CN106908423A - The method that urea is determined based on nano cupric oxide - Google Patents
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Abstract
The present invention discloses a kind of method that urea is determined based on nano cupric oxide, alkaline peroxide enzymatic activity is simulated based on nano cupric oxide, ammonia and carbon dioxide can be generated with selective hydrolysis urea with reference to urase, the process that nano cupric oxide catalyzing hydrogen peroxide aoxidizes para hydroxybenzene propionic acid is suppressed by ammonia, reduce the generation of dimer fluorescence-causing substance, there is provided a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide.It is that urea, urase and phosphate buffer three are mixed into warm bath to carry out urase enzymatic reaction, then being added thereto to para hydroxybenzene propionic acid, hydrogen peroxide, nano cupric oxide and phosphate buffer continuation warm bath carries out fluorescence reaction, and the maximum excitation wavelength and launch wavelength of fluorescence reaction product are respectively 320 nm and 409 nm.The range of linearity for determining urea is 0.0375 ~ 0.3 mmol/L, and its detection is limited to 27 μm of ol/L.The method can be used for the measure of urea in urine.
Description
Technical field
Hydrolyzed under urase catalysis the present invention relates to urea and produce ammonia, suppress nano cupric oxide simulation alkaline peroxide enzyme
Catalyzing hydrogen peroxide oxidation para hydroxybenzene propionic acid forms the activity of fluorescence-causing substance in the basic conditions, sets up the fluorescence for determining urea
Analysis method, belongs to analytical chemistry and field of nanometer technology.
Background technology
Peroxidase is primarily present in the peroxisome of cell, can catalyzing hydrogen peroxide oxidation phenols and amine
Compound, then removes the effect of hydrogen peroxide, phenols and amine toxicity.Natural peroxide enzyme content is few, expensive, holds
Easily lost catalysis activity by external environmental interference, nano artificial analogue enztme due to its Stability Analysis of Structures, economy, be easy to extensive preparation
And the advantages of adjustable catalysis activity, have been widely used for bioanalysis and biotechnology applications.Natural peroxide enzyme and its
Analogies generally play optimum performance under acidic or neutral conditions.However, the research for carrying out in the basic conditions is little.Cause
This, the discovery of alkaline peroxide enzyme mimics is significant for practical application.
Urea is a kind of organic molecule being made up of carbon, oxygen, nitrogen and protium, is basic group of blood and other body fluid
Into composition.Urea is the final product of amino acid metabolism, is synthesized in liver, is typically excreted with urine.But 40% ~ 50%
In the reabsorption that the medullary substance portion of glomerulus is passive, after the content of human body urea has exceeded the absorbability of itself, it will right
Liver, kidney, alveolar of human body etc. cause harm.Urea plays an important role in diagnosis kidney and liver diseases.Kidney failure（It is acute or
It is chronic）, urethremphraxis, dehydration, shock, the disease such as burn and hemorrhage of gastrointestinal tract can cause the urea level to increase, hepatic failure, ephritis
Syndrome, cachexia（Low albumen and high carbohydrate meals）The urea level can be also caused to reduce etc. serious disease.Therefore, urinate
The detection of element is significant in clinical diagnosis.
The present invention is based on nano cupric oxide as simulation alkaline peroxide enzymatic hydrogen peroxide oxidation para hydroxybenzene third
Acid, the reaction of ammonia is produced with reference to the hydrolysis of urase catalyzing urea, there is provided a kind of to determine the glimmering of urea by catalyst of nano cupric oxide
Light analysis method.
The content of the invention
The purpose of the present invention be based on the good simulation alkaline peroxide enzymatic activity of nano cupric oxide, can be with reference to urase
Selective hydrolysis urea generates ammonia and carbon dioxide, then suppresses nano cupric oxide catalyzing hydrogen peroxide oxidation para hydroxybenzene by ammonia
The process of propionic acid, reduces the generation of dimer fluorescence-causing substance, there is provided a kind of to determine the glimmering of urea by catalyst of nano cupric oxide
Light analysis method.
To achieve these goals, the present invention uses following technical scheme：Described one kind is with nano cupric oxide as catalyst
The fluorescence analysis method of urea is determined, it is characterized in that urea, urase and phosphate buffer three are blended under warm bath first
Urase enzymatic reaction is carried out, para hydroxybenzene propionic acid, hydrogen peroxide, nano oxidized copper solution and phosphate is then added thereto to and is delayed
Fliud flushing continues warm bath carries out fluorescence reaction, with the fluorescence intensity of the above-mentioned fluorescence reaction product of fluorescent spectrophotometer assay；It is described
Nano cupric oxide be obtained by following steps：1）The ml of acetic acid copper solution 150 and 0.5 ml glacial acetic acid for taking 0.02 mol/L are added
To in the three-necked bottle equipped with condenser pipe, boiling is heated with stirring to；2）The ml of sodium hydroxide solution 10 of 0.04 g/ml is rapidly joined,
Continue to stir 5 minutes after adding, obtain cupric oxide precipitation；3）The cupric oxide pelleting centrifugation for obtaining will be reacted, with nothing
Water-ethanol is washed three times, and drying under reduced pressure obtains final product nano-cupric oxide powder.
Described a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide, it is characterized in that fluorescence reaction
The maximum excitation wavelength and launch wavelength of product are respectively 320 nm and 409 nm.
Described a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide, it is characterized in that urase enzymatic
PH value of reaction system is 7.0, and urase concentration is 0.3 U/mL, and reaction temperature is 37 DEG C, and the reaction time is 30 minutes.
The pH value of fluorescence reaction system is 10.25, and oxidation copper concentration is 0.4 mg/L, and para hydroxybenzene propionate concentration is 4
Mmol/L, reaction temperature is 65 DEG C, and the reaction time is 15 minutes.
Described a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide, it is characterized in that by 150 μ L
Be 10mmol/L with concentration, pH be 7.0 phosphate buffered saline into various concentrations urea liquid and 50 μ L concentration be
The urase solution mixing of 6 U/mL, mixed liquor in 37 DEG C of warm bath hydrolysis 30 minutes, then to sequentially adding 50 in mixed liquor
μ L concentration is the hydrogenperoxide steam generator of 120 mmol/L, and 50 μ L concentration are the para hydroxybenzene propionic acid of 80 mmol/L, and 200 μ L are dense
Spend during the nano oxidized copper solution and 500 μ L concentration for 2mg/L be 10.25 PBS for 100mmol/L, pH,
Mixed liquor determines its fluorescence intensity at 409 nm in 65 DEG C of warm bath, after 15 minutes respectively, and excitation wavelength is 320 nm, with
Fluorescence intensity difference obtains standard curve for determining urea to the mapping of urea concentration logarithm value.
Described a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide, it is characterized in that urea is detected
The range of linearity is 0.0375 ~ 0.3 mmol/L, and detection is limited to 27 μm of ol/L.
The present invention determines the fluorescence analysis method of urine urea content using nano cupric oxide for catalyst, it is characterized in that it
Comprise the steps of：（1）The urine and urase being separately added into EP pipes after being diluted with phosphate buffer mix warm bath；
（2）To phosphate buffer is added in above-mentioned EP pipes, hydrogen peroxide, para hydroxybenzene propionic acid and nano oxidized copper solution continue temperature
Bath；（3）Product is placed in fluorescence intensity in sepectrophotofluorometer, is urinated according in urea standard curve determination urine
Plain concentration；Described nano cupric oxide is obtained by following steps：1）Take the ml of acetic acid copper solution 150 and 0.5 of 0.02 mol/L
Ml glacial acetic acid is added in the three-necked bottle equipped with condenser pipe, is heated with stirring to boiling；2）Rapidly join the hydroxide of 0.04 g/ml
The ml of sodium solution 10, continues to stir 5 minutes after adding, and obtains cupric oxide precipitation；3）The cupric oxide for obtaining will be reacted to sink
Form sediment centrifugation, with absolute ethanol washing three times, drying under reduced pressure obtains final product nano-cupric oxide powder.
Described utilization nano cupric oxide is the fluorescence analysis method that catalyst determines urine urea content, it is characterized in that
It is the urine sample that the phosphate buffer that 10 mmol/L, pH are 7.0 dilutes 600 times that 150 μ L concentration are separately added into EP pipes
Product and the mixing of urase solution that 50 μ L concentration are 6 U/mL, mixed liquor in 37 DEG C of warm bath hydrolysis 30 minutes, then to mixed
The hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L is sequentially added in conjunction liquid, 50 μ L concentration are 80 mmol/L to hydroxyl
Base benzenpropanoic acid, 200 μ L concentration are the nano oxidized copper solution and 500 μ L concentration of 2 mg/L for 100mmol/L, pH are 10.25
PBS in, mixed liquor determines its fluorescence intensity at 409 nm in 65 DEG C of warm bath, after 15 minutes respectively,
Excitation wavelength is 320 nm.
Described urase enzymatic reaction system pH value is preferably 7.0, and urase concentration is preferably 0.3U/mL.
Described urase enzymatic reaction temperature is preferably 37 DEG C, and the reaction time is preferably 30 minutes.
The pH value of described fluorescence reaction system is preferably 10.25, and nano oxidized copper concentration is preferably 0.4mg/L, to hydroxyl
Base phenylpropyl alcohol acid concentration is preferably 4mmol/L.
Described fluorescence reaction temperature is preferably 65 DEG C, and the reaction time is preferably 15 minutes.
Technical scheme is comprised the following steps that：
（One）The preparation of nano cupric oxide：
Take acetic acid copper solution and glacial acetic acid is added in the three-necked bottle equipped with condenser pipe, be heated with stirring to boiling, rapidly join hydrogen
Sodium hydroxide solution, after adding, after continuing to stir, obtains cupric oxide.The cupric oxide for obtaining will be reacted to be centrifuged immediately, used
Absolute ethanol washing, drying under reduced pressure obtains final product nano-cupric oxide powder.Nano-cupric oxide powder is scattered in redistilled water and is obtained
To brown nano cupric oxide colloidal solution.
The specific preparation process of nano cupric oxide is as follows：
（1）The ml of acetic acid copper solution 150 and 0.5 ml glacial acetic acid for taking 0.02 mol/L are added to the three-necked bottle equipped with condenser pipe
In, it is heated with stirring to boiling；
（2）The ml of sodium hydroxide solution 10 of 0.04 g/ml is rapidly joined, continues to stir 5 minutes after adding, obtain black oxidation
Copper is precipitated；
（3）The cupric oxide pelleting centrifugation that obtains will be reacted, with absolute ethanol washing three times, drying under reduced pressure obtains final product nano oxygen
Change copper powder body.
（Two）The measure of urea
Urea, urase and phosphate buffer three are mixed first, mixed liquor is put into 37 DEG C of warm bath and carries out urase enzyme in 30 minutes
Promote reaction, be then added thereto to 65 DEG C of temperature after para hydroxybenzene propionic acid, hydrogen peroxide, nano cupric oxide and phosphate buffer
Bath reaction 15 minutes, determine fluorescence intensity, with fluorescence intensity difference to urea concentration logarithm value mapping obtain standard curve with
In measure urea.
（Three）The measure of urea in urine
Urea is replaced with into the urine sample repeat step two after dilution, is that can obtain by gained fluorescence intensity substitution standard curve
The concentration of urea in urine.
Advantages of the present invention：
The present invention successfully constructs a kind of fluorescence for detecting urea using the simulation alkaline peroxide enzyme characteristic of nano cupric oxide
Analysis method.The range of linearity that the technology determines urea is 0.0375 ~ 0.3 mmol/L, and its detection is limited to 27 μm of ol/L.This hair
Bright high with sensitivity, sample requirements are few, favorable reproducibility, low cost and other advantages, can be applied to the survey of urea in urine sample
It is fixed.
Brief description of the drawings
Fig. 1 is the fluorescence spectra of fluorescence system.
Fig. 2 is influence figure of the pH value to fluorescence intensity.
Fig. 3 is influence figure of the nano oxidized copper concentration to fluorescence intensity.
Fig. 4 is influence figure of the para hydroxybenzene propionate concentration to fluorescence intensity.
Fig. 5 is influence figure of the urea concentration to fluorescence intensity.
Fig. 6 is influence figure of the urase concentration to fluorescence intensity.
Fig. 7 is the canonical plotting of urea.
Specific embodiment
The present invention will be further described by the following examples, but the present invention is not limited only to this.
Example 1：
The specific preparation process of nano cupric oxide is as follows：（1）Take the ml of acetic acid copper solution 150 and 0.5 ml ice vinegar of 0.02 mol/L
Acid is added in the three-necked bottle equipped with condenser pipe, is heated with stirring to boiling；（2）The NaOH for rapidly joining 0.04 g/ml is molten
The ml of liquid 10, continues to stir 5 minutes after adding, and obtains cupric oxide precipitation；（3）The cupric oxide precipitation for obtaining will be reacted
Centrifugation, with absolute ethanol washing three times, drying under reduced pressure obtains final product the nano-cupric oxide powder of a diameter of 6 nm.
Example 2：
By the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L, 50 μ L concentration are the para hydroxybenzene propionic acid of 80 mmol/L
With 200 μ L concentration for nano oxidized copper solution prepared by the example 1 of 2mg/L is added to the phosphorus that 700 μ L concentration are 100mmol/L
Hydrochlorate cushioning liquid（pH 10.25）In, mixing is placed in 65 DEG C of warm bath after shaking up, and its fluorescence spectrum is determined after 15 minutes.Such as Fig. 1
Shown, fluorescence-causing substance excitation wavelength is 320 nm, and launch wavelength is 409 nm.
Example 3：
By the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L, 50 μ L concentration are the para hydroxybenzene propionic acid of 80 mmol/L
With 200 μ L concentration for nano oxidized copper solution prepared by the example 1 of 2mg/L is added to 700 μ L concentration for 100mmol/L is different
The PBS of pH（PH 9~11）In, mixing is placed in 65 DEG C of warm bath after shaking up, and it is determined after 15 minutes in 409 nm
The fluorescence intensity at place（Excitation wavelength is 320 nm）.As shown in Fig. 2 fluorescence intensity reaches maximum between being 10.1 ~ 10.3 in pH
Value.
Example 4：
By the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L, 50 μ L concentration are the para hydroxybenzene propionic acid of 80 mmol/L
The nano oxidized copper solution prepared with the example 1 of 200 μ L various concentrations（0~5 mg/L）Being added to 700 μ L concentration is
The PBS of 100mmol/L（pH 10.25）In, mixing shake up after be placed in 65 DEG C of warm bath, determined after 15 minutes its
Fluorescence intensity at 409 nm（Excitation wavelength is 320 nm）.As shown in figure 3, fluorescence intensity is dense with nano cupric oxide in mixed liquor
Degree increases and increases and reach maximum in final concentration of 0.4 mg/L.
Example 5：
By the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L, the para hydroxybenzene propionic acid of 50 μ L various concentrations（0~80
mmol/L）It is for the nano oxidized copper solution of the preparation of example 1 of 2mg/L is added to 700 μ L concentration with 200 μ L concentration
The PBS of 100mmol/L（pH 10.25）In, mixing shake up after be placed in 65 DEG C of warm bath, determined after 15 minutes its
Fluorescence intensity at 409 nm（Excitation wavelength is 320 nm）.As shown in figure 4, fluorescence intensity is with para hydroxybenzene propionic acid in mixed liquor
Concentration increases and increases, and in final concentration of 3.5 mmol/L, fluorescence intensity reaches maximum.
Example 6：
By the urea liquid of 150 μ L various concentrations（0~15 mmol/L, is 10mmol/L with concentration, and pH is 7.0 phosphate
Buffer）With the mixing of urase solution that 50 μ L concentration are 6 U/mL, mixed liquor in 37 DEG C of warm bath hydrolysis 30 minutes,
Then to the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L is sequentially added in mixed liquor, 50 μ L concentration are 80 mmol/
The para hydroxybenzene propionic acid of L, 200 μ L concentration are that the nano oxidized copper solution and 500 μ L concentration of the preparation of example 1 of 2mg/L are
The PBS of 100mmol/L（pH 10.25）In, mixed liquor determined respectively in 65 DEG C of warm bath, after 15 minutes its
Fluorescence intensity at 409 nm, excitation wavelength is 320 nm, as shown in figure 5, fluorescence intensity difference is with urea concentration in mixed liquor
Increase and increase, in final concentration of 0.75 mmol/L, fluorescence intensity reaches stationary value.
Example 7：
By the urea liquid that 150 μ L concentration are 15 mmol/L（It is 10mmol/L with concentration, pH is 7.0 phosphate buffer
Prepare）With the urase solution of 50 μ L various concentrations（0.04~6 U/mL）Mixing, mixed liquor is in 37 DEG C of warm bath hydrolysis 30
Minute, then to the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L is sequentially added in mixed liquor, 50 μ L concentration are 80
The para hydroxybenzene propionic acid of mmol/L, nano oxidized copper solution and 500 μ L concentration that 200 μ L concentration are prepared for the example 1 of 2mg/L
It is the PBS of 100mmol/L（pH 10.25）In, mixed liquor determines it respectively in 65 DEG C of warm bath after 15 minutes
Fluorescence intensity at 409 nm, excitation wavelength is 320 nm, as shown in fig. 6, fluorescence intensity difference is dense with urase in mixed liquor
Degree increases and increases, and in final concentration of 0.1 U/mL, fluorescence intensity reaches stationary value.
Example 8：
By the urea liquid of 150 μ L various concentrations（0.25~2 mmol/L, is 10mmol/L with concentration, and pH is 7.0 phosphoric acid
Salt buffer is prepared）With the urase solution mixing that 50 μ L concentration are 6 U/mL, mixed liquor is in 37 DEG C of 30 points of warm bath hydrolysis
Clock, then to the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L is sequentially added in mixed liquor, 50 μ L concentration are 80
The para hydroxybenzene propionic acid of mmol/L, nano oxidized copper solution and 500 μ L concentration that 200 μ L concentration are prepared for the example 1 of 2mg/L
It is the PBS of 100mmol/L（pH 10.25）In, mixed liquor determines it respectively in 65 DEG C of warm bath after 15 minutes
Fluorescence intensity at 409 nm, excitation wavelength is 320 nm, and the mapping of urea concentration logarithm value is obtained with fluorescence intensity difference
Urea standard curve.As shown in fig. 7, fluorescence intensity is linear in the range of 0.0375 ~ 0.3 mmol/L with urea concentration,
Detection is limited to 27 μm of ol/L.
Example 9：
It is 10mmol/L that 150 μ L concentration are separately added into EP pipes, and pH is the urine of 7.0 600 times of phosphate buffer dilution
Liquid sample and the mixing of urase solution that 50 μ L concentration are 6 U/mL, mixed liquor in 37 DEG C of warm bath hydrolysis 30 minutes, then
To the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L is sequentially added in mixed liquor, 50 μ L concentration are 80 mmol/L
Para hydroxybenzene propionic acid, 200 μ L concentration are the phosphate of 100mmol/L for the nano oxidized copper solution and 500 μ L concentration of 2mg/L
Cushioning liquid（pH 10.25）In, it is strong that mixed liquor determines its fluorescence at 409 nm in 65 DEG C of warm bath, after 15 minutes respectively
Degree, excitation wavelength is 320 nm.Urea content, this numerical value in urine sample are calculated through the gained urea standard curve of example 8
It is consistent with the data that diacetyl monoxime method is obtained.Sample recovery rate 93.5 ~ 105.6%, relative standard deviation 1.0% ~ 3.1%, thus
It can be seen that this method is reliably applicable.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification made within god and principle, equivalent and improvement etc., should be included within the scope of the present invention.
Claims (8)
1. it is a kind of with nano cupric oxide as catalyst determine urea fluorescence analysis method, it is characterized in that first by urea, urase
Being blended under warm bath with phosphate buffer three carries out urase enzymatic reaction, is then added thereto to para hydroxybenzene propionic acid, mistake
Hydrogen oxide, nano oxidized copper solution and phosphate buffer continue warm bath carries out fluorescence reaction, uses fluorescent spectrophotometer assay
The fluorescence intensity of above-mentioned fluorescence reaction product；Described nano cupric oxide is obtained by following steps：1）Take the vinegar of 0.02 mol/L
The ml of sour copper solution 150 and 0.5 ml glacial acetic acid are added in the three-necked bottle equipped with condenser pipe, are heated with stirring to boiling；2）Quickly
The ml of sodium hydroxide solution 10 of 0.04 g/ml is added, continues to stir 5 minutes after adding, obtain cupric oxide precipitation；3）Will
The cupric oxide pelleting centrifugation that obtains of reaction, with absolute ethanol washing three times, drying under reduced pressure obtains final product nano-cupric oxide powder.
2. a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide according to claim 1, it is special
Levy is that the maximum excitation wavelength and launch wavelength of fluorescence reaction product is respectively 320 nm and 409 nm.
3. a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide according to claim 1, it is special
It is that urase enzymatic reaction system pH value is 7.0 to levy, and urase concentration is 0.3 U/mL, and reaction temperature is 37 DEG C, and the reaction time is 30
Minute.
4. a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide according to claim 1, it is special
Levy be fluorescence reaction system pH value for 10.25, oxidation copper concentration is 0.4 mg/L, and para hydroxybenzene propionate concentration is 4 mmol/
L, reaction temperature is 65 DEG C, and the reaction time is 15 minutes.
5. a kind of fluorescence analysis method for determining urea as catalyst with nano cupric oxide according to claim 1, it is special
Levy be by 150 μ L concentration be 10mmol/L, pH be 7.0 phosphate buffered saline into various concentrations urea liquid
With the mixing of urase solution that 50 μ L concentration are 6 U/mL, mixed liquor in 37 DEG C of warm bath hydrolysis 30 minutes, then to mixing
The hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L is sequentially added in liquid, 50 μ L concentration are 80 mmol/L to hydroxyl
Benzenpropanoic acid, 200 μ L concentration for 2mg/L nano oxidized copper solution and 500 μ L concentration be 100mmol/L, pH be 10.25 phosphorus
In hydrochlorate cushioning liquid, mixed liquor determines its fluorescence intensity at 409 nm in 65 DEG C of warm bath, after 15 minutes respectively, excites
Wavelength is 320 nm, and standard curve is obtained for determining urea to the mapping of urea concentration logarithm value with fluorescence intensity difference.
6. according to claim 1 or 5 it is a kind of with nano cupric oxide as catalyst determine urea fluorescence analysis method, its
It is characterized in that the urea detection range of linearity is 0.0375 ~ 0.3 mmol/L, detection is limited to 27 μm of ol/L.
7. a kind of utilization nano cupric oxide be catalyst determine urine urea content fluorescence analysis method, it is characterized in that it by with
Lower step composition：（1）The urine and urase being separately added into EP pipes after being diluted with phosphate buffer mix warm bath；（2）To
Phosphate buffer is added in above-mentioned EP pipes, hydrogen peroxide, para hydroxybenzene propionic acid and nano oxidized copper solution continue warm bath；（3）
Product is placed in fluorescence intensity in sepectrophotofluorometer, it is dense according to urea in urea standard curve determination urine
Degree；Described nano cupric oxide is obtained by following steps：1）Take the ml of acetic acid copper solution 150 and 0.5 ml ice of 0.02 mol/L
Acetic acid is added in the three-necked bottle equipped with condenser pipe, is heated with stirring to boiling；2）The NaOH for rapidly joining 0.04 g/ml is molten
The ml of liquid 10, continues to stir 5 minutes after adding, and obtains cupric oxide precipitation；3）To react the cupric oxide that obtains precipitate from
The heart, with absolute ethanol washing three times, drying under reduced pressure obtains final product nano-cupric oxide powder.
8. utilization nano cupric oxide according to claim 7 is the fluorescence analysis side that catalyst determines urine urea content
Method, it is characterized in that it is the phosphate buffer dilution that 10 mmol/L, pH are 7.0 that 150 μ L concentration are separately added into EP pipes
600 times of urine sample and the urase solution mixing that 50 μ L concentration are 6 U/mL, mixed liquor is in 37 DEG C of warm bath hydrolysis 30
Minute, then to the hydrogenperoxide steam generator that 50 μ L concentration are 120 mmol/L is sequentially added in mixed liquor, 50 μ L concentration are 80
The para hydroxybenzene propionic acid of mmol/L, 200 μ L concentration are that the nano oxidized copper solution and 500 μ L concentration of 2 mg/L are 100mmol/
During L, pH are 10.25 PBS, mixed liquor determines it in 65 DEG C of warm bath, after 15 minutes at 409 nm respectively
Fluorescence intensity, excitation wavelength be 320 nm.
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