CN111992224B - Two-dimensional cobalt oxide stable rhodium nano mimic enzyme and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of catalytic detection of a nano mimic enzyme, in particular to a two-dimensional cobalt oxide stable rhodium nano mimic enzyme and a preparation method and application thereof. Based on the ultra-large specific surface area of the two-dimensional cobalt oxide, the good catalytic performance of the rhodium nanoparticles and the excellent synergistic enhancement effect of the two-dimensional cobalt oxide and the good catalytic performance of the rhodium nanoparticles, the cobalt ions are reduced into the rhodium nanoparticles and adsorbed on the surface of the two-dimensional cobalt oxide while forming the ultra-thin two-dimensional cobalt oxide by the aid of a surfactant CTAB (cetyl trimethyl ammonium bromide) assisted one-pot method, and the two-dimensional cobalt oxide stable rhodium nano mimic enzyme, 2D CoO @ Rh NC is obtained. The 2D CoO @ Rh NC is successfully applied to visible colorimetric detection of p-aminophenol in an environmental water sample or urea in a water sample, soil and urine, and has the advantages of simplicity in operation, good selectivity, high sensitivity, strong visibility and the like.

Description

Two-dimensional cobalt oxide stable rhodium nano mimic enzyme and preparation method and application thereof

Technical Field

The invention relates to the technical field of catalytic detection of nano mimic enzyme, in particular to a two-dimensional cobalt oxide stable rhodium nano mimic enzyme and a preparation method and application thereof.

Background

The nano mimic enzyme serving as an efficient biomimetic catalyst has low cost and high stability, can catalyze various reactions under mild conditions, has high substrate specificity, selectivity and catalysis efficiency, and is widely applied to the fields of photodynamic therapy, biomimetic catalysis, biosensing, environmental analysis and detection and the like.

Urea is an important nitrogen-containing organic compound and is widely applied to the field of agriculture. P-aminophenol is used in the pharmaceutical industry for the synthesis of paracetamol, clofibrate, etc., and the united states and chinese pharmacopoeias limit the maximum content of p-aminophenol in a medicament to 50ppm. Therefore, the development of a method for detecting urea and p-aminophenol efficiently, conveniently and sensitively has important significance.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problem to be solved by the invention is to provide a two-dimensional cobalt oxide stable rhodium nano mimic enzyme and a preparation method and application thereof, wherein the two-dimensional cobalt oxide stable rhodium nano mimic enzyme (2D CoO @ Rh NC) is prepared by a surfactant CTAB assisted one-pot reduction method, and the catalytic activity of the oxidation mimic enzyme and the visibility and sensitivity of colorimetric detection of urea and p-aminophenol are improved.

The technical scheme adopted by the invention for realizing the purpose is as follows: based on the ultra-large specific surface area of two-dimensional cobalt oxide, the good catalytic performance of rhodium nanoparticles and the excellent synergistic enhancement effect of the two, the two-dimensional cobalt oxide-stabilized rhodium nano mimic enzyme is prepared by a surfactant CTAB (cetyl trimethyl ammonium bromide) assisted one-pot method reduction, so that when cobalt ions form ultra-thin two-dimensional cobalt oxide, the rhodium ions are reduced into rhodium nanoparticles and adsorbed on the surface of the two-dimensional cobalt oxide, and the two-dimensional cobalt oxide-stabilized rhodium nano mimic enzyme, 2D CoO @ Rh NC, is obtained.

The invention comprises a preparation method of a two-dimensional cobalt oxide stable rhodium nanometer mimic enzyme, which comprises the following steps:

(1) Weighing Co (NO) ₃ ) ₂ ·9H ₂ O、RhCl ₃ ·3H ₂ Adding O and hexadecyl trimethyl ammonium bromide into a beaker, adding water, and then, carrying out ultrasonic stirring to fully dissolve;

(2) At room temperature, naBH is added ₄ Dissolving in water, slowly dripping into the mixed solution obtained in the step (1), and fully stirring for reaction;

(3) And (3) centrifugally separating the reaction mixed solution, fully washing the obtained solid with ethanol and deionized water, and drying in vacuum to obtain black two-dimensional cobalt oxide stable rhodium nano mimic enzyme, 2D CoO @ Rh NC.

Further, the synthetic mechanism of the 2D CoO @ Rh NC is as follows:

the high concentration surfactant CTAB is easy to form a positively charged double-layer CTAB micelle, and is prepared from NaBH ₄ Hydrolysis to give negatively charged OH ^– And positively charged Co ²⁺ By virtue of being strongThe electrostatic action is absorbed on the surface of the bilayer in sequence to form a curved Co oxide nanosheet; due to Rh ³⁺ /Rh ⁰ Has a much higher redox potential than Co ²⁺ Co0, in the presence of a reducing agent sodium borohydride (NaBH) ₄ ) In the presence of Rh ³⁺ Is reduced into rhodium nano particles and is uniformly adsorbed on the surface of the Co oxide nanosheet to form the two-dimensional cobalt oxide stable rhodium nano mimic enzyme.

Further, said Co (NO) ₃ ) ₂ ·9H ₂ O，RhCl ₃ ·3H ₂ O, CTAB and NaBH ₄ The molar ratio of (1), (0.1-0.3) to (1-2) to (2-3), and the reaction time of the step (2) is 5-15min.

Further, the preparation method specifically comprises the following steps:

(1) Weighing 1mmol of Co (NO) ₃ ) ₂ ·9H ₂ O、0.2mmol RhCl ₃ ·3H ₂ O and 1.4mmol CTAB in a small 100mL beaker, 25mLH was added ₂ O, ultrasonic stirring for 30min to fully dissolve;

(2) 2.6mmol of NaBH ₄ Dissolving in 15mLH ₂ Slowly dripping the mixture into the mixed solution obtained in the step (1), and fully stirring and reacting for 10min at room temperature;

(3) After centrifugal separation, the solid is fully washed by ethanol and deionized water, and vacuum drying is carried out at 60 ℃ to obtain black 2D CoO @ Rh NC mimic enzyme.

The invention comprises application of two-dimensional cobalt oxide stable rhodium nano mimic enzyme, and the two-dimensional cobalt oxide stable rhodium nano mimic enzyme can be used for colorimetric detection of urea and p-aminophenol simultaneously.

Further, the detecting step is:

(1) Preparing a standard solution: preparing 2D CoO @ Rh NC mimic enzyme;

(2) Preparation of actual samples:

randomly measuring 1 part of each water sample in different environments, filtering the water samples for three times by a 4-micron microporous filtering membrane, and distilling and concentrating to obtain water samples in different environments;

randomly weighing 1 part of each soil sample in different environments, ultrasonically leaching for 1 week by using water, and filtering for three times by using a microporous filtering membrane with the diameter of 4 mu m to prepare different soil samples;

sampling to obtain one part of urine of adults at different time, and filtering for three times by a 4-micron microporous filtering membrane for later use;

(3) Determination of urea and p-aminophenol in the sample:

putting a 2D CoO @ Rh NC mimic enzyme standard solution into a centrifuge tube, adding a sample to be tested and TMB, fixing the volume by using a disodium hydrogen phosphate-citric acid buffer solution with the pH =3.5, uniformly mixing, standing at room temperature for a period of time, and measuring an absorption spectrum within the range of 250-800 nm;

calculating the urea content in the sample to be detected according to the linear relation between the logarithm of the absorbance ratio at the wavelength of 652nm and 436nm and the urea concentration; and calculating the concentration of the p-aminophenol in the sample to be detected according to a linear equation between the logarithm of the absorbance ratio at 283nm and at 652nm or at 371nm and the concentration of the p-aminophenol.

Further, the reaction conditions in the step (3) are as follows: c. C _{2D CoO@Rh NC} ＝20μg/mL，pH＝3.5，c _TMB =0.05mM, reaction time 50min;

the 2D CoO @ Rh NC-TMB reaction system in the step (3) has specific selective spectral response to urea and p-aminophenol and is accompanied by color change from visible blue to brown yellow or from blue to colorless;

in the step (3), the linear ratio relationship exists between the absorption spectrum intensity of the 2D CoO @ Rh NC-TMB reaction system and the concentration of urea or p-aminophenol.

Further, the reaction mechanism of the detection is as follows:

the CoO @ Rh NC mimic enzyme has high-efficiency oxidation mimic enzyme activity, can catalyze and oxidize TMB in the presence of air at room temperature to generate blue oxTMB, has special reduction performance on aminophenol, and can selectively reduce the blue oxide oxTMB into colorless TMB; the urea can reduce the blue oxide oxTMB into colorless TMB and further form brown yellow imine through the action of free amino and carbonyl; thereby realizing the colorimetric detection of urea and p-aminophenol simultaneously by enzyme catalysis.

The two-dimensional cobalt oxide stable rhodium nano mimic enzyme, the preparation method and the application thereof have the beneficial effects that:

based on the ultra-large specific surface area of the two-dimensional cobalt oxide and the good catalytic performance of the rhodium nanoparticles, especially the excellent synergistic enhancement effect between the two, the invention prepares a two-dimensional cobalt oxide stabilized rhodium nano mimic enzyme (2D CoO @ Rh NC) by the reduction of a surfactant CTAB assisted one-pot method, optimizes the experimental conditions on the basis of discussing the catalytic activity mechanism thereof, applies the 2D CoO @ Rh NC mimic enzyme to the colorimetric detection of urea and p-aminophenol, effectively detects the visibility of the urea in water environment, soil and urine and the p-aminophenol in an environmental water sample, avoids the interference of other components in the sample, and has strong visibility, simple and convenient operation, good selectivity and high sensitivity. The dual-mode ratiometric probe can also effectively reduce background interference absorption, and improve the accuracy and precision of detection results.

Drawings

FIG. 1 is a schematic diagram of the synthetic route of 2D CoO @ Rh NC according to the embodiment of the present invention;

FIG. 2 is a schematic diagram of the mechanism for simultaneously colorimetric detection of urea and p-aminophenol in the embodiment of the invention under 2D CoO @ Rh NC;

in FIG. 3:

(a) A TEM image of the structure representation of the 2D CoO @ Rh NC of the embodiment of the invention;

(b) The invention discloses an HR-TEM image of structural representation of 2D CoO @ Rh NC;

(c) The EDS diagram represents the structure of the 2D CoO @ Rh NC of the embodiment of the invention;

(d) The invention is a 3RD diagram of the structural representation of 2D CoO @ Rh NC of the embodiment of the invention;

in fig. 4:

(a) The ultraviolet-visible absorption spectrogram and the corresponding color change chart of the 2D CoO @ Rh NC-TMB system of the embodiment of the invention;

(b) The time response curve diagram of the 2D CoO @ Rh NC-TMB system of the embodiment of the invention at 652nm is shown;

in fig. 5:

(a) Is a line graph of the influence of different pH values on the 2D CoO @ Rh NC catalytic oxidation TMB in the embodiment of the invention;

(b) Is a bar graph of the influence of the rhodium-cobalt molar ratio in different composite materials of the embodiment of the invention on the catalytic oxidation of TMB by 2D CoO @ Rh NC;

(c) Is a line graph of the influence of different temperatures on the 2D CoO @ Rh NC catalytic oxidation TMB in the embodiment of the invention;

(d) Is a bar graph of the effect of different temperatures on the 2D CoO @ Rh NC catalytic oxidation TMB in the examples of the invention;

(e) Is a line graph of the influence of different TMB contents on the 2D CoO @ Rh NC catalytic oxidation TMB in the embodiment of the invention;

(f) Is a line graph of the influence of different reaction times on the catalytic oxidation of the 2D CoO @ Rh NC TMB in the embodiment of the invention;

FIG. 6 shows the effect of a common interfering substance on colorimetric identification of urea and p-aminophenol in a 2D CoO @ Rh NC-TMB system and the corresponding color change thereof in an embodiment of the present invention;

in fig. 7:

(a) UV-vis spectral titration curves for colorimetric identification of urea and corresponding color changes (c) for embodiments of the invention _Urea ：6,9,15,30,45,60,75,90,105,120,135,150,165μM)；

(b) The linear relation curve of lg (A652/A436) and urea with different concentrations in the embodiment of the invention is shown;

(c) UV-vis spectral titration curves for colorimetric identification of p-aminophenol and the corresponding color changes (c) for the inventive examples _{P-aminophenol} ：1.7,3.3,15,25,35,45,55,65,75,85,95,105μM)；

(d) Is a linear relation curve of Lg (A283/A652) and Lg (A283/A371) and c-aminophenol in the embodiment of the invention.

Detailed Description

The invention is further explained in detail with reference to the drawings and the specific embodiments;

example 1:

a two-dimensional cobalt oxide stabilized rhodium nanometer mimic enzyme is prepared by a surfactant CTAB assisted one-pot reduction method based on the ultra-large specific surface area of two-dimensional cobalt oxide, the good catalytic performance of rhodium nanoparticles and the excellent synergistic enhancement effect of the two-dimensional cobalt oxide and enables cobalt ions to form ultra-thin two-dimensional cobalt oxide, and meanwhile, the rhodium ions are reduced into rhodium nanoparticles and adsorbed on the surface of the two-dimensional cobalt oxide, so that the two-dimensional cobalt oxide stabilized rhodium nanometer mimic enzyme is obtained, and the 2D CoO @ Rh NC is obtained.

The invention comprises a preparation method of a two-dimensional cobalt oxide stable rhodium nano mimic enzyme, which comprises the following preparation steps:

(1) Weighing Co (NO) ₃ ) ₂ ·9H ₂ O、RhCl ₃ ·3H ₂ Adding O and hexadecyl trimethyl ammonium bromide into a beaker, adding water, and then, carrying out ultrasonic stirring to fully dissolve;

(2) At room temperature, naBH is added ₄ Dissolving in water, slowly dripping into the mixed solution in the step (1), and fully stirring for reaction;

(3) And (3) centrifugally separating the reaction mixed solution, fully washing the obtained solid with ethanol and deionized water, and drying in vacuum to obtain black two-dimensional cobalt oxide stable rhodium nano mimic enzyme, 2D CoO @ Rh NC.

The synthetic mechanism of the 2D CoO @ Rh NC is as follows:

the high concentration surfactant CTAB easily forms a positively charged double-layer CTAB micelle from NaBH ₄ Hydrolysis to give negatively charged OH ^– And positively charged Co ²⁺ The nano-particles are sequentially adsorbed on the surfaces of the double molecular layers by virtue of strong electrostatic action to form curved Co oxide nano-sheets; due to Rh ³⁺ /Rh ⁰ Has a much higher redox potential than Co ²⁺ /Co ⁰ In the reducing agent sodium borohydride (NaBH) ₄ ) In the presence of Rh ³⁺ Is reduced into rhodium nano particles and is uniformly adsorbed on the surface of the Co oxide nano sheet to form the two-dimensional cobalt oxide stable rhodium nano mimic enzyme.

The Co (NO) ₃ ) ₂ ·9H ₂ O，RhCl ₃ ·3H ₂ O, CTAB and NaBH ₄ The molar ratio of (1), (0.1-0.3) to (1-2) to (2-3), and the reaction time of the step (2) is 5-15min.

The invention comprises application of two-dimensional cobalt oxide stable rhodium nano mimic enzyme, and the two-dimensional cobalt oxide stable rhodium nano mimic enzyme can be used for colorimetric detection of urea and p-aminophenol simultaneously.

The detection steps are as follows:

(1) Preparing a standard solution: preparing 2D CoO @ Rh NC mimic enzyme, urea, p-aminophenol and TMB standard solution;

(2) Preparation of actual samples:

randomly measuring 1 part of each water sample in different environments, filtering the water samples for three times by a 4-micron microporous filtering membrane, and distilling and concentrating to obtain water samples in different environments;

randomly weighing 1 part of each soil sample in different environments, ultrasonically leaching the soil samples with water for 1 week, and filtering the soil samples for three times through a 4-micron microporous filtering membrane to obtain different soil samples;

sampling to obtain urine of adult at different time, and filtering with 4 μm microporous filter membrane for three times;

(3) Determination of urea and p-aminophenol in the sample:

putting a 2D CoO @ Rh NC mimic enzyme standard solution into a centrifuge tube, adding a sample to be tested and TMB, fixing the volume by using a disodium hydrogen phosphate-citric acid buffer solution with the pH =3.5, uniformly mixing, standing at room temperature for a period of time, and measuring an absorption spectrum within the range of 250-800 nm;

calculating the urea content in the sample to be detected according to the linear relation between the logarithm of the absorbance ratio at the wavelength of 652nm and 436nm and the urea concentration; and calculating the concentration of the p-aminophenol in the sample to be detected according to a linear equation between the logarithm of the absorbance ratio at 283nm and at 652nm or at 371nm and the concentration of the p-aminophenol.

The reaction conditions in the step (3) are as follows: c. C _{2D CoO@Rh NC} ＝20μg/mL，pH＝3.5，c _TMB =0.05mM, reaction time 50min;

the 2D CoO @ Rh NC-TMB reaction system in the step (3) has specific selective spectral response to urea and p-aminophenol and is accompanied by color change from visible blue to brown yellow or from blue to colorless;

in the step (3), the linear ratio relationship exists between the absorption spectrum intensity of the 2D CoO @ Rh NC-TMB reaction system and the concentration of urea or p-aminophenol.

The reaction mechanism of the detection is as follows:

the CoO @ Rh NC mimic enzyme has high-efficiency oxidation mimic enzyme activity, can catalyze and oxidize TMB in the presence of air at room temperature to generate blue oxTMB, has special reduction performance on aminophenol, and can selectively reduce the blue oxide oxTMB into colorless TMB; the urea can reduce the blue oxide oxTMB into colorless TMB and further form brown yellow imine through the action of free amino and carbonyl; thereby realizing the colorimetric detection of urea and p-aminophenol simultaneously by enzyme catalysis.

Example 2:

a method for preparing two-dimensional cobalt oxide stable rhodium nano mimic enzyme,

preparation of 2D CoO @ Rh NC:

0.291g (1 mmol) of Co (NO) was weighed ₃ ) ₂ ·9H ₂ O、0.0418g(0.2mmol)RhCl ₃ ·3H ₂ O and 0.5g (1.4 mmol) cetyltrimethylammonium bromide (CTAB) in a small 100mL beaker was added 25mLH ₂ O, ultrasonic stirring for 30min for full dissolution; 0.1g (2.6 mmol) of NaBH ₄ Dissolving in 15mLH ₂ And slowly dripping the solution into the mixed solution, fully stirring and reacting for 10min at room temperature, performing centrifugal separation, fully washing the solid with ethanol and deionized water, and performing vacuum drying at 60 ℃ to obtain the black 2D CoO stable Rh nano mimic enzyme (the synthetic route is shown in figure 1).

The structure of 2D CoO @ Rh NC is characterized by TEM, EDS, 3RD, etc., as shown in FIG. 3. TEM shows that rhodium nanoparticles are uniformly modified on the ultrathin two-dimensional cobalt oxide, and further EDS proves that elements such as Rh, co, O and the like exist in the composite material; the main characteristic diffraction peaks of the (220) (311) (400) (511) (400) surface and the (111) surface of the simple substance rhodium, which are attributed to the cobalt oxide, appear in a 3RD spectrogram, and further prove that the rhodium is nano-modified on the two-dimensional cobalt oxide nanosheet.

A two-dimensional cobalt oxide stable rhodium nanometer mimic enzyme for colorimetric detection of urea and p-aminophenol simultaneously,

a detection step:

(1) Preparation of 2D CoO @ Rh NC mimic enzyme standard solution

Ultrasonically dispersing the 2D CoO @ Rh NC mimic enzyme into deionized water, preparing a 2D CoO @ Rh NC mimic enzyme standard solution with the concentration of 0.1mg/mL, and storing at room temperature for later use.

(2) Preparation of actual samples

Randomly measuring 1 part of each 1000.0mL environmental water sample (such as Yiyi river water, polygon river water, apartment tap water and the like), filtering for three times by a 4-micrometer microporous filtering membrane, distilling and concentrating to 10.0mL to prepare different environmental water samples, and storing at room temperature for later use;

randomly weighing 1 part of each of 10.0g of soil samples (field soil and the like in different places), ultrasonically leaching for 1 week by using 100.0mL of water, filtering for three times by using a microporous filtering membrane with the diameter of 4 mu m to prepare different soil samples, respectively marking as a soil sample 1 and a soil sample 2, and storing at room temperature for later use;

sampling to obtain urine of adult at different time (morning, noon and evening), filtering with 4 μm microporous filter membrane for three times, and storing at room temperature.

(3) Determination of urea and p-aminophenol in a sample

Taking 600 mu L (0.1 mg/mL) of 2D CoO @ Rh NC mimic enzyme standard solution, adding 200 mu L of a sample to be detected and 100 mu L (1.5 mM) of TMB into a 5mL centrifuge tube, then adding a disodium hydrogen phosphate-citric acid buffer solution with the pH =3.5 to a constant volume of 3mL, uniformly mixing, standing at room temperature for 50min, and measuring the absorption spectrum within the range of 250-800 nm.

FIG. 2 is a reaction mechanism diagram of detection.

The results show that: for the colorimetric detection of urea, the log lg of the absorbance ratio of the system at 652nm and 436nm (A) ₆₅₂ /A ₄₃₆ ) With urea concentration (c) _Urea ) In the range of 6.0-165.0 mu M, a good linear relation is shown, the correlation coefficient (R) is 0.9988, and the detection limit is 1.1 mu M. By the linear equation lg (A) ₆₅₂ /A ₄₃₆ )＝0.00898c _Urea 0.41603, the urea content in the sample to be tested can be calculated. Under the optimal test condition, the method is successfully applied to the detection of urea in an environmental water sample and a soil sample, as shown in table 1, the recovery rate of urea in the sample is 96.7-100.9%, and the relative error (RSD) is less than 4.2%; further applied to the non-simultaneousIn the detection of urea in urine at morning, noon and evening, as shown in table 2, the recovery rate of urea in the sample is 96.1-103.6%, and the relative error (RSD) is less than 3.3%.

As shown in FIG. 7, for the colorimetric detection of p-aminophenol, the log lg (A) of the absorbance ratio at 283nm,371nm and 652nm of the system ₂₈₃ /A ₆₅₂ ) Or lg (A) ₂₈₃ /A ₃₇₁ ) And concentration of p-aminophenol (c) _{P-aminophenol} ) In the range of 1.7-105.0. Mu.M, a good linear relationship is shown, with detection limits of 0.68. Mu.M and 0.72. Mu.M. According to the linear equation: lg (A) ₂₈₃ /A ₆₅₂ )＝-0.03632+0.01219c _{P-aminophenol} And lg (A) ₂₈₃ /A ₃₇₁ )＝-0.20103+0.01254c _{P-aminophenol} And the concentration of the p-aminophenol in the sample to be detected can be calculated. Under the best test condition, the method is successfully applied to the detection of the p-aminophenol in an environmental water sample (table 3), the recovery rate of the p-aminophenol in the sample is 96.0-105.8%, and the relative error (RSD) is less than 2.9%.

Table 1 detection of urea in environmental and soil samples (n = 5) by system ^a

^a pH 3.5,c _{2D CoO@Rh NC} ＝20μg/mL.

^b The actual value is the measured value in the table multiplied by 10 ^-2 (the sample is concentrated 100 times before the detection)

TABLE 2 detection of urea in urine at different times (n = 5) using the system ^a

^a pH 3.5,c _{2D CoO@Rh NC} ＝20μg/mL.

^b Actual value is measured in Table X10 (sample is diluted 10 times before measurement)

Table 3 detection of p-aminophenol in environmental water sample by system (n = 5) ^a

^a pH 3.5,c _{2D CoO@Rh NC} ＝20μg/mL.

^b The actual value is the measured value in the table multiplied by 10 ^-2 (the sample was concentrated 100-fold before testing).

Example 3:

and (3) detecting the activity of the 2D CoO @ Rh NC mimic enzyme catalytic oxidation TMB standard sample:

(1) Preparation of 2D CoO @ Rh NC mimic enzyme standard solution

Ultrasonically dispersing the 2D CoO @ Rh NC mimic enzyme into deionized water, preparing a 2D CoO @ Rh NC mimic enzyme standard solution with the concentration of 0.1mg/mL, and storing at room temperature for later use.

(2) Preparation of TMB Standard solution

0.3651g (1.5 mmol) of TMB is weighed and dissolved in 1000.0mL of deionized water to prepare 1.5mM TMB standard solution, and the TMB standard solution is stored at room temperature and diluted to the required concentration when in use.

(3) Activity of 2D CoO @ Rh NC mimic enzyme for catalytic oxidation of TMB standard sample

Taking 600 mu L (0.1 mg/mL) of 2D CoO @ Rh NC mimic enzyme standard solution, adding 100 mu L (1.5 mM) of TMB into a 5mL centrifuge tube, fixing the volume to 3mL by using pH =3.5 disodium hydrogen phosphate-citric acid buffer solution, mixing uniformly, standing at room temperature for 50min, measuring an absorption spectrum in a range of 250-800 nm, and observing the change of the solution color.

As shown in fig. 4: the 2D CoO @ Rh NC mimic enzyme is capable of catalyzing oxidation of TMB, generating obvious ultraviolet-visible characteristic absorption peaks at 652nm and 371nm, and changing from colorless to blue along with obvious color change.

Example 4:

and (2) carrying out simultaneous colorimetric detection on urea and p-aminophenol standard solutions by using two-dimensional cobalt oxide stabilized rhodium nano mimic enzyme:

(1) Preparation of 2D CoO @ Rh NC mimic enzyme standard solution

Ultrasonically dispersing the 2D CoO @ Rh NC mimic enzyme into deionized water, preparing a 2D CoO @ Rh NC mimic enzyme standard solution with the concentration of 0.1mg/mL, and storing at room temperature for later use.

(2) Preparation of urea, p-aminophenol and TMB standard solution

0.0601g (1.0 mmol) of urea, 0.1091g (1.0 mmol) of p-aminophenol and 0.3651g (1.5 mmol) of TMB are weighed and respectively dissolved in 1000.0mL of deionized water to respectively prepare 1.0mM urea, 1.0mM p-aminophenol and 1.5mM TMB standard solution, and the solution is stored at room temperature and diluted to the required concentration when in use.

(3) Determination of urea and p-aminophenol in standard solutions

Adding 600 mu L (0.1 mg/mL) of a 2D CoO @ Rh NC mimic enzyme standard solution into a 5mL centrifuge tube, adding 1.0mM urea or p-aminophenol standard sample and 100 mu L (1.5 mM) of TMB with different volumes, diluting to 3mL by using a disodium hydrogen phosphate-citric acid buffer solution with pH =3.5, uniformly mixing, standing at room temperature for 50min, measuring an absorption spectrum within the range of 250-800 nm, calculating the relation between the spectral intensity and the concentration of the urea or p-aminophenol standard sample, and recording different color change characteristics.

Example 5:

the optimization of the experimental conditions for colorimetric detection of urea and p-aminophenol simultaneously by using the two-dimensional cobalt oxide stabilized rhodium nano mimic enzyme,

in order to obtain the optimal catalytic activity of the 2D CoO @ Rh NC, the influence of factors such as different pH values, the molar ratio of rhodium to cobalt of the composite material, temperature, TMB concentration, reaction time and the like on the oxidation mimic enzyme is measured,

as shown in fig. 5: the optimized test conditions were determined to be: c. C _{2D CoO@Rh NC} ＝20μg/mL，pH＝3.5，Mol _Rh /Mol _Co ＝1:5，c _TMB =0.05mM, room temperature, 50min.

Example 6:

exploration of common coexisting interferent K ⁺ ,Ca ²⁺ ,Mg ²⁺ ,Cl ^- Phenylalanine, alanine, glycine, threonine, glucose, acetone, formaldehyde, naphthaldehyde, uric acid, phenol, aniline, para-urea and para-aminophenol detectionInfluence of (2)

As shown in fig. 6: the result shows that common coexisting interference substances have no obvious influence on colorimetric identification of urea and p-aminophenol in a 2D CoO @ Rh NC-TMB system.

The invention starts from the reaction principle and discusses the mechanism of the two-dimensional cobalt oxide-rhodium nano composite material (2D CoO @ Rh NC) for imitating enzyme catalytic oxidation of TMB and the specific visible colorimetric action of the added urea and the p-aminophenol. On the basis, the optimal test conditions are further determined to be influenced by factors such as pH value, rhodium-cobalt molar ratio of the composite material, temperature, TMB concentration, reaction time and the like: c. C _{2D CoO@Rh NC} ＝20μg/mL，pH＝3.5，Mol _Rh /Mol _Co ＝1:5，c _TMB =0.05mM, room temperature, 50min. Common coexisting interferents have no obvious interference on the detection of urea and p-aminophenol, and can be used for visually detecting the p-aminophenol and water samples in environmental water samples, soil samples and urea in urine samples. Wherein, the linear regression equation for the colorimetric detection of urea is lg (A) ₆₅₂ /A ₄₃₆ ) =0.00898-0.41603, the linear range of detection is 6.0-165.0 μ M, the correlation coefficient (R) is 0.9988, the detection limit is 1.1 μ M, the recovery rate of urea in the sample is 96.1-103.6%, and the relative error (RSD) is less than 4.2%.

The linear regression equation of colorimetric detection of p-aminophenol is lg (A) ₂₈₃ /A ₆₅₂ )＝-0.03632+0.01219c(R ² = 0.9727) and lg (a) ₂₈₃ /A ₃₇₁ )＝-0.20103+0.01254c(R ² = 0.9827), the linear range of detection is 1.7-105.0 μ M, the detection limit is 0.68 μ M and 0.72 μ M, the recovery rate of p-aminophenol in the sample is 96.0-105.8%, and the relative error (RSD) is less than 2.9%.

The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. All equivalent changes and modifications made according to the spirit of the present disclosure should be covered within the scope of the present disclosure.

Claims (9)

1. A method for preparing two-dimensional cobalt oxide stable rhodium nano mimic enzyme is characterized in that,

the two-dimensional cobalt oxide stabilized rhodium nanometer mimic enzyme is prepared by reducing cobalt ions into ultrathin two-dimensional cobalt oxide through a surfactant CTAB assisted one-pot method based on the overlarge specific surface area of the two-dimensional cobalt oxide, the good catalytic performance of rhodium nanoparticles and the excellent synergistic enhancement effect of the two-dimensional cobalt oxide and the rhodium nanoparticles, and the rhodium ions are reduced into rhodium nanoparticles and adsorbed on the surface of the two-dimensional cobalt oxide to obtain the two-dimensional cobalt oxide stabilized rhodium nanometer mimic enzyme, 2D CoO @ RhNC,

the preparation steps comprise:

(1) Weighing Co (NO) ₃ ) ₂ ·9H ₂ O、RhCl ₃ ·3H ₂ Adding O and hexadecyl trimethyl ammonium bromide into a beaker, adding water, and then, carrying out ultrasonic stirring to fully dissolve;

(2) At room temperature, naBH is added ₄ Dissolving in water, slowly dripping into the mixed solution obtained in the step (1), and fully stirring for reaction;

(3) And (3) centrifugally separating the reaction mixed solution, fully washing the obtained solid with ethanol and deionized water, and drying in vacuum to obtain black two-dimensional cobalt oxide stable rhodium nano mimic enzyme, 2D CoO @ Rh NC.

2. The method for preparing the two-dimensional cobalt oxide stable rhodium nanometer mimic enzyme according to claim 1, wherein the synthetic mechanism of the 2D CoO @ Rh NC is as follows:

the high concentration surfactant CTAB easily forms a positively charged double-layer CTAB micelle from NaBH ₄ Hydrolysis of the resulting negatively charged OH ^– And positively charged Co ²⁺ The nano-particles are sequentially adsorbed on the surfaces of the double molecular layers by virtue of strong electrostatic action to form curved Co oxide nano-sheets; due to Rh ³⁺ /Rh ⁰ Much higher redox potential than Co ²⁺ /Co ⁰ Rh in the presence of a reducing agent sodium borohydride ³⁺ Is reduced into rhodium nano particles and is uniformly adsorbed on the surface of the Co oxide nanosheet to form the two-dimensional cobalt oxide stable rhodium nano mimic enzyme.

3. The method for preparing the two-dimensional cobalt oxide stable rhodium nanometer mimic enzyme according to claim 1, which is characterized in that: the Co (NO) ₃ ) ₂ ·9H ₂ O，RhCl ₃ ·3H ₂ O, CTAB and NaBH ₄ The molar ratio of (1), (0.1-0.3) to (1-2) to (2-3), and the reaction time of the step (2) is 5-15min.

4. The method for preparing the two-dimensional cobalt oxide stable rhodium nano mimic enzyme according to claim 1, wherein the preparation method comprises the following specific steps:

(1) Weighing 1mmol Co (NO) ₃ ) ₂ ·9H ₂ O、0.2mmol RhCl ₃ ·3H ₂ Adding 25mL of H2O into a small 100mL beaker together with O and 1.4mmol of CTAB, and ultrasonically stirring for 30min for full dissolution;

(2) 2.6mmol of NaBH ₄ Dissolved in 15mL of H ₂ Slowly dripping the mixture into the mixed solution obtained in the step (1), and fully stirring and reacting for 10min at room temperature;

(3) After centrifugal separation, the solid was washed thoroughly with ethanol and deionized water, and dried under vacuum at 60 ℃ to obtain black 2DCoO @ RhNC mimic enzyme.

5. Use of a two-dimensional cobalt oxide-stabilized rhodium nanomodulase prepared by the method of any one of claims 1 to 4, wherein: the two-dimensional cobalt oxide stable rhodium nano mimic enzyme is used for colorimetric detection of urea and p-aminophenol simultaneously.

6. The use of a two-dimensional cobalt oxide-stabilized rhodium biomimetic as in claim 5, wherein said simultaneous colorimetric detection of urea and p-aminophenol comprises the steps of:

(1) Preparing a standard solution: preparing 2D CoO @ Rh NC mimic enzyme;

(2) Preparation of actual samples:

randomly measuring 1 part of each water sample in different environments, filtering the water samples for three times by a 4-micron microporous filtering membrane, and distilling and concentrating to obtain water samples in different environments;

randomly weighing 1 part of each soil sample in different environments, ultrasonically leaching the soil samples with water for 1 week, and filtering the soil samples for three times through a 4-micron microporous filtering membrane to obtain different soil samples;

sampling to obtain one part of urine of adults at different time, and filtering for three times by a 4-micron microporous filtering membrane for later use;

(3) Determination of urea and p-aminophenol in the sample:

putting a 2D CoO @ Rh NC mimic enzyme standard solution into a centrifuge tube, adding a sample to be tested and TMB, fixing the volume by using a disodium hydrogen phosphate-citric acid buffer solution with the pH =3.5, uniformly mixing, standing at room temperature for a period of time, and measuring an absorption spectrum within the range of 250-800 nm;

calculating the urea content in the sample to be detected according to the linear relation between the logarithm of the absorbance ratio at the wavelength of 652nm and 436nm and the urea concentration; and calculating the concentration of the p-aminophenol in the sample to be detected according to a linear equation between the logarithm of the absorbance ratio between 283nm and 652nm or 371nm and the concentration of the p-aminophenol.

7. The use of a two-dimensional cobalt oxide-stabilized rhodium nanomodulase as claimed in claim 6, wherein:

the reaction conditions in the step (3) are as follows: c. C _2DCoO@RhNC ＝20μg/mL，pH＝3.5，c _TMB =0.05mM, reaction time 50min.

8. The use of a two-dimensional cobalt oxide-stabilized rhodium nanomodulase of claim 6, wherein:

the 2D CoO @ Rh NC-TMB reaction system in the step (3) has specific selective spectral response to urea and p-aminophenol and is accompanied by color change from visible blue to brown yellow or from blue to colorless;

in the step (3), the linear ratio relationship exists between the absorption spectrum intensity of the 2D CoO @ Rh NC-TMB reaction system and the concentration of urea or p-aminophenol.

9. Use of a two-dimensional cobalt oxide-stabilized rhodium nanomodulase according to any one of claims 5 or 6, wherein the reaction mechanism for detection is:

the CoO @ Rh NC mimic enzyme has high-efficiency oxidation mimic enzyme activity, can catalyze and oxidize TMB in the presence of air at room temperature to generate blue oxTMB, has special reduction performance on aminophenol, and can selectively reduce the blue oxide oxTMB into colorless TMB; the urea can reduce the blue oxide oxTMB into colorless TMB and further form brown yellow imine through the action of free amino and carbonyl; thereby realizing the colorimetric detection of urea and p-aminophenol simultaneously by enzyme catalysis.

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