyze color reaction to realize trace detection of fluorine ions.
Another aspect of the embodiments of the present invention also provides a method for preparing a nanoenzyme-surface enhanced raman substrate, including:
to MnCo2O4The nano tube is subjected to chemical reduction treatment to obtain MnCo with oxygen vacancy-rich surface2O4A nanotube;
making the MnCo2O4Mixing and reacting the nanotubes and the gold source to form a mixture in MnCo2O4Growing Au nano particles in situ at oxygen vacancies of the nano tube to form R-MnCo2O4And @ Au compound, namely obtaining the nano enzyme-surface enhanced Raman substrate.
In some embodiments, the preparation method comprises: subjecting the MnCo to heat treatment2O4Uniformly mixing the nanotube and sodium borohydride, and reacting at room temperature (25 ℃) for 10-20 hours to obtain MnCo with oxygen vacancy-rich surface2O4A nanotube.
Further, the MnCo2O4The mass ratio of the nanotube to the sodium borohydride is 1-3: 50-200.
Further, the MnCo2O4The nanotube is synthesized by electrostatic spinning technology and roasting method.
In some embodiments, the preparation method comprises: dropwise adding a gold source solution to the MnCo with the oxygen vacancy-rich surface2O4Reacting in a nanotube at room temperature (25 ℃) for 15-60 min to obtain R-MnCo2O4The @ Au class (per) oxide enzyme complex.
Further, the gold source solution includes a chloroauric acid solution, but is not limited thereto.
Further, the MnCo2O4The mass ratio of the nanotube to the gold source is 1: 1-2: 1.
Further, the dropping rate of the gold source solution is 10-30 seconds per drop.
In a more specific embodiment, the preparation method specifically includes:
weighing 0.05-0.2 g of sodium borohydride solid and 1-3 mg of MnCo synthesized by electrostatic spinning technology and roasting method2O4Slightly grinding the nanotube in an agate mortar, placing the nanotube in a room temperature for 3-6 h, adding 1ml of ultrapure water, carrying out ultrasonic treatment for 30s, standing for 10-20 h, carrying out centrifugal washing (5-10 times), dispersing the washed nanotube in 3-6 ml of ultrapure water, placing the solution on a magnetic stirring table for stirring, and dropwise adding 0.3-0.5 ml of ultrapure water with the concentration of 4 multiplied by 10 by using a syringe-3Dropwise adding the chloroauric acid solution at the dropping speed of 10-30 seconds per drop, stirring for 15-60 min, and then centrifuging (8000-10000 rad/min) to wash (5-10 times) to obtain R-MnCo2O4The @ Au class (per) oxide enzyme complex.
The MnCo in the nanoenzyme-surface enhanced Raman substrate provided by the invention2O4The preparation of the nano tube adopts an electrostatic spinning technology and a roasting method, the reaction is simple and convenient, the time and the efficiency are saved, the problems of high reaction temperature and long reaction time of a high-temperature solid phase method and a sol-gel method are solved, sodium borohydride is used to ensure that the surface of the nano tube is rich in oxygen vacancies, and reduced MnCo is generated2O4Nanotube (R-MnCo)2O4) The abundant electrons generated by the oxygen vacancy are beneficial to reducing metal ions into metal nano particles, and the difficulty that the cobalt-manganese composite oxide synthesized by the traditional method is difficult to be further compounded with other substances is overcome.
In another aspect of the embodiments of the present invention, an application of the nanoenzyme-surface enhanced raman substrate in the field of fluorine ion detection is also provided.
In another aspect of the embodiments of the present invention, there is also provided a reagent for detecting fluoride ions, which comprises the aforementioned nanoenzyme-surface enhanced raman substrate and a 3,3',5,5' -Tetramethylbenzidine (TMB) color developing solution.
Further, the fluorinion detection reagent consists of 3,3',5,5' -tetramethyl benzidine (TMB) color development liquid and R-MnCo2O4@ Au-based (per) oxide enzyme complex base solution, and R-MnCo2O4The @ Au (per) oxidase complex substrate serving as the nanoenzyme not only has oxidase-like and peroxidase-like activities, but also has strong SERS activity.
The fluorine ion detection reagent has the properties of similar oxidases and similar peroxidases, can catalyze a color reaction, and realizes trace detection of fluorine ions.
In another aspect of the embodiments of the present invention, a fluorine ion detection kit is further provided, which includes the nanoenzyme-surface enhanced raman substrate or the fluorine ion detection reagent.
Further, the fluorine ion detection kit comprises: the surface enhanced Raman scattering system comprises a nano enzyme-surface enhanced Raman substrate, 3',5,5' -tetramethylbenzidine color development liquid, hydrogen peroxide and acetic acid-sodium acetate buffer solution.
Further, the pH value of the acetic acid-sodium acetate buffer solution is 4.0-4.2.
Furthermore, the concentration of the 3,3',5,5' -tetramethylbenzidine color development solution is 0.2-1 mmol/L.
Further, firstly, preparing dimethyl sulfoxide solution of 3,3',5,5' -tetramethylbenzidine, and then diluting with acetic acid-sodium acetate buffer solution with pH value of 4.0-4.2 to obtain 3,3',5,5' -tetramethylbenzidine color developing solution with concentration of 0.2-1 mmol/L.
Furthermore, the invention is characterized in that TMB color development liquid, acetic acid-sodium acetate buffer solution and R-MnCo2O4Mixing the substrate solution of the @ Au (per) oxide enzyme complex and hydrogen peroxide in sequence, wherein the volume ratio of the acetic acid-sodium acetate buffer solution to the hydrogen peroxide to the TMB color developing solution to the substrate solution is 1:1:1:1, the excitation wavelength is 633nm at 25 ℃, and the test range is 1000--1And monitoring the SERS time under the conditions that the integration time is 10s and the integration frequency is 1, and determining the time required by the time to reach the reaction equilibrium as the time for later-stage SERS detection.
Furthermore, the minimum detection concentration of the fluorine ion detection kit on fluorine ions is 0.1nmol/L, and the detection concentration range is 0.1-100 nmol/L.
The fluorine ion detection kit provided by the invention has in-situ non-destructive property, can be recycled for other tests, has small dosage, short reaction time and low cost, and can monitor the reaction process in situ in real time.
Accordingly, another aspect of the embodiments of the present invention provides a method for detecting fluoride ions, which is implemented by the kit as described above, the method comprising:
and in the presence of hydrogen peroxide and 3,3',5,5' -tetramethylbenzidine color developing solution, applying the solution to be detected possibly containing fluorine ions to the nano enzyme-surface enhanced Raman substrate for reaction, detecting by using a Raman spectrometer, recording the detection result, and realizing the detection of the solution to be detected.
Further, the fluorine ion detection method of the present invention can be used for rapidly detecting fluorine ions and their contents in water, food, toothpaste, living organisms, and the like.
Further, the reaction temperature is room temperature, and the reaction time is 10-30 min.
In some embodiments, the fluoride ion detection method comprises: sequentially adding 3,3',5,5' -tetramethyl benzidine color developing solution and R-MnCo2O4The method comprises the following steps of (1) @ Au (per) oxide enzyme complex substrate solution, hydrogen peroxide and solution to be detected containing fluorine ions, wherein the volume ratio is 1:1:1:1, and SERS detection is carried out after the substrate solution is soaked for 10-30 min at the temperature of 25 ℃.
In conclusion, the invention prepares MnCo by adopting the electrostatic spinning technology at room temperature2O4Making the surface of the nano tube rich in oxygen vacancy by a chemical reduction method, and finally growing Au nano particles in situ at the oxygen vacancy to form R-MnCo2O4@ Au-based (per) oxide enzyme complex SERS substrate; fluoride ion inhibits TMB reaction and enables in situ monitoring of the reaction. The kit for rapidly detecting the fluorine ions by the nanoenzyme-surface enhanced Raman system is prepared through the steps, and the MnCo rich in oxygen vacancies2O4The nano tube loaded with Au nano particles not only has activities of an oxidase-like enzyme and a peroxidase-like enzyme, but also has strong SERS activity, and can react with TMB to generate a blue oxidation state TMB visible to naked eyes, and fluorine ions inhibit the oxidation of the TMB to enable the color of a product to be coloredIt became light blue and even colorless, which was monitored by SERS. The kit prepared by the method is simple and convenient in method, time-saving and efficient, and the ultralow detection limit of the fluoride ion detection is realized. Prepared R-MnCo2O4The @ Au (per) oxide enzyme complex SERS substrate has stable property, can be repeatedly used, is easy to store and is convenient to detect. The nano enzyme-surface enhanced Raman substrate provided by the invention has very important significance for detecting fluorine ions.
In some more specific embodiments, the kit of the present invention for rapidly detecting fluorine ions by nanoenzyme-surface enhanced raman substrate comprises the following specific steps: (a) preparing fluorine ion solutions to be detected with different concentrations; (b) preparation of R-MnCo2O4@ Au-based (per) oxide enzyme complex SERS substrate solution; (c) the optimal reaction time is obtained by monitoring the SERS time by using the kit, wherein a composite SERS substrate suspension is added into TMB color developing solution and hydrogen peroxide solution to obtain an SERS substrate-TMB color developing solution system, and surface enhanced Raman is used for in-situ real-time monitoring to obtain the time required for the reaction balance, and the time required for determining the time required for detecting fluorine ions is used for obtaining an SERS spectrogram; (d) the method realizes ultra-sensitive detection and ultra-low detection limit of fluorine ions, and adds the fluorine ions to be detected into an SERS substrate-TMB color developing liquid system for detecting the fluorine ions to obtain an SERS spectrogram.
The process of collecting the analyte containing fluoride ions described in the step (a) above: preparing a series of sodium fluoride solutions with different concentrations (0.1-100 nmol/L) for later use;
preparing R-MnCo in the step (b)2O4@ Au-based (per) oxide enzyme complex SERS substrate solution process: weighing 0.05-0.2 g of sodium borohydride solid and 1-3 mg of MnCo synthesized by electrostatic spinning technology and roasting method2O4Slightly grinding the nanotube in an agate mortar, placing the nanotube in a room temperature for 3-6 h, adding 1ml of ultrapure water, carrying out ultrasonic treatment for 30s, standing for 10-20 h, carrying out centrifugal washing (5-10 times), dispersing the washed nanotube in 3-6 ml of ultrapure water, placing the solution on a magnetic stirring table for stirring, and dropwise adding 0.3-0.5 ml of ultrapure water with the concentration of 4 multiplied by 10 by using a syringe-3The dropping speed of the mol/L chloroauric acid solution is about 10-30 seconds per dropStirring for 15-60 min, and centrifuging (8000-10000 rad/min) to wash (5-10 times) to obtain R-MnCo2O4The @ Au (per) oxide enzyme complex;
using the substrate for SERS time monitoring in step (c) above to obtain the optimal reaction time: preparing 2-5 mg/mL R-MnCo2O4@ Au (per) oxidase complex water solution, adding 30 mu L of the water solution into 970 mu L, pH ═ 4.0-4.2 acetic acid-sodium acetate buffer solution, and performing ultrasonic treatment for 30s to obtain R-MnCo with the concentration of 0.003mg/mL2O4@ Au-based (per) oxide enzyme complex solution. Preparing a dimethyl sulfoxide solution of 15 mmol/L3, 3',5,5' -Tetramethylbenzidine (TMB), and diluting with an acetic acid-sodium acetate buffer solution with the pH value of 4.0-4.2 to obtain a TMB color development solution with the concentration of 0.2-1 mmol/L; preparing 10 by using acetic acid-sodium acetate solution with pH of 4.0-4.2-3mol/L hydrogen peroxide;
mixing the R-MnCo prepared in the step (c)2O4Mixing the @ Au (per) oxide enzyme complex solution with TMB (tetramethylbenzidine) color developing solution and hydrogen peroxide solution which are equal in volume to obtain an SERS substrate-TMB color developing solution system; carrying out in-situ time monitoring by using surface enhanced Raman spectroscopy to obtain the optimal reaction time of 10-30 min;
the fluorine ion detection process in the step (d): taking multiple parts of 50-100 mu L of SERS substrate-TMB color developing liquid system solution, respectively and fully mixing with 10-50 mu L of fluoride ion aqueous solution with different concentrations (the concentration is 100nmol/L-0.1 nmol/L), reacting for 10-30 min at room temperature, and enabling a typical oxidases-like substrate TMB to generate a color-developed oxidation state TMB based on the property of the compound SERS substrate of oxidases-like enzyme and peroxidase-like enzyme.
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the technical solutions of the present invention are further explained below with reference to the drawings and several preferred embodiments, but the experimental conditions and the setting parameters therein should not be construed as limiting the basic technical solutions of the present invention. And the scope of the present invention is not limited to the following examples.
Example 1
1) 0.08g of sodium borohydride (NaBH) is weighed out4) Solid and 1mg MnCo synthesized by electrostatic spinning technology and roasting method2O4Slightly grinding the nanotube in agate mortar, standing at 25 deg.C for 6 hr, adding 1ml ultrapure water, ultrasonically treating for 30s, standing at 25 deg.C for 12 hr, centrifugally washing with ultrapure water (10 times), dispersing in 5ml ultrapure water, stirring on a magnetic stirring table, and adding 0.5ml of 4 × 10-3mol/L chloroauric acid (HAuCl)4) Dripping the solution at the speed of about 20 seconds per drop, stirring for 30min, centrifuging (10000rad/min), and washing (5 times) to obtain R-MnCo2O4The @ Au (per) oxidase complex was prepared as an aqueous solution at 3 mg/ml.
2) Preparing 3mg/mL R-MnCo2O4@ Au (per) oxidase complex water solution, adding 30 μ L of the water solution into 970 μ L, pH value of 4.2 acetic acid-sodium acetate buffer solution, and subjecting to ultrasonic treatment for 30s to obtain 0.003mg/mL R-MnCo2O4@ Au-based (per) oxide enzyme complex solution; preparing 15mmol/L dimethyl sulfoxide solution of 3,3',5,5' -Tetramethylbenzidine (TMB), adding 30 mu L of the aqueous solution into 970 mu L, pH value 4.2 acetic acid-sodium acetate buffer solution, and performing ultrasonic treatment for 30s to obtain 0.45mmol/L TMB color development solution; diluting the hydrogen peroxide solution to a concentration of 10 with an acetic acid-sodium acetate solution having a pH of 4.2-3A hydrogen peroxide solution of mol/L, and keeping the solution protected from light.
3) The prepared R-MnCo2O4And mixing the @ Au (per) oxide enzyme complex solution, the TMB color developing solution, the hydrogen peroxide solution and the acetic acid-sodium acetate solution according to the volume of 20 mu L, 20 mu L and 20 mu L respectively to obtain the SERS substrate-TMB color developing solution system. The obtained SERS substrate-TMB color developing liquid system is immediately placed into a LabRAMAN Ramis intelligent full-automatic Raman spectrometer for in-situ time monitoring, and the spectrum detection range is 1000-plus-one 1800cm-1Under the conditions of 633nm excitation wavelength, 10s integration time and 1 integration frequency, the initial reaction rate is slower, the detection is carried out once every 5min, and the reaction rate begins to increase after 10minAnd adding the mixture, and reacting until the reaction reaches the reaction equilibrium after 15min to obtain the optimal reaction time of 15 min. As shown in FIG. 1a, three characteristic peaks 1191, 1337 and 1611cm which are peculiar to TMB in oxidation state at 1, 5, 10, 14, 15, 16 and 17 minutes are respectively shown-1The intensity of (3) is changed, as shown in FIG. 1b, and the strongest peak 1611cm is selected-1And (3) making an intensity-time curve to obtain that the reaction equilibrium is reached in 15min, and taking the reaction time of 15min as the reaction time for detecting the fluorine ions by using the SERS substrate-TMB system.
Example 2
1) 0.05g of sodium borohydride (NaBH) is weighed out4) Solid and 1mg MnCo synthesized by electrostatic spinning technology and roasting method2O4Slightly grinding the nanotube in agate mortar, standing at 25 deg.C for 5 hr, adding 1ml ultrapure water, ultrasonically treating for 30s, standing at 25 deg.C for 10 hr, centrifugally washing with ultrapure water (5 times), dispersing in 3ml ultrapure water, stirring on a magnetic stirring table, and adding 0.35ml of 4 × 10-3mol/L chloroauric acid (HAuCl)4) The dropping speed of the solution is about 10 seconds per drop, the solution is stirred for 15min and then centrifuged (9000rad/min) to be washed (8 times), and R-MnCo is obtained2O4@ Au (per) oxidase complex was prepared as an aqueous solution of 2 mg/ml.
2) Preparing sodium fluoride aqueous solution with the concentration of 0, 0.1, 1, 10 and 100nmol/L respectively; preparing 2mg/mL R-MnCo2O4@ Au (per) oxidase complex water solution, adding 30 μ L of the water solution into 970 μ L, pH value of 4.1 acetic acid-sodium acetate buffer solution, and subjecting to ultrasonic treatment for 30s to obtain 0.003mg/mL R-MnCo2O4@ Au-based (per) oxide enzyme complex solution; preparing 15mmol/L dimethyl sulfoxide solution of 3,3',5,5' -Tetramethylbenzidine (TMB), adding 30 mu L of the aqueous solution into 970 mu L, pH value 4.1 acetic acid-sodium acetate buffer solution, and performing ultrasonic treatment for 30s to obtain 0.2mmol/L TMB color development solution; diluting the hydrogen peroxide solution to a concentration of 10 with an acetic acid-sodium acetate solution having a pH of 4.1-3M in hydrogen peroxide.
3) Respectively taking 20 mu L of R-MnCo2O4@ Au (per) oxidase complex solution, 20. mu.L, TMB color developing solutionMixing with 20 mu L of hydrogen peroxide solution to obtain an SERS substrate-TMB color developing solution system, and preparing 5 parts of SERS substrate-TMB color developing solution; respectively adding 20 μ L of fluoride ion aqueous solution with different concentrations (concentration of 0, 0.1, 1, 10, 100nmol/L) into the solution, mixing, reacting at 25 deg.C for 10min, and monitoring in situ with LabRAMAN Raman spectrometer with spectrum detection range of 1000 plus 1800cm-1The surface enhanced raman spectrum was measured at an excitation wavelength of 633nM, an integration time of 10s, and an integration frequency of 1, as shown in fig. 2a, where 0nM is a blank control spectrum of the raman intensity of the SERS substrate-TMB color developing solution system when an equal volume of aqueous solution containing no fluoride ion was added, and the other curves respectively show that the concentrations of the added fluoride ion were 0.1, 1, 10, and 100 nmol/L. The results show that with increasing fluoride ion concentration, three characteristic peaks 1191, 1337 and 1611cm of TMB in oxidation state-1The intensity of (2) is gradually reduced, as shown in fig. 2b, in the range of 0-100nmol/L, the intensities of the three characteristic peaks and the fluorine ion concentration show excellent linear relation, so that the fluorine ion concentration can be detected in situ by using SERS, and the ultra-sensitive detection of the fluorine ions is realized. As can be seen from the figure, the lower limit concentration of the detection on the fluorine ions can reach 0.1nmol/L, which indicates that the kit has extremely high sensitivity on the detection of the fluorine ions.
Example 3
1) 0.2g of sodium borohydride (NaBH) is weighed out4) Solid and 3mg MnCo synthesized by electrostatic spinning technology and roasting method2O4Slightly grinding the nanotube in agate mortar, standing at 25 deg.C for 3 hr, adding 1ml ultrapure water, ultrasonically treating for 30s, standing at 25 deg.C for 20 hr, centrifugally washing with ultrapure water (8 times), dispersing in 6ml ultrapure water, stirring on a magnetic stirring table, and adding 0.7ml of 4 × 10-3mol/L chloroauric acid (HAuCl)4) The dropping speed of the solution is about 30 seconds per drop, the solution is stirred for 60min and then is centrifugally washed (10 times) at 8000rad/min to obtain R-MnCo2O4The @ Au (per) oxidase complex was prepared as an aqueous solution of 5 mg/ml.
2) Preparing sodium fluoride aqueous solution with the concentration of 0, 0.1, 1, 10 and 100nmol/L respectively; preparing 5mg/mL R-MnCo2O4@ Au (per) oxidase complex water solution, adding 30 μ L of the water solution into 970 μ L, pH value of 4.0 acetic acid-sodium acetate buffer solution, and subjecting to ultrasonic treatment for 30s to obtain 0.003mg/mL R-MnCo2O4@ Au-based (per) oxide enzyme complex solution; preparing 15mmol/L dimethyl sulfoxide solution of 3,3',5,5' -Tetramethylbenzidine (TMB), adding 30 mu L of the aqueous solution into 970 mu L, pH value 4.0 acetic acid-sodium acetate buffer solution, and performing ultrasonic treatment for 30s to obtain 1mmol/L TMB color development solution; diluting the hydrogen peroxide solution to a concentration of 10 with an acetic acid-sodium acetate solution having a pH of 4.0-3M in hydrogen peroxide.
3) Respectively taking 20 mu L of R-MnCo2O4Mixing the @ Au (per) oxide enzyme complex solution, 20 mu L of TMB (tetramethylbenzidine) color developing solution and 20 mu L of hydrogen peroxide solution to obtain an SERS substrate-TMB color developing solution system, and preparing 5 parts of SERS substrate-TMB color developing solution; respectively adding 20 μ L of fluoride ion aqueous solution with different concentrations (concentration of 0, 0.1, 1, 10, 100nmol/L) into the solution, mixing, reacting at 25 deg.C for 30min, and monitoring in situ with LabRAMAN Raman spectrometer with spectrum detection range of 1000 plus 1800cm-1The surface enhanced raman spectrum was measured at 633nm excitation wavelength, 10s integration time, 1 integration times and was similar to fig. 2a and 2 b.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
In addition, the inventors of the present invention have also made experiments with other materials, process operations, and process conditions described in the present specification with reference to the above examples, and have obtained preferable results.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. A nanoenzyme-surface enhanced Raman substrate is characterized by comprising R-MnCo2O4@ Au complex.
2. The nanoenzyme-surface enhanced raman substrate of claim 1, wherein: the R-MnCo2O4The @ Au complex is in MnCo2O4R-MnCo generated by chemically reducing nano tube2O4The oxygen vacancy is prepared after Au nano particles grow in situ; and/or the diameter of the Au nano particles is 10-30 nm.
3. A preparation method of a nanoenzyme-surface enhanced Raman substrate is characterized by comprising the following steps:
to MnCo2O4Nanotube feedingPerforming chemical reduction treatment to obtain MnCo with oxygen vacancy-rich surface2O4A nanotube;
making the MnCo2O4Mixing and reacting the nanotubes and the gold source to form a mixture in MnCo2O4Growing Au nano particles in situ at oxygen vacancies of the nano tube to form R-MnCo2O4And @ Au compound, namely obtaining the nano enzyme-surface enhanced Raman substrate.
4. The production method according to claim 3, characterized by comprising: subjecting the MnCo to heat treatment2O4Uniformly mixing the nanotube and sodium borohydride, and reacting at room temperature for 10-20 h to obtain MnCo with the surface rich in oxygen vacancies2O4A nanotube; preferably, the MnCo2O4The mass ratio of the nanotube to the sodium borohydride is 1-3: 50-200; preferably, the MnCo2O4The nanotube is synthesized by electrostatic spinning technology and roasting method.
5. The production method according to claim 3, characterized by comprising: dropwise adding a gold source solution to the MnCo with the oxygen vacancy-rich surface2O4Reacting in a nanotube at room temperature for 15-60 min to obtain R-MnCo2O4@ Au complex; preferably, the gold source solution comprises a chloroauric acid solution; preferably, the MnCo2O4The mass ratio of the nanotube to the gold source is 1: 1-2: 1; preferably, the dropping rate of the gold source solution is 10-30 seconds per drop.
6. Use of the nanoenzyme-surface enhanced raman substrate of any one of claims 1-2 in the field of fluoride ion detection.
7. A fluorine ion detecting reagent, characterized by comprising the nanoenzyme-surface enhanced raman substrate according to any one of claims 1 to 2 and a 3,3',5,5' -tetramethylbenzidine color developing solution.
8. A fluorine ion detection kit, characterized by comprising the nanoenzyme-surface enhanced raman substrate of any one of claims 1 to 2 or the fluorine ion detection reagent of claim 7.
9. The fluoride ion detection kit according to claim 8, characterized by comprising: the method comprises the following steps of (1) preparing a nano enzyme-surface enhanced Raman substrate, 3',5,5' -tetramethylbenzidine color development liquid, hydrogen peroxide and acetic acid-sodium acetate buffer solution; preferably, the pH value of the acetic acid-sodium acetate buffer solution is 4.0-4.2; preferably, the concentration of the 3,3',5,5' -tetramethylbenzidine color developing solution is 0.2-1 mmol/L; and/or the lowest detection concentration of the fluorine ion detection kit on fluorine ions is 0.1 nmol/L; preferably, the detection concentration of the fluorine ion detection kit on fluorine ions is 0.1-100 nmol/L.
10. A method for detecting fluoride ions, which is carried out using the kit according to claim 8 or 9, said method comprising:
in the presence of hydrogen peroxide and 3,3',5,5' -tetramethylbenzidine color developing solution, applying a solution to be detected possibly containing fluorine ions on the nanoenzyme-surface enhanced Raman substrate for reaction, detecting by using a Raman spectrometer, recording the detection result, and realizing the detection of the solution to be detected; preferably, the reaction temperature is room temperature, and the reaction time is 10-30 min.
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