CN105628700B - Hydrogen peroxide detection kit based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles - Google Patents
Hydrogen peroxide detection kit based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Download PDFInfo
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Abstract
The invention discloses a kind of hydrogen peroxide detection kits based on bismuth bovine serum albumin(BSA) Platinum Nanoparticles.The present invention aoxidizes 3,3 ', 5,5 ' tetramethyl biphenyl amine hydrochlorates using bismuth bovine serum albumin(BSA) Platinum Nanoparticles catalyzing hydrogen peroxide and develops the color, and provides a kind of quick, easy, sensitive hydrogen peroxide detection method.Bismuth bovine serum albumin(BSA) Platinum Nanoparticles used in the detection method and kit prepare easy.Differentiation of the light absorption value realization to tumour cell and normal cell can be observed by the naked eye and be measured, and the burst size and rate of release of hydrogen peroxide can be calculated according to light absorption value.
Description
Technical field
The present invention provides a kind of novel extracellular content of hydrogen peroxide rapid detection method and kit.It is related to one kind
The detection method of hydrogen peroxide based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, belongs to analytical chemistry, receives
Rice technology and cell biology.
Background technology
Hydrogen peroxide is very important reactive intermediate in a kind of vital movement, while being bleached in food production, paper
The fields such as agent, disinfectant, fuel cell, enzymatic analysis play an important role as in, therefore are carried out to hydrogen peroxide highly sensitive
Degree, highly selective analysis and detection are extremely necessary.In recent years the study found that some tumour cells such as breast cancer is thin
How the amount of hydrogen peroxide that born of the same parents, kidney cancer cell, lung carcinoma cell etc. are discharged rapidly and accurately passes through far more than normal cell
The content for detecting extracellular hydrogen peroxide carrys out tumor cell and detects production of the quantity of tumour cell for research tumour
Raw development mechanism and antitumor drug mechanism of action have great significance.
Enzyme is efficient biocatalyst, almost all of in organism to react the participation for having enzyme.Enzymatic analysis is facing
The fields such as bed diagnosis, biology, food, chemistry, environment are all widely used.Enzymic catalytic reaction is because it is with efficient, single-minded, item
The features such as part is mild and be concerned.However native enzyme limited source, purification difficult is expensive, and in order to keep its work
Property require experiment condition and operating environment all more harsh, so that its application is greatly limited, therefore the exploitation of analogue enztme
And application study has been to be concerned by more and more people.
The present invention is based on the Mimetic enzyme characteristics of bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles, use hydrogen peroxide as
Electron acceptor participates in the chromogenic reaction of bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme catalysis, provides a kind of colorimetric
Detect the new method and detection kit of extracellular hydrogen peroxide.
Invention content
It is an object of the present invention to provide one kind being based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
The method of the active extracellular hydrogen peroxide of colorimetric detection.
In order to realize that the purpose of above-mentioned detection method, the present invention use following technical scheme：
A kind of hydrogen peroxide inspection based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme of the present invention
Survey method, it is characterized in that by hydrogen peroxide, 3,3',5,5'-tetramethylbenzidine hydrochloride, bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles
Constant-temperature incubation after aqueous solution mixing, the sulfuric acid that 0.5 mol/L is added terminate reaction, and reaction product is yellow, maximum absorption wavelength
For 450 nm；Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles the Mimetic enzyme is made by following steps：In 5 mL concentration
A concentration of 16 mmol/L platinum acid chloride solutions of 5 mL and 0.5 mL concentration are added in Bovine Serum Albumin in Aqueous Solution for 50 mg/mL
For 1.5 mol/L sodium hydroxide solutions, 2 h are reacted at 80 DEG C of water-bath after mixing, it is 3k that solution, which is packed into cutoff, after reaction
Super filter tube, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is obtained, by cow's serum
Albumin-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bovine serum albumin(BSA)-Platinum Nanoparticles powder, in a concentration of 23.8 mg/ of 0.6 mL
Bismuth nitrate solution and 1.8 mL that a concentration of 0.5 mmol/L of 0.6 mL are added in the bovine serum albumin(BSA) of mL-platinum aqueous solution are dense
It is 10 mmol/L to spend, the phosphate buffer of pH=7, reacts 2.5 h at 80 DEG C of water-bath after mixing, and solution, which is packed into, after reaction cuts
Only molecular weight be 3k super filter tube, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, obtain bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles
Aqueous solution；Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bismuth-bovine serum albumin(BSA)-nanometer platinum powder
End.
The hydrogen peroxide detection method based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, it is special
Sign is that the concentration of hydrogen peroxide can be judged according to color development system solution colour.
The hydrogen peroxide detection method based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, it is special
Sign is can be according to the absorbance at 450 nm of microplate reader to detect the concentration of hydrogen peroxide.
The hydrogen peroxide detection method based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, it is special
Sign is linear ranging from 0.5 ~ 30 μm of ol/L of detection, and detection is limited to 0.068 μm of ol/L.
The hydrogen peroxide detection method based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, it is special
Sign is that 100 μ L hydrogenperoxide steam generators are added in 96 orifice plates, 3,3 ', 5, the 5 '-tetramethyls connection of a concentration of 8 mmol/L of 10 μ L
Anilinechloride, a concentration of 4.76 mg/mL bismuths-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solutions of 10 μ L and 80 μ L are a concentration of
50 μ L a concentration of 0.5 are added in 37 DEG C of 15 min of constant-temperature incubation after mixing in 100 mmol/L, the phosphate buffer of pH=5
The sulfuric acid of mol/L terminates reaction, visually observes the variation of color or measures the absorbance at 450 nm wavelength by microplate reader.
The hydrogen peroxide detection method based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, it is special
Sign is to can be used for measuring extracellular concentration of hydrogen peroxide.
Specifically,（One）The preparation of bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles：In the ox blood of a concentration of 50 mg/mL of 5 mL
A concentration of 16 mmol/L platinum acid chloride solutions of 5 mL and a concentration of 1.5 mol/L hydrogen-oxygens of 0.5 mL are added in pure protein solution
Change sodium solution, reacts 2 h at 80 DEG C of water-bath after mixing.The super filter tube that solution loading cutoff is 3k after reaction, 6000
R/min centrifugal ultrafiltrations, and wash 3 times, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is obtained, by bovine serum albumin(BSA)-Platinum Nanoparticles water
Solution is freeze-dried to obtain bovine serum albumin(BSA)-Platinum Nanoparticles powder.In the bovine serum albumin of a concentration of 23.8 mg/mL of 0.6 mL
The bismuth nitrate solution and a concentration of 10 mmol/L of 1.8 mL of a concentration of 0.5 mmol/L of 0.6 mL are added in vain-platinum aqueous solution,
The phosphate buffer of pH=7 reacts 2.5 h after mixing at 80 DEG C of water-bath, and it is 3k's that solution, which is packed into cutoff, after reaction
Super filter tube, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, obtain the aqueous solution of bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles.By bismuth-
Bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles powder.Make in preparation process
All glasswares pass through chloroazotic acid and impregnate, and are used in combination distilled water thoroughly to clean, dry.
（Two）Peroxide standard curve：It is added the Hydrogen peroxide standard liquid of 100 μ L various concentrations in 96 orifice plates, 10
The 3,3',5,5'-tetramethylbenzidine hydrochloride of a concentration of 8 mmol/L of μ L, a concentration of 4.76 mg/mL bismuths-ox bloods of 10 μ L
Pure albumen-Platinum Nanoparticles aqueous solution, a concentration of 100 mmol/L of 80 μ L, the phosphate buffer of pH=5.In 37 DEG C of perseverances after mixing
Temperature is incubated 15 min, and the sulfuric acid that a concentration of 0.5 mol/L of 50 μ L are added terminates reaction.It visually observes the variation of color or passes through
Microplate reader measures the absorbance at 450 nm wavelength.
（Three）The hydrogen peroxide determination method that cell generates：Culture medium is the RPMI-1640 containing 6 % fetal calf serums, culture
Environment is 5% carbon dioxide incubator of 37 DEG C of constant temperature.Adherent cell is digested with pancreatin, culture medium is added to blow and beat mixing, is calculated
Cell quantity.It is diluted to cell suspension to be added into 96 orifice plates with 200 μ L of every hole later, cultivates 24 hours and waited in incubator
It is complete adherent.Wait for that adherent complete cell hole a concentration of 10 mmol/L of 200 μ L each later, the phosphate buffer of pH 7.4 are light
Flushing 3 times is played in featheriness, removes extra culture solution with for use.100 μ L propylene glycol methyl ether acetates are added in each cell hole
The 3,3',5,5'-tetramethylbenzidine hydrochloride of a concentration of 8 mmol/L of 10 μ L, 10 μ are added after stimulation in each hole
Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution of a concentration of 4.76 mg/mL of L, a concentration of 100 mmol/L of 80 μ L, pH=5
Phosphate buffer.It is terminated instead in 37 DEG C of 15 min of constant-temperature incubation, the sulfuric acid that a concentration of 0.5 mol/L of 50 μ L are added after mixing
It answers.It visually observes the variation of color or the absorbance at 450 nm wavelength is measured by microplate reader, according to Hydrogen peroxide standard song
Line computation concentration of hydrogen peroxide.
It is another object of the present invention to provide one kind simulating peroxide based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles
The extracellular hydrogen peroxide detection kit of enzymatic activity.Kit includes providing 3,3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorates
Solution（A liquid）, bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles solution（B liquid）, sulfuric acid solution（Terminate liquid）, hydrogenperoxide steam generator（Standard
Liquid）.
In order to realize that the purpose of above-mentioned detection method, the present invention use following technical scheme：
（Four）Hydrogen peroxide detection kit：A liquid includes 3,3 ', the 5,5 '-tetramethyl benzidines of a concentration of 8 mmol/L
Hydrochloric acid saline solution.B liquid includes above-mentioned technical proposal（One）With a concentration of 100 mmol/L, the phosphate-buffered of pH=5 after preparation
Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles the solution for a concentration of 0.53 mg/mL that liquid is prepared.Terminate liquid includes a concentration of 0.5 mol/
The sulfuric acid solution of L.Titer includes a concentration of 1, the aqueous hydrogen peroxide solution of 2,10,20,30,40,60 μm of ol/L.
（Five）The detection method of hydrogen peroxide：100 μ L technical solutions are added in 96 orifice plates（Four）Titer, 10 μ l
Technical solution（Four）A liquid, 90 μ l technical solutions（Four）B liquid 50 μ L are added in 37 DEG C of 15 min of constant-temperature incubation after mixing
Technical solution（Four）Terminate liquid terminate reaction.The absorbance at 450 nm wavelength is measured with microplate reader, draws hydrogen peroxide mark
Directrix curve calculates regression equation.100 μ l sample solutions, 10 μ l technical solutions are added in 96 orifice plates（Four）A liquid, 90 μ
L technical solutions（Four）B liquid 50 μ L technical solutions are added in 37 DEG C of 15 min of constant-temperature incubation after mixing（Four）Terminate liquid
Terminate reaction.The absorbance at 450 nm wavelength is measured with microplate reader, according to peroxide standard curve or regression equation calculation
Concentration of hydrogen peroxide.Linear ranging from 0.5 ~ 30 μm of ol/L of detection, detection are limited to 0.068 μm of ol/L.
Advantages of the present invention：
（1）The present invention is catalyzed peroxidating using the Mimetic enzyme catalytic activity of bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles
Hydroxide 3,3',5,5'-tetramethylbenzidine hydrochloride develops the color, can be with to show the variation of solution colour and light absorption value
Detection for hydrogen peroxide.
（2）The not excessive pipetting of the present invention, reduces the loss of hydrogen peroxide, shortens detection time, make it
Detection work can be completed in 15 min.
（3）It is not necessary that stabilizer is added during detecting hydrogen peroxide, detection reagent used is easy to get the present invention,
Testing cost is low.
（4）The present invention is low to the processing requirement of sample, high to the detection specificity of hydrogen peroxide.
（5）Hydrogen peroxide detection kit provided by the invention, detection process are easy, and stability is good, have it is easy to operate,
The advantages that detection time is short, high sensitivity, high specificity, use easy to spread.
Description of the drawings
Fig. 1 is the canonical plotting of hydrogen peroxide.
Fig. 2 is peroxide caused by MCF-7 the and HEK293 cells that stimulate by 250 ng/mL propylene glycol methyl ether acetates
Change hydrogen and compares figure.
Fig. 3 is influence diagram of the propylene glycol methyl ether acetate concentration to generated hydrogen peroxide.
Fig. 4 is influence diagram of the propylene glycol methyl ether acetate stimulation time to generated hydrogen peroxide.
Specific implementation mode
Embodiment 1：
A concentration of 16 mmol/L chlorine platinum of 5 mL is added in the Bovine Serum Albumin in Aqueous Solution of a concentration of 50 mg/mL of 5 mL
Acid solution and a concentration of 1.5 mol/L sodium hydroxide solutions of 0.5 mL react 2 h after mixing at 80 DEG C of water-bath.Solution after reaction
The super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations are packed into, and are washed 3 times, bovine serum albumin(BSA)-nanometer is obtained
Bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bovine serum albumin(BSA)-Platinum Nanoparticles powder by platinum aqueous solution.0.6
The bismuth nitrate of a concentration of 0.5 mmol/L of 0.6 mL is added in bovine serum albumin(BSA)-platinum aqueous solution of a concentration of 23.8 mg/mL of mL
Solution and a concentration of 10 mmol/L of 1.8 mL, the phosphate buffer of pH=7 react 2.5 h after mixing, instead at 80 DEG C of water-bath
Solution is packed into the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations after answering, and washes 3 times, obtains bismuth-cow's serum
The aqueous solution of albumin-Platinum Nanoparticles.It is freeze-dried bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution to obtain bismuth-bovine serum albumin
In vain-Platinum Nanoparticles powder.All glasswares used in above procedure pass through chloroazotic acid and impregnate, and distilled water is used in combination thoroughly to clean,
It dries.
Embodiment 2：
It is added the Hydrogen peroxide standard liquid of 100 μ L various concentrations in 96 orifice plates, the 3 of a concentration of 8 mmol/L of 10 μ L,
3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorates, a concentration of 4.76 mg/mL bismuths-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solutions of 10 μ L
And 80 a concentration of 100 mmol/L of μ L, the phosphate buffer of pH=5.In 37 DEG C of 15 min of constant-temperature incubation after mixing, it is added
The sulfuric acid of a concentration of 0.5 mol/L of 50 μ L terminates reaction.The absorbance at 450 nm wavelength is measured with microplate reader and draws mark
Directrix curve（Fig. 1）.It detects linear ranging from 0.5 ~ 30 μm of ol/L, and detection is limited to 0.068 μm of ol/L.
Embodiment 3：
Culture medium is the RPMI-1640 containing 6 % fetal calf serums, and culture environment is 5% carbon dioxide culture of 37 DEG C of constant temperature
Case.Adherent cell is digested with pancreatin, culture medium is added to blow and beat mixing, calculates cell quantity.Be diluted to after cell suspension with
Be added into 96 orifice plates per 200 μ L of hole, cultivate in incubator wait within 24 hours it is complete adherent.Wait for adherent complete each cell later
The phosphate buffer of hole a concentration of 10 mmol/L of 200 μ L, pH 7.4 gently blow and beat flushing 3 times, remove extra culture
Liquid is with for use.The propylene glycol methyl ether acetate of a concentration of 250 ng/mL of 100 μ L is added in each cell hole stimulates 10 min
The 3,3',5,5'-tetramethylbenzidine hydrochloride of a concentration of 8 mmol/L of 10 μ L is added in each hole later, 10 μ L are dense
Degree is the bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution and a concentration of 100 mmol/L of 80 μ L of 4.76 mg/mL, pH=5
Phosphate buffer.It is terminated instead in 37 DEG C of 15 min of constant-temperature incubation, the sulfuric acid that a concentration of 0.5 mol/L of 50 μ L are added after mixing
It answers.It visually observes the variation of color or passes through the absorbance at microplate reader 450 nm wavelength of measurement.Human breast carcinoma as shown in Figure 2 is thin
The light absorption value measured by hydrogen peroxide that born of the same parents MCF-7 is generated is far above people's renal epithelial cell HEK293.
Embodiment 4：
Culture medium is the RPMI-1640 containing 6 % fetal calf serums, and culture environment is 5% carbon dioxide culture of 37 DEG C of constant temperature
Case.Adherent cell is digested with pancreatin, culture medium is added to blow and beat mixing, calculates cell quantity.Be diluted to after cell suspension with
Be added into 96 orifice plates per 200 μ L of hole, cultivate in incubator wait within 24 hours it is complete adherent.Wait for adherent complete each cell later
The phosphate buffer of hole a concentration of 10 mmol/L of 200 μ L, pH 7.4 gently blow and beat flushing 3 times, remove extra culture
Liquid is with for use.100 μ L various concentrations are added in each cell hole（0-500 ng/mL）Propylene glycol methyl ether acetate stimulation
It is added the 3,3',5,5'-tetramethylbenzidine hydrochloride of a concentration of 8 mmol/L of 10 μ L after 10 min in each hole, 10
Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles the aqueous solution and 80 μ L a concentration of 100 mmol/L, pH of a concentration of 4.76 mg/mL of μ L=
5 phosphate buffer.In 37 DEG C of 15 min of constant-temperature incubation after mixing, the sulfuric acid that a concentration of 0.5 mol/L of 50 μ L are added is whole
Only react.It visually observes the variation of color or passes through the absorbance at microplate reader 450 nm wavelength of measurement.Human milk gland as shown in Figure 3
The light absorption value measured by hydrogen peroxide that cancer cell MCF-7 is generated increases with the increase of propylene glycol methyl ether acetate concentration, and
Significant change does not occur for people's renal epithelial cell HEK293.
Embodiment 5：
Culture medium is the RPMI-1640 containing 6 % fetal calf serums, and culture environment is 5% carbon dioxide culture of 37 DEG C of constant temperature
Case.Adherent cell is digested with pancreatin, culture medium is added to blow and beat mixing, calculates cell quantity.Be diluted to after cell suspension with
Be added into 96 orifice plates per 200 μ L of hole, cultivate in incubator wait within 24 hours it is complete adherent.Wait for adherent complete each cell later
The phosphate buffer of hole a concentration of 10 mmol/L of 200 μ L, pH 7.4 gently blow and beat flushing 3 times, remove extra culture
Liquid is with for use.When the propylene glycol methyl ether acetate of a concentration of 250 ng/mL of 100 μ L is added in each cell hole stimulates different
Between（0-20 min）3,3 ', the 5,5 '-tetramethyl biphenyl amine salt of a concentration of 8 mmol/L of 10 μ L are added in each hole later
Hydrochlorate, bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution of a concentration of 4.76 mg/mL of 10 μ L and 80 μ L a concentration of 100
Mmol/L, the phosphate buffer of pH=5.In 37 DEG C of 15 min of constant-temperature incubation after mixing, a concentration of 0.5 mol/ of 50 μ L are added
The sulfuric acid of L terminates reaction.It visually observes the variation of color or passes through the absorbance at microplate reader 450 nm wavelength of measurement.Such as Fig. 4
The light absorption value measured by hydrogen peroxide that shown human breast cancer cell line Bcap-37 generates increases with propylene glycol methyl ether acetate stimulation time
Add and increases, and significant change does not occur for people's renal epithelial cell HEK293.
Embodiment 6：
Culture medium is the RPMI-1640 containing 6 % fetal calf serums, and culture environment is 5% carbon dioxide culture of 37 DEG C of constant temperature
Case.Adherent cell is digested with pancreatin, culture medium is added to blow and beat mixing, calculates cell quantity.Be diluted to after cell suspension with
Be added into 96 orifice plates per 200 μ L of hole, cultivate in incubator wait within 24 hours it is complete adherent.Wait for adherent complete each cell later
The phosphate buffer of hole a concentration of 10 mmol/L of 200 μ L, pH 7.4 gently blow and beat flushing 3 times, remove extra culture
Liquid is with for use.The propylene glycol methyl ether acetate of a concentration of 250 ng/mL of 100 μ L is added in each cell hole stimulates 10 min
The 3,3',5,5'-tetramethylbenzidine hydrochloride of a concentration of 8 mmol/L of 10 μ L is added in each hole later, 10 μ L are dense
Degree is the bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution and a concentration of 100 mmol/L of 80 μ L of 4.76 mg/mL, pH=5
Phosphate buffer.It is terminated instead in 37 DEG C of 15 min of constant-temperature incubation, the sulfuric acid that a concentration of 0.5 mol/L of 50 μ L are added after mixing
It answers.The absorbance at 450 nm wavelength is measured with microplate reader, calculating MCF-7 cells according to the standard curve of 2 gained of example releases
The speed for letting off hydrogen oxide is 2.66 × 10-16 mol/cell/s。
Embodiment 7：
One kind is based on the active extracellular hydrogen peroxide detection of bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
Kit.Kit includes providing 3,3 ', 5,5 '-tetramethyl biphenyl amide hydrochlorides（A liquid）, bismuth-bovine serum albumin(BSA)-
Platinum Nanoparticles solution（B liquid）, sulfuric acid solution（Terminate liquid）, hydrogenperoxide steam generator（Titer）.A liquid includes a concentration of 8 mmol/L
3,3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate aqueous solutions.B liquid includes after prepared by embodiment 1 with a concentration of 100 mmol/L,
Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles solution of a concentration of 0.53 mg/mL of the phosphate buffered saline of pH=5.Terminate liquid packet
Include the sulfuric acid solution of a concentration of 0.5 mol/L.Titer includes a concentration of 1, the peroxidating of 2,10,20,30,40,60 μm of ol/L
Aqueous solution of hydrogen.
Embodiment 8：
The titer of 100 μ L embodiments 7, a liquid of 10 μ L embodiments 7, the b of 90 μ L embodiments 7 are added in 96 orifice plates
Liquid terminates reaction after mixing in 37 DEG C of 15 min of constant-temperature incubation, the terminate liquid that 50 μ L embodiments 7 are added.It is measured with microplate reader
Absorbance at 450 nm wavelength draws peroxide standard curve or calculates regression equation.100 μ L are added in 96 orifice plates
Sample solution, a liquid of 10 μ L embodiments 7, the b liquid of 90 μ L embodiments 7 are added in 37 DEG C of 15 min of constant-temperature incubation after mixing
The terminate liquid of 50 μ L embodiments 7 terminates reaction.The absorbance at 450 nm wavelength is measured with microplate reader, according to hydrogen peroxide mark
Directrix curve or regression equation calculation concentration of hydrogen peroxide.Linear ranging from 0.5 ~ 30 μm of ol/L of detection, detection are limited to 0.068 μ
mol/L。
The foregoing is merely the exemplary embodiments of the present invention, are not intended to limit the invention, all essences in the present invention
Any modification made by within refreshing and principle, equivalent replacement and improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of hydrogen peroxide detection method based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, it is characterized in that
By hydrogen peroxide, 3,3',5,5'-tetramethylbenzidine hydrochloride is permanent after bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution mixing
Temperature is incubated, and the sulfuric acid that 0.5 mol/L is added terminates reaction, and reaction product is yellow, a length of 450 nm of maximum absorption wave；Described
Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme is made by following steps：In the ox blood of a concentration of 50 mg/mL of 5 mL
A concentration of 16 mmol/L platinum acid chloride solutions of 5 mL and a concentration of 1.5 mol/L hydrogen-oxygens of 0.5 mL are added in pure protein solution
To change sodium solution, reacts 2 h at 80 DEG C of water-bath after mixing, solution is packed into the super filter tube that cutoff is 3k after reaction, and 6000
R/min centrifugal ultrafiltrations, and wash 3 times, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is obtained, by bovine serum albumin(BSA)-Platinum Nanoparticles water
Solution is freeze-dried to obtain bovine serum albumin(BSA)-Platinum Nanoparticles powder, in the bovine serum albumin of a concentration of 23.8 mg/mL of 0.6 mL
The bismuth nitrate solution and a concentration of 10 mmol/L of 1.8 mL of a concentration of 0.5 mmol/L of 0.6 mL are added in vain-platinum aqueous solution,
The phosphate buffer of pH=7 reacts 2.5 h after mixing at 80 DEG C of water-bath, and it is 3k's that solution, which is packed into cutoff, after reaction
Super filter tube, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, obtain the aqueous solution of bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles；By bismuth-
Bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles powder.
2. the hydrogen peroxide inspection according to claim 1 based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
Survey method, it is characterized in that can judge the concentration of hydrogen peroxide according to color development system solution colour.
3. the hydrogen peroxide inspection according to claim 1 based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
Survey method, it is characterized in that can be according to the absorbance at 450 nm of microplate reader to detect the concentration of hydrogen peroxide.
4. the peroxidating according to claim 1 or 3 based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
Hydrogen detection method, it is characterized in that linear ranging from 0.5 ~ 30 μm of ol/L of detection, detection are limited to 0.068 μm of ol/L.
5. the peroxide according to claim 1 or 2 or 3 based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
Change hydrogen detection method, it is characterized in that 100 μ L hydrogenperoxide steam generators are added in 96 orifice plates, a concentration of 8 mmol/L's of 10 μ L
3,3',5,5'-tetramethylbenzidine hydrochloride, a concentration of 4.76 mg/mL bismuths-bovine serum albumin(BSA)-Platinum Nanoparticles of 10 μ L are water-soluble
Liquid and a concentration of 100 mmol/L of 80 μ L, the phosphate buffer of pH=5 add in 37 DEG C of 15 min of constant-temperature incubation after mixing
The sulfuric acid for entering a concentration of 0.5 mol/L of 50 μ L terminates reaction, visually observes the variation of color or measures 450 by microplate reader
Absorbance at nm wavelength.
6. the peroxide according to claim 1 or 2 or 3 based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
Change hydrogen detection method, it is characterized in that can be used for measuring extracellular concentration of hydrogen peroxide.
7. a kind of hydrogen peroxide detection kit based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme, feature
It is to contain 3,3 ', 5,5 '-tetramethyl biphenyl amide hydrochlorides, bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles solution, sulphur in kit
Acid solution and hydrogenperoxide steam generator, the 3,3',5,5'-tetramethylbenzidine HCI solution is as a liquid, bismuth-cow's serum
Albumin-Platinum Nanoparticles solution is as b liquid, and sulfuric acid solution is as terminate liquid, and hydrogenperoxide steam generator is as titer.
8. the hydrogen peroxide inspection according to claim 7 based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme
Test agent box, it is characterized in that containing the 3,3',5,5'-tetramethylbenzidine hydrochloric acid brine of a concentration of 8 mmol/L in a liquid
Solution；Contain useful a concentration of 100 mmol/L in the b liquid, the phosphate buffered salines of pH=5 at a concentration of 0.53 mg/
Bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles solution of mL；Contain the sulfuric acid solution of a concentration of 0.5 mol/L in the terminate liquid；It is described
Contain a concentration of 1 in titer, the aqueous hydrogen peroxide solution of 2,10,20,30,40,60 μm of ol/L.
9. a kind of mistake based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme according to claim 7 or 8
Hydrogen oxide detection kit the, it is characterized in that bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme is by following steps
It is made：A concentration of 16 mmol/L chloroplatinic acids of 5 mL are added in the Bovine Serum Albumin in Aqueous Solution of a concentration of 50 mg/mL of 5 mL
Solution and a concentration of 1.5 mol/L sodium hydroxide solutions of 0.5 mL react 2 h after mixing at 80 DEG C of water-bath, and solution fills after reaction
Enter the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, obtains bovine serum albumin(BSA)-Platinum Nanoparticles
Bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bovine serum albumin(BSA)-Platinum Nanoparticles powder, 0.6 by aqueous solution
The bismuth nitrate of a concentration of 0.5 mmol/L of 0.6 mL is added in bovine serum albumin(BSA)-platinum aqueous solution of a concentration of 23.8 mg/mL of mL
Solution and a concentration of 10 mmol/L of 1.8 mL, the phosphate buffer of pH=7 react 2.5 h after mixing, instead at 80 DEG C of water-bath
Solution is packed into the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations after answering, and washes 3 times, obtains bismuth-cow's serum
The aqueous solution of albumin-Platinum Nanoparticles is freeze-dried bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution to obtain bismuth-bovine serum albumin
In vain-Platinum Nanoparticles powder.
10. a kind of any mistakes based on bismuth-bovine serum albumin(BSA)-Platinum Nanoparticles Mimetic enzyme of claim 7-9
The application of hydrogen oxide detection kit, it is characterized in that 100 μ L titers are added in 96 orifice plates, 10 μ L a liquid, 90 μ L b
Liquid is added 50 μ L terminate liquids and terminates reaction in 37 DEG C of 15 min of constant-temperature incubation after mixing;450 nm wavelength are measured with microplate reader
The absorbance at place draws peroxide standard curve or calculates regression equation；It is added 100 μ l sample solutions in 96 orifice plates, 10
μ L a liquid, 90 μ L b liquid are added 50 μ L terminate liquids and terminate reaction, use microplate reader in 37 DEG C of 15 min of constant-temperature incubation after mixing
The absorbance at 450 nm wavelength is measured, according to peroxide standard curve or regression equation calculation concentration of hydrogen peroxide, detection
The range of linearity is 0.5 ~ 30 μm of ol/L, and detection is limited to 0.068 μm of ol/L.
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| CN103433484A (en) * | 2013-08-22 | 2013-12-11 | 福建医科大学 | Bovine serum albumin-platinum composite nanomaterial mimetic peroxidase |
| CN104990914A (en) * | 2015-06-02 | 2015-10-21 | 蒋春亮 | Tumor tissue cell protoheme quantitative detection reagent and preparation method thereof |
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| CN104990914A (en) * | 2015-06-02 | 2015-10-21 | 蒋春亮 | Tumor tissue cell protoheme quantitative detection reagent and preparation method thereof |
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| Title |
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| Peroxidase-mimic bismuth–gold nanoparticles for determining the activity of thrombin and drug screening;Chia-Wen Lien et al.;《ChemComm.》;20120621;第48卷;第7953页左栏第1段;Supporting Information第1页第1段,第2页第2段 * |
| Synthesis and Peroxidase-Like Activity of Salt-Resistant Platinum Nanoparticles by Using Bovine Serum Albumin as the Scaffold;Shao-Bin He et al.;《ChemCatChem》;20140415;第6卷;第1543-1548页 * |
| 食品中残留过氧化氢的测定;马春华等;《分析实验室》;20130430;第32卷(第4期);第113-117页 * |
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