CN105572065A - Glucose detection kit based on platinum-bismuth nano mimic peroxidase - Google Patents
Glucose detection kit based on platinum-bismuth nano mimic peroxidase Download PDFInfo
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Abstract
The invention discloses a glucose detection kit based on platinum-bismuth nano mimic peroxidase. Glucose oxidase catalyzes glucose to be oxidized to generate hydrogen peroxide, the platinum-bismuth nano mimic peroxidase catalyzes hydrogen peroxide to oxidize 3-methyl-2-benzothiazolinone hydrazone hydrochloride and N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt for coupling color development, and then a new quick, simple, convenient and sensitive glucose detection method is presented. The platinum-bismuth nano mimic peroxidase used in the new detection method is easy and convenient to prepare and easy to obtain, can achieve visualized analysis on glucose in serum, has the advantages of being easy and convenient to operate, high in sensitivity and specificity and the like and is easy to use and popularize.
Description
Technical field
The present invention relates to a kind of novel glucose content method for quick and detection kit, particularly relate to a kind of glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme and detection kit thereof, belong to analytical chemistry and field of nanometer technology.
Background technology
Glucose is that one can directly absorb, and supplements the carbohydrates of heat energy, is the main source of needed by human body energy, is oxidized to carbon dioxide and water in vivo, and supplies heat simultaneously, or store with glycogen form.Can promote the function of detoxification of liver, have protective effect to liver, be energy goods and materials the most common in biosome.And the sugar in serum is called blood sugar, be all glucose in most cases.Energy major part in body needed for each histocyte activity is from glucose, and therefore blood in human body in sugar must keep certain level could maintain the needs of each Organ and tissue in body.Along with the growth in the living standard of people and increasing the weight of of aging population, increasing people easily suffers from hyperglycaemia, the too high meeting of blood glucose value cause thirsty, weak, urine is many, eat more, serious then can cause dizziness, diabetes, kidney failure, the every disorder of health, does not take timely remedy measures even to cause death.Now be used for the treatment of the insulin price of diabetes, dependence is strong, therefore accurately detecting fast of blood glucose value is had great significance for the hypoglycemic of hyperglycaemia and the prevention of diabetes.
The present invention is based on platinum bismuth nanometer Mimetic enzyme, provide a kind of method and detection kit of colorimetric detection glucose.
Summary of the invention
One object of the present invention is to provide a kind of glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme.Comprising utilizing glucose oxidase enzymatic glucose oxidase Hydrogen Peroxide, platinum bismuth nanometer Mimetic enzyme catalyzing hydrogen peroxide oxidation 3-methyl-2-benzothiazolinone hydrazone hydrochloride and N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt coupling colour development, the maximum absorption wavelength of color development system is 590nm, can be drawn the content of glucose by 590nm absorbance.
In order to realize the object of above-mentioned detection method, the present invention by the following technical solutions:
a kind of glucose sensing approach based on platinum bismuth nanometer Mimetic enzyme of the present invention,it is characterized in that glucose oxidase, glucose solution, phosphate buffer mixes, and adds phosphate buffer, 3-methyl-2-benzothiazolinone hydrazone hydrochloride after temperature bath
,n-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt, platinum bismuth nanometer Mimetic enzyme aqueous solution, visualization or mensuration ultravioletvisible absorption after temperature bath, described platinum bismuth nanometer Mimetic enzyme is obtained by following steps: be that to add 5mL concentration in the Bovine Serum Albumin in Aqueous Solution of 50mg/mL be 16mmol/L platinum acid chloride solution and 0.5mL concentration in 5mL concentration be 1.5mol/L sodium hydroxide solution, water-bath 80 DEG C reaction 2h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution freeze drying is obtained bovine serum albumin(BSA)-Platinum Nanoparticles powder, be add bismuth nitrate solution that 0.6mL concentration is 0.5mmol/L in the bovine serum albumin(BSA)-platinum aqueous solution of 23.8mg/mL and 1.8mL concentration is 10mmol/L in 0.6mL concentration, the phosphate buffer of pH=7, water-bath 80 DEG C reaction 2.5h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain the aqueous solution of platinum bismuth nanometer Mimetic enzyme, the freeze drying of platinum bismuth nanometer Mimetic enzyme aqueous solution is obtained platinum bismuth nanometer Mimetic enzyme powder.
Described
based on the glucose sensing approach of platinum bismuth nanometer Mimetic enzymeit is characterized in that being the glucose oxidase of 180U/mL and the Glucose standards solution of 640 μ L variable concentrations by 80 μ L concentration, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of temperature bath 30min, add the phosphate buffer that 1.3mL concentration is 10mmol/L, pH=7,1mL concentration is the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.5mmol/L
,800 μ L concentration are the N-ethyl-N-(2-hydroxyl-3-sulfopropyl of 3mmol/L)-3-methylaniline sodium salt, 100 μ L concentration are 4.76mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution, 37 DEG C of temperature bath 30min, visualization solution colour or mensuration ultravioletvisible absorption.
Described
based on the glucose sensing approach of platinum bismuth nanometer Mimetic enzyme, it is characterized in that color product solution colour is purple, along with the increase of concentration of glucose, solution colour is deepened, and the detection of visualization solution colour is limited to 5 μm of ol/L.
Described
based on the glucose sensing approach of platinum bismuth nanometer Mimetic enzyme, it is characterized in that color product maximum absorption wavelength is 590nm, make typical curve according to the absorbance that 590nm wavelength place records, its range of linearity detected is 1 ~ 100 μm of ol/L, detects and is limited to 0.2 μm of ol/L.
The present invention realizes
glucose detectionthe object of kit, the present invention by the following technical solutions:
Of the present invention
based on a glucose determination reagent box for platinum bismuth nanometer Mimetic enzyme,it is characterized in that kit comprises a liquid, b liquid, titer, enzyme liquid and sample diluting liquid, containing 3-methyl-2-benzothiazolinone hydrazone hydrochloride and N-ethyl-N-(2-hydroxyl-3-sulfopropyl in described a liquid)-3-methylaniline sodium salt, containing platinum bismuth nanometer Mimetic enzyme solution in b liquid, titer is Glucose standards solution, and enzyme liquid is glucose oxidase solution.
N-ethyl-N-(2-hydroxyl-3-sulfopropyl containing concentration to be the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.28mmol/L and concentration be 1.33mmol/L in above-mentioned a liquid)-3-methylaniline sodium salt; Be the platinum bismuth nanometer Mimetic enzyme solution of 0.34mg/mL containing the concentration of the phosphate buffered saline of useful 10mmol/L, pH=7 in b liquid; It is 6.25,31.25 that titer comprises concentration, 62.5,125,250,375, and the glucose solution of 500,625 μm of ol/L; It is the glucose oxidase solution of 90U/mL that enzyme liquid comprises by the concentration of the phosphate buffered saline of 10mmol/L, pH=7; Sample diluting liquid comprises 10mmol/L, the phosphate buffer of pH=7.
Described platinum bismuth nanometer Mimetic enzyme is obtained by following steps: be that to add 5mL concentration in the Bovine Serum Albumin in Aqueous Solution of 50mg/mL be 16mmol/L platinum acid chloride solution and 0.5mL concentration in 5mL concentration be 1.5mol/L sodium hydroxide solution, water-bath 80 DEG C reaction 2h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution freeze drying is obtained bovine serum albumin(BSA)-Platinum Nanoparticles powder, be add bismuth nitrate solution that 0.6mL concentration is 0.5mmol/L in the bovine serum albumin(BSA)-platinum aqueous solution of 23.8mg/mL and 1.8mL concentration is 10mmol/L in 0.6mL concentration, the phosphate buffer of pH=7, water-bath 80 DEG C reaction 2.5h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain the aqueous solution of platinum bismuth nanometer Mimetic enzyme, the freeze drying of platinum bismuth nanometer Mimetic enzyme aqueous solution is obtained platinum bismuth nanometer Mimetic enzyme powder.
Of the present invention
a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzymeapplication, it is characterized in that conduct
glucose detection.
Described
a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzymeapplication, it is characterized in that in 640 μ L titers, add 160 μ L enzyme liquid, 37 DEG C of temperature bath 30min, add 1.8mLa liquid and 1.4mLb liquid, and 37 DEG C of temperature bath 30min, measure the absorbance at 590nm place, draw glucose standard curve or calculating regression equation; 600 μ L sample diluting liquids are added, 160 μ L enzyme liquid, 37 DEG C of temperature bath 30min in 40 μ L human serum samples, add 1.8mLa liquid and 1.4mLb liquid, 37 DEG C of temperature bath 30min, measure the absorbance at 590nm place, according to typical curve or regression equation, calculate the content of glucose in blood serum sample.
Detection method of the present invention adopts following concrete technical scheme:
(1) in 5mL concentration be 50mg/mL Bovine Serum Albumin in Aqueous Solution in add 5mL concentration be 16mmol/L platinum acid chloride solution and 0.5mL concentration is 1.5mol/L sodium hydroxide solution, water-bath 80 DEG C reaction 2h after mixing.After reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and washes 3 times, obtains bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution freeze drying is obtained bovine serum albumin(BSA)-Platinum Nanoparticles powder.Be add bismuth nitrate solution that 0.6mL concentration is 0.5mmol/L in the bovine serum albumin(BSA)-platinum aqueous solution of 23.8mg/mL and 1.8mL concentration is 10mmol/L in 0.6mL concentration, the phosphate buffer of pH=7, water-bath 80 DEG C reaction 2.5h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain platinum bismuth nanometer Mimetic enzyme aqueous solution.The freeze drying of platinum bismuth nanometer Mimetic enzyme aqueous solution is obtained platinum bismuth nanometer Mimetic enzyme powder.The all glasswares used in preparation process all soak through chloroazotic acid, and thoroughly clean with distilled water, dry.
(2) glucose sensing approach of the present invention: be the glucose oxidase of 180U/mL by 80 μ L concentration, the Glucose standards solution of 640 μ L variable concentrations, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7, after 37 DEG C of temperature bath 30min, add the phosphate buffer that 1.3mL concentration is 10mmol/L, pH=7,1mL concentration is the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.5mmol/L
,800 μ L concentration are the N-ethyl-N-(2-hydroxyl-3-sulfopropyl of 3mmol/L)-3-methylaniline sodium salt, 100 μ L concentration are 4.76mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution, 37 DEG C of temperature bath 30min, the change of visualization color or the absorbance at mensuration 590nm place.
(3) based on the glucose sensing approach of horseradish peroxidase: be the glucose oxidase of 180U/mL by 80 μ L concentration, the Glucose standards solution of 640 μ L variable concentrations, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7, after 37 DEG C of temperature bath 30min, adding 2.9mL concentration is 10mmol/L, the phosphate buffer of pH=7, 0.2mL concentration is 3 of 16mmol/L, 3 ', 5, 5 '-tetramethyl biphenyl amine hydrochlorate, 100 μ L concentration are the horseradish peroxidase of 0.5U/mL, 37 DEG C of temperature bath 10min, the change of visualization color or the absorbance at mensuration 652nm place.
Another object of the present invention is to provide a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzyme.Kit comprises 3-methyl-2-benzothiazolinone hydrazone hydrochloride and N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt solution (a liquid), platinum bismuth nanometer Mimetic enzyme solution (b liquid), Glucose standards solution (titer), glucose oxidase solution (enzyme liquid), sample diluting liquid.
In order to realize the object of mentioned reagent box, the present invention adopts following concrete technical scheme:
(4) glucose determination reagent box: a liquid comprises the N-ethyl-N-(2-hydroxyl-3-sulfopropyl that 3-methyl-2-benzothiazolinone hydrazone hydrochloride that concentration is 0.28mmol/L and concentration are 1.33mmol/L)-3-methylaniline sodium-salt aqueous solution.The concentration of phosphate buffered saline that b liquid comprises technique scheme () preparation rear 10mmol/L, pH=7 is the platinum bismuth nanometer Mimetic enzyme solution of 0.34mg/mL.It is 6.25,31.25 that titer comprises concentration, 62.5,125,250,375, and the glucose solution of 500,625 μm of ol/L.It is the glucose oxidase solution of 90U/mL that enzyme liquid comprises by the concentration of the phosphate buffered saline of 10mmol/L, pH=7.Sample diluting liquid comprises 10mmol/L, the phosphate buffer of pH=7.
(5) glucose determination reagent box using method: the enzyme liquid adding 160 μ L technical schemes (four) in the titer of 640 μ L technical schemes (four), 37 DEG C of temperature bath 30min, add a liquid of 1.8mL technical scheme (four) and the b liquid of 1.4mL technical scheme (four), 37 DEG C of temperature bath 30min, the change of visualization color or measure the absorbance at 590nm place and draw glucose standard curve or calculating regression equation.The sample diluting liquid of 600 μ L technical schemes (four) is added in 40 μ L human serum samples, the enzyme liquid of 160 μ L technical schemes (four), 37 DEG C of temperature bath 30min, add a liquid of 1.8mL technical scheme (four) and the b liquid of 1.4mL technical scheme (four), 37 DEG C of temperature bath 30min, the change of visualization color or the absorbance at mensuration 590nm place.Judge according to color development system solution colour or carry out quantitatively according to absorbance standard curve.
Advantage of the present invention:
(1) the present invention utilizes glucose oxidase enzymatic glucose oxidase Hydrogen Peroxide, platinum bismuth nanometer Mimetic enzyme catalyzing hydrogen peroxide oxidation 3-methyl-2-benzothiazolinone hydrazone hydrochloride and N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt coupling colour development, thus show the change of solution colour and ultra-violet absorption spectrum feature, may be used for the content detection of glucose.
(2) detection specificity of the present invention to glucose is high, can be used for serum glucose level and measures.
(3) the present invention detects the highly sensitive of the detection of glucose, and the detection of visualization color change is limited to 5 μm of ol/L, and the spectrophotometry range of linearity is 1 ~ 100 μm of ol/L, detects and is limited to 0.2 μm of ol/L.
(4) glucose determination reagent box provided by the invention, has good stability, and the advantage such as easy and simple to handle, highly sensitive, high specificity, is easy to promote the use of.
Accompanying drawing explanation
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 is that fructose, lactose, maltose are for the effect diagram of glucose detection.
Embodiment
Embodiment 1:
Be that to add 5mL concentration in the Bovine Serum Albumin in Aqueous Solution of 50mg/mL be 16mmol/L platinum acid chloride solution and 0.5mL concentration in 5mL concentration be 1.5mol/L sodium hydroxide solution, water-bath 80 DEG C reaction 2h.After reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and washes 3 times, obtains bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution freeze drying is obtained bovine serum albumin(BSA)-Platinum Nanoparticles powder.Be add bismuth nitrate solution that 0.6mL concentration is 0.5mmol/L in the bovine serum albumin(BSA)-platinum aqueous solution of 23.8mg/mL and 1.8mL concentration is 10mmol/L in 0.6mL concentration, the phosphate buffer of pH=7, water-bath 80 DEG C reaction 2.5h, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain the aqueous solution of platinum bismuth nanometer Mimetic enzyme catalysis.The freeze drying of platinum bismuth nanometer Mimetic enzyme catalytic water solution is obtained platinum bismuth nanometer Mimetic enzyme catalytic powder.
Embodiment 2:
The Glucose standards solution of to be the glucose oxidase of 180U/mL and 640 μ L concentration by 80 μ L concentration be 800 μm of ol/L, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of temperature bath 30min, adding 1.3mL concentration is 10mmol/L, the phosphate buffer of pH=7,1mL concentration is the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.5mmol/L
,800 μ L concentration are the N-ethyl-N-(2-hydroxyl-3-sulfopropyl of 3mmol/L)-3-methylaniline sodium salt, concentration prepared by 100 μ L embodiments 1 is 4.76mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution, 37 DEG C of temperature bath 30min, measure ultravioletvisible absorption collection of illustrative plates, as shown in Figure 1, color product maximum absorption wavelength is 590nm.
Embodiment 3:
Be the glucose oxidase of 180U/mL and the Glucose standards solution of 640 μ L variable concentrations by 80 μ L concentration, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of temperature bath 30min, adding 1.3mL concentration is 10mmol/L, the phosphate buffer of pH=7,1mL concentration is the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.5mmol/L
,800 μ L concentration are the N-ethyl-N-(2-hydroxyl-3-sulfopropyl of 3mmol/L)-3-methylaniline sodium salt, the concentration prepared by 100 μ L embodiments 1 is 4.76mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution, and 37 DEG C of temperature bath 30min, observe solution colour.As shown in Figure 2, along with the increasing of concentration of glucose, the purple that solution manifests is more and more darker, and the detection changed by visualization color is limited to 5 μm of ol/L.
Embodiment 4:
Be the glucose oxidase of 180U/mL and the Glucose standards solution of 640 μ L variable concentrations by 80 μ L concentration, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of temperature bath 30min, adding 1.3mL concentration is 10mmol/L, the phosphate buffer of pH=7,1mL concentration is the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.5mmol/L
,800 μ L concentration are the N-ethyl-N-(2-hydroxyl-3-sulfopropyl of 3mmol/L)-3-methylaniline sodium salt, concentration prepared by 100 μ L embodiments 1 is 4.76mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution, 37 DEG C of temperature bath 30min, measure 590nm wavelength place absorbance, drawing standard curve.As shown in Figure 3, the range of linearity of spectrophotometry is 1 ~ 100 μm of ol/L, detects and is 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 comprises 3-methyl-2-benzothiazolinone hydrazone hydrochloride and N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt solution (a liquid), platinum bismuth nanometer Mimetic enzyme solution (b liquid), Glucose standards solution (titer), glucose oxidase solution (enzyme liquid), sample diluting liquid.A liquid comprises the N-ethyl-N-(2-hydroxyl-3-sulfopropyl that 3-methyl-2-benzothiazolinone hydrazone hydrochloride that concentration is 0.28mmol/L and concentration are 1.33mmol/L)-3-methylaniline sodium-salt aqueous solution.B liquid comprises the platinum bismuth nanometer Mimetic enzyme solution that concentration that embodiment 1 prepares the phosphate buffered saline of rear 10mmol/L, pH=7 is 0.34mg/mL.It is 6.25,31.25 that titer comprises concentration, 62.5,125,250,375, and the glucose solution of 500,625 μm of ol/L.It is the glucose oxidase solution of 90U/mL that enzyme liquid comprises by the concentration of the phosphate buffered saline of 10mmol/L, pH=7.Sample diluting liquid comprises 10mmol/L, the phosphate buffer of pH=7.
Embodiment 6:
Be the glucose oxidase of 180U/mL and the Glucose standards solution of 640 μ L variable concentrations by 80 μ L concentration, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of temperature bath 30min, adding 2.9mL concentration is 10mmol/L, the phosphate buffer of pH=7,0.2mL concentration is 3 of 16mmol/L, 3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate and 100 μ L concentration are the horseradish peroxidase of 0.5U/mL, 37 DEG C of temperature bath 30min, measure the absorbance at 652nm place, drawing standard curve.Be the glucose oxidase of 180U/mL and 40 μ L human serum samples and 680 μ L concentration by 80 μ L concentration be 10mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of temperature bath 30min, adding 2.9mL concentration is 10mmol/L, the phosphate buffer of pH=7,0.2mL concentration is 3 of 16mmol/L, 3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate and 100 μ L concentration are the horseradish peroxidase of 0.5U/mL, 37 DEG C of temperature bath 10min, measure the absorbance at 652nm place, calculate the content of glucose in blood serum sample according to typical curve.
Embodiment 7:
The enzyme liquid of 160 μ L embodiments 5 is added in the titer of 640 μ L embodiments 5,37 DEG C of temperature bath 30min, add a liquid of 1.8mL embodiment 5 and the b liquid of 1.4mL embodiment 5,37 DEG C of temperature bath 30min, measure the absorbance at 590nm place, draw glucose standard curve or calculate regression equation.The sample diluting liquid of 600 μ L embodiments 5 is added in 40 μ L human serum samples, the enzyme liquid of 160 μ L embodiments 5,37 DEG C of temperature bath 30min, add a liquid of 1.8mL embodiment 5 and the b liquid of 1.4mL embodiment 5,37 DEG C of temperature bath 30min, measure the absorbance at 590nm place, according to typical curve or regression equation, calculate the content of glucose in blood serum sample.Result is as shown in the table, and with the Measures compare in example 6, the data consistent that two kinds of methods record within the scope of hypoglycemia value 3.18mmol/L to hyperglycaemia value 10.24mmol/L, relative error is-6.6% ~ 4.9%;
Embodiment 8:
The glucose solution of the sample diluting liquid preparation of 600 μ L embodiments 5 is added in 40 μ L human serum sample solution, the enzyme liquid of 160 μ L embodiments 5,37 DEG C of temperature bath 30min, add a liquid of 1.8mL embodiment 5 and the b liquid of 1.4mL embodiment 5,37 DEG C of temperature bath 30min, measure the absorbance at 590nm wavelength place, the recovery of standard addition in conjunction with example 7 calculation sample is 94.5% ~ 104.5%, and relative standard deviation is 2.9% ~ 7.2%.
Embodiment 10:
Getting 640 μ L concentration is respectively the glucose standard of 0.5mmol/L, 640 μ L concentration are the fructose titer of 0.5mmol/L, 640 μ L concentration are the lactose titer of 0.5mmol/L, 640 μ L concentration are the maltose titer of 0.2mmol/L, add the enzyme liquid of 160 μ L embodiments 5,37 DEG C of temperature bath 30min, add a liquid of 1.8mL embodiment 5 and the b liquid of 1.4mL embodiment 5,37 DEG C of temperature bath 30min, measure the absorbance at 590nm wavelength place.As shown in Figure 4, fructose, lactose, maltose can not produce interference to glucose sensing approach of the present invention.
The foregoing is only exemplary embodiments of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1.
based on a glucose sensing approach for platinum bismuth nanometer Mimetic enzyme,it is characterized in that glucose oxidase, glucose solution, phosphate buffer mixes, and adds phosphate buffer, 3-methyl-2-benzothiazolinone hydrazone hydrochloride after temperature bath
,n-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt, platinum bismuth nanometer Mimetic enzyme aqueous solution, visualization or mensuration ultravioletvisible absorption after temperature bath, described platinum bismuth nanometer Mimetic enzyme is obtained by following steps: be that to add 5mL concentration in the Bovine Serum Albumin in Aqueous Solution of 50mg/mL be 16mmol/L platinum acid chloride solution and 0.5mL concentration in 5mL concentration be 1.5mol/L sodium hydroxide solution, water-bath 80 DEG C reaction 2h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution freeze drying is obtained bovine serum albumin(BSA)-Platinum Nanoparticles powder, be add bismuth nitrate solution that 0.6mL concentration is 0.5mmol/L in the bovine serum albumin(BSA)-platinum aqueous solution of 23.8mg/mL and 1.8mL concentration is 10mmol/L in 0.6mL concentration, the phosphate buffer of pH=7, water-bath 80 DEG C reaction 2.5h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain the aqueous solution of platinum bismuth nanometer Mimetic enzyme, the freeze drying of platinum bismuth nanometer Mimetic enzyme aqueous solution is obtained platinum bismuth nanometer Mimetic enzyme powder.
2. according to claim 1
based on the glucose sensing approach of platinum bismuth nanometer Mimetic enzymeit is characterized in that being the glucose oxidase of 180U/mL and the Glucose standards solution of 640 μ L variable concentrations by 80 μ L concentration, 80 μ L concentration are 10mmol/L, the phosphate buffer mixing of pH=7,37 DEG C of temperature bath 30min, add the phosphate buffer that 1.3mL concentration is 10mmol/L, pH=7,1mL concentration is the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.5mmol/L
,800 μ L concentration are the N-ethyl-N-(2-hydroxyl-3-sulfopropyl of 3mmol/L)-3-methylaniline sodium salt, 100 μ L concentration are 4.76mg/mL platinum bismuth nanometer Mimetic enzyme aqueous solution, 37 DEG C of temperature bath 30min, visualization solution colour or mensuration ultravioletvisible absorption.
3. according to claim 1 and 2
based on the glucose sensing approach of platinum bismuth nanometer Mimetic enzyme, it is characterized in that color product solution colour is purple, along with the increase of concentration of glucose, solution colour is deepened, and the detection of visualization solution colour is limited to 5 μm of ol/L.
4. according to claim 1 and 2
based on the glucose sensing approach of platinum bismuth nanometer Mimetic enzyme, it is characterized in that color product maximum absorption wavelength is 590nm, make typical curve according to the absorbance that 590nm wavelength place records, its range of linearity detected is 1 ~ 100 μm of ol/L, detects and is limited to 0.2 μm of ol/L.
5.
based on a glucose determination reagent box for platinum bismuth nanometer Mimetic enzyme,it is characterized in that kit comprises a liquid, b liquid, titer, enzyme liquid and sample diluting liquid, containing 3-methyl-2-benzothiazolinone hydrazone hydrochloride and N-ethyl-N-(2-hydroxyl-3-sulfopropyl in described a liquid)-3-methylaniline sodium salt, containing platinum bismuth nanometer Mimetic enzyme solution in b liquid, titer is Glucose standards solution, and enzyme liquid is glucose oxidase solution.
6. according to claim 5
based on a glucose determination reagent box for platinum bismuth nanometer Mimetic enzyme,it is characterized in that the N-ethyl-N-(2-hydroxyl-3-sulfopropyl containing concentration to be the 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 0.28mmol/L and concentration be 1.33mmol/L in a liquid)-3-methylaniline sodium salt; Be the platinum bismuth nanometer Mimetic enzyme solution of 0.34mg/mL containing the concentration of the phosphate buffered saline of useful 10mmol/L, pH=7 in b liquid; It is 6.25,31.25 that titer comprises concentration, 62.5,125,250,375, and the glucose solution of 500,625 μm of ol/L; It is the glucose oxidase solution of 90U/mL that enzyme liquid comprises by the concentration of the phosphate buffered saline of 10mmol/L, pH=7; Sample diluting liquid comprises 10mmol/L, the phosphate buffer of pH=7.
7. according to claim 5 or 6
based on a glucose determination reagent box for platinum bismuth nanometer Mimetic enzyme,it is characterized in that what described platinum bismuth nanometer Mimetic enzyme was obtained by following steps: be that to add 5mL concentration in the Bovine Serum Albumin in Aqueous Solution of 50mg/mL be 16mmol/L platinum acid chloride solution and 0.5mL concentration in 5mL concentration be 1.5mol/L sodium hydroxide solution, water-bath 80 DEG C reaction 2h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution, bovine serum albumin(BSA)-Platinum Nanoparticles aqueous solution freeze drying is obtained bovine serum albumin(BSA)-Platinum Nanoparticles powder, be add bismuth nitrate solution that 0.6mL concentration is 0.5mmol/L in the bovine serum albumin(BSA)-platinum aqueous solution of 23.8mg/mL and 1.8mL concentration is 10mmol/L in 0.6mL concentration, the phosphate buffer of pH=7, water-bath 80 DEG C reaction 2.5h after mixing, after reaction, solution loads cutoff is the super filter tube of 3k, 6000r/min centrifugal ultrafiltration, and wash 3 times, obtain the aqueous solution of platinum bismuth nanometer Mimetic enzyme, the freeze drying of platinum bismuth nanometer Mimetic enzyme aqueous solution is obtained platinum bismuth nanometer Mimetic enzyme powder.
8. claim 5-7 is arbitrary described
a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzymeapplication, it is characterized in that conduct
glucose detection.
9. according to claim 8
a kind of glucose determination reagent box based on platinum bismuth nanometer Mimetic enzymeapplication, it is characterized in that in 640 μ L titers, add 160 μ L enzyme liquid, 37 DEG C of temperature bath 30min, add 1.8mLa liquid and 1.4mLb liquid, and 37 DEG C of temperature bath 30min, measure the absorbance at 590nm place, draw glucose standard curve or calculating regression equation; 600 μ L sample diluting liquids are added, 160 μ L enzyme liquid, 37 DEG C of temperature bath 30min in 40 μ L human serum samples, add 1.8mLa liquid and 1.4mLb liquid, 37 DEG C of temperature bath 30min, measure the absorbance at 590nm place, according to typical curve or regression equation, calculate the content of glucose in blood serum sample.
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| CN111239125A (en) * | 2020-03-06 | 2020-06-05 | 中国药科大学 | Glucose colorimetric detection method by using platinum disulfide |
| CN113155927A (en) * | 2021-04-01 | 2021-07-23 | 华东师范大学 | Glucose potential sensor based on SDBA-Au composite nanoenzyme |
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