CN105572065B - Glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme - Google Patents
Glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme Download PDFInfo
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Abstract
The invention discloses a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme.The present invention is catalyzed the glycoxidative generation hydrogen peroxide of grape using glucose oxidase, and platinum bismuth nanometer Mimetic enzyme catalyzing hydrogen peroxide aoxidizes 3 methyl, 2 benzothiazolinone hydrazone hydrochloride and N ethyl ns（2 hydroxyl, 3 sulfopropyl）3 methylaniline sodium salt coupling colour developments provide a kind of quick, easy, sensitive glucose detection new method.Platinum bismuth nanometer Mimetic enzyme used in the new detecting method prepares simplicity and is easy to get, and may be implemented to carry out visual analyzing to the glucose in serum, has many advantages, such as easy to operate, high sensitivity, high specificity, use easy to spread.
Description
Technical field
The present invention relates to a kind of novel glucose content rapid detection method and detection kit more particularly to a kind of bases
In the glucose sensing approach and its detection kit of platinum bismuth nanometer Mimetic enzyme, belong to analytical chemistry and nanotechnology
Field.
Background technology
Glucose be it is a kind of can directly be absorbed and utilized, supplement the carbohydrate of thermal energy, be the main of needed by human body energy
Source is oxidized to carbon dioxide and water, and supplies heat simultaneously in vivo, or is stored in the form of glycogen.It can promote liver
Function of detoxification has protective effect to liver, is energy goods and materials most commonly seen in organism.And the sugar in serum is known as blood glucose,
All it is glucose in most cases.The energy needed for each tissue cellular activity largely comes from glucose in vivo, because of this person
Internal blood glucose must keep certain horizontal needs that could maintain internal each organ and tissue.With people’s lives level
Improve and the exacerbation of aging of population, more and more people are easy to suffer from hyperglycemia, and blood glucose value it is excessively high can cause it is thirsty, weary
Power, urine are more, eat more, is serious, can lead to dizziness, diabetes, kidney failure, body is every in disorder, does not take and controls in time
Treatment measure even results in death.Now insulin price, the dependence for being used to treat diabetes are strong, therefore for blood glucose value
Accurate quickly detection has great significance for the hypoglycemic of hyperglycemia and the prevention of diabetes.
The present invention is based on platinum bismuth nanometer Mimetic enzyme, method and the detection of a kind of colorimetric detection glucose are provided
Kit.
Invention content
It is an object of the present invention to provide a kind of glucose detection sides based on platinum bismuth nanometer Mimetic enzyme
Method.It is catalyzed the glycoxidative generation hydrogen peroxide of grape, platinum bismuth nanometer Mimetic enzyme including using glucose oxidase
Catalyzing hydrogen peroxide aoxidizes 3- methyl-2-benzothiazolinone hydrazones hydrochloride and N- ethyls-N-（2- hydroxyl -3- sulfopropyls）-3-
Methylaniline sodium salt coupling colour development, a length of 590 nm of maximum absorption wave of color development system, Portugal can be obtained by 590 nm absorbances
The content of grape sugar.
In order to realize that the purpose of above-mentioned detection method, the present invention use following technical scheme：
A kind of glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme of the present invention, it is characterized in that will
Glucose oxidase, glucose solution, phosphate buffer mix, and phosphate buffer, 3- methyl -2- benzene is added after warm bath
And thiazolinone hydrazone hydrochloride, N- ethyls-N-（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts, platinum bismuth nanometer were simulated
Ultravioletvisible absorption is visually observed or measured to oxide enzyme aqueous solution after warm bath；The platinum bismuth nanometer Mimetic enzyme
Made from following steps：5 mL a concentration of 16 are added in the Bovine Serum Albumin in Aqueous Solution of a concentration of 50 mg/mL of 5 mL
Mmol/L platinum acid chloride solutions and a concentration of 1.5 mol/L sodium hydroxide solutions of 0.5 mL, 80 DEG C of 2 h of reaction of water-bath after mixing,
Solution is packed into the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations after reaction, and washes 3 times, obtains cow's serum
Albumin-Platinum Nanoparticles aqueous solution is freeze-dried bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution to obtain bovine serum albumin(BSA)-nanometer
Platinum powder end, 0.6 mL a concentration of 0.5 is added in bovine serum albumin(BSA)-platinum aqueous solution of a concentration of 23.8 mg/mL of 0.6 mL
The bismuth nitrate solution of mmol/L and a concentration of 10 mmol/L of 1.8 mL, the phosphate buffer of pH=7,80 DEG C of water-bath after mixing
2.5 h are reacted, solution is packed into the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations after reaction, and washes 3 times,
The aqueous solution of platinum bismuth nanometer Mimetic enzyme is obtained, platinum bismuth nanometer Mimetic enzyme aqueous solution is freeze-dried to obtain
Platinum bismuth nanometer Mimetic enzyme powder.
The glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme, it is characterized in that by 80 μ L concentration
For the glucose standards solution of the glucose oxidase and 640 μ L various concentrations of 180 U/mL, a concentration of 10 mmol/ of 80 μ L
A concentration of 10 mmol/L of 1.3 mL, the phosphoric acid of pH=7 is added in L, the phosphate buffer mixing of pH=7,37 DEG C of 30 min of warm bath
Salt buffer, the 3- methyl-2-benzothiazolinone hydrazone hydrochlorides of a concentration of 0.5 mmol/L of 1 mL, 800 μ L a concentration of 3
N- ethyls-the N- of mmol/L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts, a concentration of 4.76 mg/mL platinum bismuths of 100 μ L
Nanometer Mimetic enzyme aqueous solution, 37 DEG C of 30 min of warm bath visually observe solution colour or measure ultravioletvisible absorption.
The glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme, it is characterized in that color product solution
Color is purple, and with the increase of concentration of glucose, solution colour is deepened, and the detection for visually observing solution colour is limited to 5 μ
mol/L。
The glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme, it is characterized in that color product is maximum
Absorbing wavelength is 590 nm, makees standard curve according to the absorbance measured at 590 nm wavelength, the range of linearity of detection is 1 ~
100 μm of ol/L, detection are limited to 0.2 μm of ol/L.
The present invention realizes that the purpose of glucose determination reagent box, the present invention use following technical scheme：
A kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme of the present invention, it is characterized in that
Kit includes a liquid, b liquid, titer, enzyme solution and sample diluting liquid, contains 3- methyl -2-[4-morpholinodithio quinoline in a liquid
Ketone hydrazone hydrochloride and N- ethyls-N-（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts are simulated containing platinum bismuth nanometer in b liquid
Peroxidase Solution, titer are glucose standards solution, and enzyme solution is glucose oxidase solution.
Contain the 3- methyl-2-benzothiazolinone hydrazones hydrochloride and concentration of a concentration of 0.28 mmol/L in above-mentioned a liquid
For the N- ethyls-N- of 1.33 mmol/L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts；Contain useful 10 mmol/ in b liquid
The platinum bismuth nanometer Mimetic enzyme solution of L, a concentration of 0.34 mg/mL of the phosphate buffered saline of pH=7；Titer
Include the glucose solution of a concentration of 6.25,31.25,62.5,125,250,375,500,625 μm of ol/L；Enzyme solution includes with 10
Mmol/L, the glucose oxidase solution of a concentration of 90 U/mL of the phosphate buffered saline of pH=7；Sample diluting liquid includes
10 mmol/L, the phosphate buffer of pH=7.
The platinum bismuth nanometer Mimetic enzyme is made from following steps：In the ox 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 of 0.5 mL are added in seralbumin aqueous solution
Sodium hydroxide solution, 80 DEG C of 2 h of reaction 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 1.8 mL a concentration of 10 mmol/L, pH of a concentration of 0.5 mmol/L of 0.6 mL are added in vain-platinum aqueous solution
=7 phosphate buffer, 80 DEG C of 2.5 h of reaction of water-bath after mixing, solution is packed into the ultrafiltration that cutoff is 3k after reaction
Pipe, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, the aqueous solution of platinum bismuth nanometer Mimetic enzyme is obtained, by platinum bismuth nanometer
Mimetic enzyme aqueous solution is freeze-dried to obtain platinum bismuth nanometer Mimetic enzyme powder.
A kind of application of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme of the present invention,
It is characterized in as glucose detection.
A kind of application of the glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme, it is characterized in that
It is added 160 μ L enzyme solutions in 640 μ L titers, 37 DEG C of 30 min of warm bath are added 1.8 mL a liquid and 1.4 mL b liquid, 37 DEG C
30 min of warm bath measures the absorbance at 590 nm, draws glucose standard curve or calculates regression equation；In 40 μ L people's blood
600 μ L sample dilutions, 160 μ L enzyme solutions are added in final proof product, 1.8 mL a liquid and 1.4 mL are added in 37 DEG C of 30 min of warm bath
B liquid, 37 DEG C of 30 min of warm bath measure the absorbance at 590 nm, according to standard curve or regression equation, calculate blood serum sample
The content of middle glucose.
The detection method of the present invention uses following specific technical solution：
（One）A concentration of 16 mmol/ of 5 mL are added in the Bovine Serum Albumin in Aqueous Solution of a concentration of 50 mg/mL of 5 mL
L platinum acid chloride solutions and a concentration of 1.5 mol/L sodium hydroxide solutions of 0.5 mL, 80 DEG C of 2 h of reaction of water-bath after mixing.After reaction
Solution is packed into the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations, and washes 3 times, obtains bovine serum albumin(BSA)-
Bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bovine serum albumin(BSA)-Platinum Nanoparticles powder by Platinum Nanoparticles aqueous solution.
It is added a concentration of 0.5 mmol/L's of 0.6 mL in bovine serum albumin(BSA)-platinum aqueous solution of a concentration of 23.8 mg/mL of 0.6 mL
Bismuth nitrate solution and a concentration of 10 mmol/L of 1.8 mL, the phosphate buffer of pH=7,80 DEG C of reactions 2.5 of water-bath after mixing
H, solution is packed into the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations after reaction, and washes 3 times, obtains platinum bismuth
Nanometer Mimetic enzyme aqueous solution.It is freeze-dried platinum bismuth nanometer Mimetic enzyme aqueous solution to obtain platinum bismuth nanometer mould
Quasi- peroxide enzyme powder.All glasswares used in preparation process pass through chloroazotic acid and impregnate, and are used in combination distilled water thoroughly clear
It washes, dries.
（Two）The glucose sensing approach of the present invention：By the glucose oxidase of a concentration of 180 U/mL of 80 μ L, 640 μ L
The glucose standards solution of various concentration, a concentration of 10 mmol/L of 80 μ L, the phosphate buffer mixing of pH=7,37 DEG C of warm bath
After 30 min, addition a concentration of 10 mmol/L of 1.3 mL, the phosphate buffer of pH=7, a concentration of 0.5 mmol/L's of 1 mL
3- methyl-2-benzothiazolinone hydrazone hydrochlorides, the N- ethyls-N- of a concentration of 3 mmol/L of 800 μ L（2- hydroxyl -3- sulphurs third
Base）- 3- methylaniline sodium salts, a concentration of 4.76 mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solutions of 100 μ L, 37 DEG C of temperature
30 min are bathed, the variation of color is visually observed or measure the absorbance at 590 nm.
（Three）Glucose sensing approach based on horseradish peroxidase：By the glucose of a concentration of 180 U/mL of 80 μ L
Oxidizing ferment, the glucose standards solution of 640 μ L various concentrations, a concentration of 10 mmol/L of 80 μ L, the phosphate buffer of pH=7
It mixes, after 37 DEG C of 30 min of warm bath, a concentration of 10 mmol/L of 2.9 mL, the phosphate buffer of pH=7,0.2 mL concentration is added
For the 3,3',5,5'-tetramethylbenzidine hydrochloride of 16 mmol/L, the horseradish peroxidase of a concentration of 0.5 U/mL of 100 μ L
Enzyme, 37 DEG C of 10 min of warm bath visually observe the variation of color or measure the absorbance at 652 nm.
It is another object of the present invention to provide a kind of glucose detections based on platinum bismuth nanometer Mimetic enzyme
Kit.Kit includes 3- methyl-2-benzothiazolinone hydrazones hydrochloride and N- ethyls-N-（2- hydroxyl -3- sulfopropyls）-
3- methylaniline sodium salt solutions（A liquid）, platinum bismuth nanometer Mimetic enzyme solution（B liquid）, glucose standards solution（Standard
Liquid）, glucose oxidase solution（Enzyme solution）, sample diluting liquid.
In order to realize the purpose of mentioned reagent box, the present invention is using technical solution in detail below：
（Four）Glucose determination reagent box：A liquid includes 3- methyl -2-[4-morpholinodithio quinoline ketone of a concentration of 0.28 mmol/L
N- ethyls-the N- of hydrazone hydrochloride and a concentration of 1.33 mmol/L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium-salt aqueous solutions.
B liquid includes above-mentioned technical proposal（One）With 10 mmol/L after preparation, a concentration of the 0.34 of the phosphate buffered saline of pH=7
The platinum bismuth nanometer Mimetic enzyme solution of mg/mL.Titer includes a concentration of 6.25,31.25,62.5,125,250,
The glucose solution of 375,500,625 μm of ol/L.Enzyme solution include with 10 mmol/L, the phosphate buffered saline of pH=7 it is dense
Degree is the glucose oxidase solution of 90 U/mL.Sample diluting liquid includes 10 mmol/L, the phosphate buffer of pH=7.
（Five）Glucose determination reagent box application method：In 640 μ L technical solutions（Four）Titer in 160 μ L are added
Technical solution（Four）Enzyme solution, 37 DEG C of 30 min of warm bath, be added 1.8 mL technical solutions（Four）A liquid and 1.4 mL technical solutions
（Four）B liquid, 37 DEG C of 30 min of warm bath, visually observe color variation or measure 590 nm at absorbance and draw grape
Standard for Sugars curve calculates regression equation.600 μ L technical solutions are added in 40 μ L human serum samples（Four）Sample dilution
Liquid, 160 μ L technical solutions（Four）Enzyme solution, 37 DEG C of 30 min of warm bath, be added 1.8 mL technical solutions（Four）A liquid and 1.4
ML technical solutions（Four）B liquid, 37 DEG C of 30 min of warm bath, visually observe color variation or measure 590 nm at absorbance.
Judged according to color development system solution colour or is quantified according to absorbance standard curve.
Advantages of the present invention：
（1）The present invention is catalyzed the glycoxidative generation hydrogen peroxide of grape using glucose oxidase, and platinum bismuth nanometer simulates peroxide
Compound enzymatic hydrogen peroxide oxidation 3- methyl-2-benzothiazolinone hydrazones hydrochloride and N- ethyls-N-（2- hydroxyl -3- sulphurs third
Base）- 3- methylaniline sodium salt coupling colour developments, to show the variation of solution colour and ultra-violet absorption spectrum feature, Ke Yiyong
In the content detection of glucose.
（2）The present invention is high to the detection specificity of glucose, can be used for serum glucose level measurement.
（3）The present invention detects the high sensitivity of the detection of glucose, and the detection for visually observing color change is limited to 5 μm of ol/
L, the spectrophotometry range of linearity are 1 ~ 100 μm of ol/L, and detection is limited to 0.2 μm of ol/L.
（4）Glucose determination reagent box provided by the invention have stability good, easy to operate, high sensitivity, specificity
The advantages that strong, use easy to spread.
Description of the drawings
Fig. 1 is the uv absorption spectra of color product.
Fig. 2 is the outside drawing of color product.
Fig. 3 is glucose standard curve figure.
Fig. 4 be fructose, lactose, maltose for glucose detection influence diagram.
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, 80 DEG C of 2 h of reaction of water-bath.Solution is packed into cut-off after reaction
Molecular weight be 3k super filter tube, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, it is water-soluble to obtain 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 by liquid.It is dense in 0.6 mL
The bismuth nitrate solution of a concentration of 0.5 mmol/L of 0.6 mL is added in bovine serum albumin(BSA)-platinum aqueous solution that degree is 23.8 mg/mL
With a concentration of 10 mmol/L of 1.8 mL, the phosphate buffer of pH=7,80 DEG C of 2.5 h of reaction of water-bath, solution is packed into after reaction
Cutoff be 3k super filter tube, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, obtain platinum bismuth nanometer simulation peroxide
The aqueous solution of enzymatic.Platinum bismuth nanometer Mimetic enzyme catalytic water solution is freeze-dried to obtain platinum bismuth nanometer simulation peroxide
Compound enzymatic powder.
Embodiment 2：
By the glucose mark of the glucose oxidase of a concentration of 180 U/mL of 80 μ L and a concentration of 800 μm of ol/L of 640 μ L
1.3 mL are added in quasi- solution, a concentration of 10 mmol/L of 80 μ L, the phosphate buffer mixing of pH=7,37 DEG C of 30 min of warm bath
A concentration of 10 mmol/L, the phosphate buffer of pH=7,3- methyl -2-[4-morpholinodithio quinoline of a concentration of 0.5 mmol/L of 1 mL
Ketone hydrazone hydrochloride, the N- ethyls-N- of a concentration of 3 mmol/L of 800 μ L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts,
A concentration of 4.76 mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution prepared by 100 μ L embodiments 1,37 DEG C of warm bath 30
Min measures ultravioletvisible absorption collection of illustrative plates, as shown in Figure 1, a length of 590 nm of color product maximum absorption wave.
Embodiment 3：
By the glucose standards solution of the glucose oxidase of a concentration of 180 U/mL of 80 μ L and 640 μ L various concentrations,
1.3 mL a concentration of 10 are added in a concentration of 10 mmol/L of 80 μ L, the phosphate buffer mixing of pH=7,37 DEG C of 30 min of warm bath
Mmol/L, the phosphate buffer of pH=7, the 3- methyl-2-benzothiazolinone hydrazone hydrochloric acid of a concentration of 0.5 mmol/L of 1 mL
Salt, the N- ethyls-N- of a concentration of 3 mmol/L of 800 μ L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts, 100 μ L are real
Apply a concentration of 4.76 mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution prepared by example 1,37 DEG C of 30 min of warm bath, observation
Solution colour.As shown in Fig. 2, with the increasing of concentration of glucose, the purple that solution shows is more and more deeper, observes face by visual observation
The detection of color change is limited to 5 μm of ol/L.
Embodiment 4：
By the glucose standards solution of the glucose oxidase of a concentration of 180 U/mL of 80 μ L and 640 μ L various concentrations,
1.3 mL a concentration of 10 are added in a concentration of 10 mmol/L of 80 μ L, the phosphate buffer mixing of pH=7,37 DEG C of 30 min of warm bath
Mmol/L, the phosphate buffer of pH=7, the 3- methyl-2-benzothiazolinone hydrazone hydrochloric acid of a concentration of 0.5 mmol/L of 1 mL
Salt, the N- ethyls-N- of a concentration of 3 mmol/L of 800 μ L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts, 100 μ L are real
A concentration of 4.76 mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution prepared by example 1 is applied, 37 DEG C of 30 min of warm bath are measured
Absorbance at 590 nm wavelength draws standard curve.As shown in figure 3, the range of linearity of spectrophotometry is 1 ~ 100 μ
Mol/L, detection are limited to 0.2 μm of ol/L.
Embodiment 5：
A kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme.Kit includes 3- methyl-
2-[4-morpholinodithio quinoline ketone hydrazone hydrochloride and N- ethyls-N-（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salt solutions（A liquid）, platinum
Bismuth nanometer Mimetic enzyme solution（B liquid）, glucose standards solution（Titer）, glucose oxidase solution（Enzyme solution）,
Sample diluting liquid.A liquid includes the 3- methyl-2-benzothiazolinone hydrazones hydrochloride of a concentration of 0.28 mmol/L and a concentration of
N- ethyls-the N- of 1.33 mmol/L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium-salt aqueous solutions.B liquid includes that embodiment 1 is made
Standby rear 10 mmol/L, the platinum bismuth nanometer of a concentration of 0.34 mg/mL of the phosphate buffered saline of pH=7 simulate peroxidating
Object enzyme solutions.Titer includes a concentration of 6.25, and the glucose of 31.25,62.5,125,250,375,500,625 μm of ol/L is molten
Liquid.Enzyme solution includes with 10 mmol/L, and the glucose oxidase of a concentration of 90 U/mL of the phosphate buffered saline of pH=7 is molten
Liquid.Sample diluting liquid includes 10 mmol/L, the phosphate buffer of pH=7.
Embodiment 6：
By the glucose standards solution of the glucose oxidase of a concentration of 180 U/mL of 80 μ L and 640 μ L various concentrations,
2.9 mL a concentration of 10 are added in a concentration of 10 mmol/L of 80 μ L, the phosphate buffer mixing of pH=7,37 DEG C of 30 min of warm bath
Mmol/L, the phosphate buffer of pH=7, the 3,3',5,5'-tetramethylbenzidine hydrochloride of a concentration of 16 mmol/L of 0.2 mL
With the horseradish peroxidase of a concentration of 0.5 U/mL of 100 μ L, 37 DEG C of 30 min of warm bath measure the absorbance at 652 nm,
Draw standard curve.By the glucose oxidase of a concentration of 180 U/mL of 80 μ L and 40 μ L human serum samples and 680 μ L
2.9 mL a concentration of 10 are added in a concentration of 10 mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of 30 min of warm bath
Mmol/L, the phosphate buffer of pH=7, the 3,3',5,5'-tetramethylbenzidine hydrochloric acid of a concentration of 16 mmol/L of 0.2 mL
The horseradish peroxidase of salt and a concentration of 0.5 U/mL of 100 μ L, 37 DEG C of 10 min of warm bath measure the extinction at 652 nm
Degree calculates the content of glucose in blood serum sample according to standard curve.
Embodiment 7：
The enzyme solution of 160 μ L embodiments 5 is added in the titer of 640 μ L embodiments 5,37 DEG C of 30 min of warm bath are added
The b liquid of a liquid of 1.8 mL embodiments 5 and 1.4 mL embodiments 5,37 DEG C of 30 min of warm bath measure the absorbance at 590 nm,
It draws glucose standard curve or calculates regression equation.The sample that 600 μ L embodiments 5 are added in 40 μ L human serum samples is dilute
Liquid, the enzyme solution of 160 μ L embodiments 5 are released, a liquid and 1.4 mL embodiments 5 of 1.8 mL embodiments 5 is added in 37 DEG C of 30 min of warm bath
B liquid, 37 DEG C of 30 min of warm bath measure the absorbance at the places 590 nm, according to standard curve or regression equation, calculating serum sample
The content of glucose in product.The results are shown in table below, compared with the method in example 6, in 3.18 mmol/L of low blood glucose level to height
The data that two methods measure within the scope of 10.24 mmol/L of blood glucose value are consistent, and relative error is -6.6% ~ 4.9%；
Embodiment 8：
The glucose solution that the sample diluting liquid of 600 μ L embodiments 5 is prepared is added in 40 μ L human serum sample's solution,
The b of a liquid and 1.4 mL embodiments 5 of 1.8 mL embodiments 5 is added in the enzyme solution of 160 μ L embodiments 5,37 DEG C of 30 min of warm bath
Liquid, 37 DEG C of 30 min of warm bath measure the absorbance value at 590 nm wavelength, and the recovery of standard addition that sample is calculated in conjunction with example 7 is
94.5% ~ 104.5%, relative standard deviation is 2. 9% ~ 7.2%.
Embodiment 10：
The glucose standard of a concentration of 0.5 mmol/L of 640 μ L, the fruit of a concentration of 0.5 mmol/L of 640 μ L are taken respectively
Standard for Sugars liquid, the lactose titer of a concentration of 0.5 mmol/L of 640 μ L, the maltose mark of a concentration of 0.2 mmol/L of 640 μ L
Quasi- liquid, is added the enzyme solution of 160 μ L embodiments 5, and 37 DEG C of 30 min of warm bath, a liquid and 1.4 mL that 1.8 mL embodiments 5 are added are real
The b liquid of example 5 is applied, 37 DEG C of 30 min of warm bath measure the absorbance at 590 nm wavelength.As shown in figure 4, fructose, lactose, maltose
Interference will not be generated to the glucose sensing approach of the present invention.
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 (9)
1. a kind of glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme, it is characterized in that by glucose oxidase,
Glucose solution, phosphate buffer mix, and phosphate buffer, 3- methyl-2-benzothiazolinone hydrazone salt is added after warm bath
Hydrochlorate, N- ethyls-N-（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts, platinum bismuth nanometer Mimetic enzyme aqueous solution,
Ultravioletvisible absorption is visually observed or measured after warm bath；The platinum bismuth nanometer Mimetic enzyme is made by following steps
's：It is molten that 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
Liquid and a concentration of 1.5 mol/L sodium hydroxide solutions of 0.5 mL, 80 DEG C of 2 h of reaction of water-bath after mixing, solution is packed into after reaction
Cutoff be 3k super filter tube, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, obtain bovine serum albumin(BSA)-Platinum Nanoparticles water
Bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution is freeze-dried to obtain bovine serum albumin(BSA)-Platinum Nanoparticles powder, in 0.6 mL by solution
The bismuth nitrate that a concentration of 0.5 mmol/L of 0.6 mL are added in the bovine serum albumin(BSA) of a concentration of 23.8 mg/mL-platinum aqueous solution is molten
Liquid and a concentration of 10 mmol/L of 1.8 mL, the phosphate buffer of pH=7,80 DEG C of 2.5 h of reaction of water-bath after mixing, after reaction
Solution is packed into the super filter tube that cutoff is 3k, 6000 r/min centrifugal ultrafiltrations, and washes 3 times, obtains the simulation of platinum bismuth nanometer
Platinum bismuth nanometer Mimetic enzyme aqueous solution is freeze-dried to obtain platinum bismuth nanometer simulation peroxide by the aqueous solution of peroxidase
Compound enzyme powder.
2. the glucose sensing approach according to claim 1 based on platinum bismuth nanometer Mimetic enzyme, it is characterized in that
By the glucose standards solution of the glucose oxidase of a concentration of 180 U/mL of 80 μ L and 640 μ L various concentrations, 80 μ L concentration
Phosphate buffer for 10 mmol/L, pH=7 mixes, and a concentration of 10 mmol/L of 1.3 mL are added in 37 DEG C of 30 min of warm bath,
The phosphate buffer of pH=7, the 3- methyl-2-benzothiazolinone hydrazone hydrochlorides of a concentration of 0.5 mmol/L of 1 mL, 800 μ
N- ethyls-the N- of a concentration of 3 mmol/L of L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts, 100 μ L a concentration of 4.76
Mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solutions, 37 DEG C of 30 min of warm bath, visually observe solution colour or measure it is ultraviolet can
See absorption.
3. the glucose sensing approach according to claim 1 or 2 based on platinum bismuth nanometer Mimetic enzyme, feature
Be color product solution colour it is purple, with the increase of concentration of glucose, solution colour is deepened, and visually observes solution colour
Detection is limited to 5 μm of ol/L.
4. the glucose sensing approach according to claim 1 or 2 based on platinum bismuth nanometer Mimetic enzyme, feature
It is a length of 590 nm of color product maximum absorption wave, standard curve is made according to the absorbance measured at 590 nm wavelength, detects
The range of linearity be 1 ~ 100 μm of ol/L, detection be limited to 0.2 μm of ol/L.
5. a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme, it is characterized in that kit includes a
Liquid, b liquid, titer, enzyme solution and sample diluting liquid, in a liquid containing 3- methyl-2-benzothiazolinone hydrazones hydrochloride and
N- ethyls-N-（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts, it is molten containing platinum bismuth nanometer Mimetic enzyme in b liquid
Liquid, titer are glucose standards solution, and enzyme solution is glucose oxidase solution.
6. a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme according to claim 5,
It is characterized in containing the 3- methyl-2-benzothiazolinone hydrazones hydrochloride and a concentration of 1.33 of a concentration of 0.28 mmol/L in a liquid
N- ethyls-the N- of mmol/L（2- hydroxyl -3- sulfopropyls）- 3- methylaniline sodium salts；Contain useful 10 mmol/L in b liquid, pH=7
The platinum bismuth nanometer Mimetic enzyme solution of a concentration of 0.34 mg/mL of phosphate buffered saline；Titer includes concentration
It is the glucose solution of 6.25,31.25,62.5,125,250,375,500,625 μm of ol/L；Enzyme solution include with 10 mmol/L,
The glucose oxidase solution of a concentration of 90 U/mL of the phosphate buffered saline of pH=7；Sample diluting liquid includes 10
Mmol/L, the phosphate buffer of pH=7.
7. a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme according to claim 5 or 6,
It is characterized in that the platinum bismuth nanometer Mimetic enzyme is made from following steps：In the ox 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 of 0.5 mL are added in seralbumin aqueous solution
Sodium hydroxide solution, 80 DEG C of 2 h of reaction 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 1.8 mL a concentration of 10 mmol/L, pH of a concentration of 0.5 mmol/L of 0.6 mL are added in vain-platinum aqueous solution
=7 phosphate buffer, 80 DEG C of 2.5 h of reaction of water-bath after mixing, solution is packed into the ultrafiltration that cutoff is 3k after reaction
Pipe, 6000 r/min centrifugal ultrafiltrations, and wash 3 times, the aqueous solution of platinum bismuth nanometer Mimetic enzyme is obtained, by platinum bismuth nanometer
Mimetic enzyme aqueous solution is freeze-dried to obtain platinum bismuth nanometer Mimetic enzyme powder.
8. a kind of any glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme of claim 5-7
Using it is characterized in that as glucose detection.
9. a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme according to claim 8 are answered
With it is characterized in that 160 μ L enzyme solutions are added in 640 μ L titers, 1.8 mL a liquid and 1.4 are added in 37 DEG C of 30 min of warm bath
ML b liquid, 37 DEG C of 30 min of warm bath measure the absorbance at 590 nm, draw glucose standard curve or calculate regression equation；
600 μ L sample dilutions, 160 μ L enzyme solutions are added in 40 μ L human serum samples, 1.8 mL are added in 37 DEG C of 30 min of warm bath
A liquid and 1.4 mL b liquid, 37 DEG C of 30 min of warm bath measure the absorbance at 590 nm, according to standard curve or regression equation,
Calculate the content of glucose in blood serum sample.
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| CN109813666A (en) * | 2019-02-12 | 2019-05-28 | 福建医科大学 | Trypsase detection kit based on pro- platinum nanocell simulations oxidizing ferment |
| CN109781719A (en) * | 2019-02-27 | 2019-05-21 | 福建医科大学 | A kind of kit detecting trypsase and its inhibitor based on platinum cluster |
| CN111239125A (en) * | 2020-03-06 | 2020-06-05 | 中国药科大学 | Glucose colorimetric detection method by using platinum disulfide |
| CN113155927B (en) * | 2021-04-01 | 2021-11-19 | 华东师范大学 | Glucose potential sensor based on SDBA-Au composite nanoenzyme |
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