CN108375573B - Method for detecting glucose by using polyethyleneimine-stabilized nano platinum particles - Google Patents
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title claims abstract description 106
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 80
- 239000008103 glucose Substances 0.000 title claims abstract description 80
- 239000002245 particle Substances 0.000 title claims abstract description 36
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 69
- 238000001514 detection method Methods 0.000 claims abstract description 22
- 229920002873 Polyethylenimine Polymers 0.000 claims abstract description 21
- 238000002835 absorbance Methods 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 11
- 239000012279 sodium borohydride Substances 0.000 claims abstract description 11
- 229910000033 sodium borohydride Inorganic materials 0.000 claims abstract description 11
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 10
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 10
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 229910052700 potassium Inorganic materials 0.000 claims abstract description 9
- 239000011591 potassium Substances 0.000 claims abstract description 9
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims abstract 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 24
- 229910052751 metal Inorganic materials 0.000 claims description 23
- 239000002184 metal Substances 0.000 claims description 23
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 6
- 239000007974 sodium acetate buffer Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000004697 Polyetherimide Substances 0.000 claims 8
- 229920001601 polyetherimide Polymers 0.000 claims 8
- 238000006243 chemical reaction Methods 0.000 abstract description 9
- 230000003647 oxidation Effects 0.000 abstract description 6
- 238000007254 oxidation reaction Methods 0.000 abstract description 6
- 230000009467 reduction Effects 0.000 abstract description 5
- 235000000346 sugar Nutrition 0.000 description 13
- 150000008163 sugars Chemical class 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 4
- 229930091371 Fructose Natural products 0.000 description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 4
- 239000005715 Fructose Substances 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
- 239000003929 acidic solution Substances 0.000 description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 4
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 238000003917 TEM image Methods 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007805 chemical reaction reactant Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Abstract
A method for detecting glucose by using polyethyleneimine-stabilized nano platinum particles mainly comprises the steps of co-incubating polyethyleneimine solution and potassium tetrachloroplatinate, adding sodium borohydride for reduction to prepare polyethyleneimine-stabilized nano platinum particles, taking a centrifugal tube, and mixing glucose oxidase with glucose solutions with different concentration gradients to generate hydrogen peroxide; adding a water solution of polyethyleneimine-stabilized nano platinum particles, tetramethyl benzidine and a buffer solution into another centrifugal tube for reaction; after the two centrifugal tubes are blended, colorless tetramethyl benzidine is oxidized into blue oxidation state tetramethyl benzidine; and establishing a linear relation between the absorbance of the oxidation state tetramethyl benzidine at 652nm and the glucose concentration when the glucose concentration is 0.1-1.0 mmol/L. The method has the advantages of simple process, mild conditions, high efficiency, low detection cost, easy control of reaction and the like.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for detecting glucose.
Background
Glucose (blood sugar) in blood is the most basic energy source substance in the living body. However, as the living standard of people is improved, hyperglycemia and even diabetes become killers of human health, so that the method for detecting the blood glucose value sensitively and at low cost has important significance for the diagnosis and treatment of diabetes.
The chromatography is a standard method for detecting the content of glucose, but has the defects of high cost, complex operation and the like. Electrochemical sensors suffer from the disadvantage of relatively short service life. The colorimetric method has the advantages of simple operation and high selectivity on glucose. Especially in recent years, the biomimetic enzyme can replace peroxidase to detect glucose, so that the colorimetric method is simpler and has low cost. The nano platinum particles can be used as bionic enzyme to simulate the characteristics of peroxidase. However, it has a high specific surface area, which results in its easy occurrence of agglomeration. The polymer template can improve the stability of the nano platinum particles, particularly the platinum particles with the particle size less than 10 nm. LorenzH et al successfully prepared platinum particles with a mean particle size of 10nm using polyethyleneimine having a molecular weight of 10000 as a template. However, the particle size of this platinum particle is still large, which results in that the catalytic efficiency per unit mass of platinum particle is still low. Therefore, it is highly desirable to prepare nano platinum particles having a smaller particle size and stability.
Disclosure of Invention
The invention aims to provide a method for detecting glucose by using polyethyleneimine-stabilized nano platinum particles, which has the advantages of simple preparation process, mild reaction conditions, good reproducibility, simple and convenient operation and low detection cost.
The method of the invention comprises the following steps:
(1) preparation of Polyethyleneimine (PEI) stabilized nano platinum particles: adding 0.5-1.0 mg of PEI (polyethyleneimine) into 1mL of deionized water, wherein the molecular weight of the polyethyleneimine is 70000, adding the PEI into the deionized water, adding 1mol/L of hydrochloric acid, adjusting the pH to 4, and mixing the PEI and potassium tetrachloroplatinate according to the molar ratio of 1: adding potassium tetrachloroplatinate into a PEI aqueous solution according to a proportion of 25-150, and then incubating for 15-25 min in a metal bath at a temperature of 20 ℃ and a rotating speed of 500 rpm; and then, according to the mol ratio of PEI to sodium borohydride of 1: 125-750, dissolving sodium borohydride in 0.3mol/L sodium hydroxide aqueous solution to prepare 1mg/mL solution, adding the sodium borohydride solution into the incubated solution, and incubating for 10-15 min under the same metal bath condition; then, according to the molar ratio of 1: 1, adding 1mol/L hydrochloric acid into the solution, adjusting the pH of the solution to be neutral, changing the color of the solution, and finally incubating the solution in a metal bath for 2-3 h to prepare the polyethyleneimine-stabilized nano platinum particles (PEI-Pt)nAnd n represents the number of stable platinum atoms per PEI molecule on average).
(2) And (3) glucose detection: according to the molar ratio of 1: 12-120, mixing glucose oxidase (GOx) with glucose solution with concentration of 0.1-1.0 mmol/L, reacting at 50 deg.C for 30min to generate hydrogen peroxide (H)2O2) (ii) a Taking another centrifuge tube, and preparing PEI-Pt with the concentration of 4-10 mu mol/LnThe solution was mixed with 6mmol/L of 3,3 ', 5, 5' -Tetramethylbenzidine (TMB)Liquid according to PEI-PtnThe molar ratio of TMB is 1: 450-180 of PEI-PtnTMB was added to a buffer solution of acetic acid-sodium acetate (400. mu.L, 0.2mol/L, pH 4.0) and reacted at 30 ℃ for 10 min. Mixing the above PEI-PtnTMB and acetic acid-sodium acetate mixed buffer solution are used as color developing agents and are respectively added into each glucose solution to react for 20min at the temperature of 30 ℃, the color developing product solution is blue, the color becomes dark along with the increase of the glucose concentration, an ultraviolet visible spectrophotometer is used for measuring the absorbance at 652nm, the absorbance is used as a vertical coordinate, the glucose concentration is used as a horizontal coordinate, a standard curve is drawn, a standard curve equation is obtained, and the linear relation is formed between the increase degree of the absorbance and the glucose concentration at 0.1-1.0 mmol/L.
Oxidation of glucose to H Using GOx2O2Adding PEI-PtnTMB and acetic acid-sodium acetate buffer solution, PEI-Pt catalyzed H2O2TMB is oxidized to generate oxidation state TMB (oxTMB), and the solution changes from colorless to blue. The maximum absorption peak of the oxTMB is at 652nm, and the more oxTMB is generated, the darker the blue color is, and the greater the absorbance at 652nm is. With PEI-PtnThe method for detecting the glucose can establish a standard curve for detecting the concentration of the glucose, and can realize sensitive detection of the glucose according to the selectivity of GOx on the catalysis of the glucose and the stabilization effect of PEI on the nano platinum particles.
Compared with the prior art, the invention has the following advantages:
1. oxidation of glucose to H by glucose oxidase2O2And then PEI-PtnCatalysis H2O2The color of the oxidation color developing agent is changed, and the glucose concentration is preliminarily judged according to the color change; the specific amount of glucose can be further judged according to the ultraviolet-visible spectrophotometer, the detection cost is low, and the operation is simple and convenient.
2. The preparation method adopts polyethyleneimine as a template, and the molecular formula is as follows: (CH)2CH2NH)nThe molecular structure is simple, and the active group is clear; structurally, the metal-coated nano particle can be prepared by complexing a plurality of ions through the amino group which has a polar group (amino group) and a hydrophobic group (vinyl group).
3. The preparation process is simple, the operation is convenient, the reaction condition is mild, the cost is low, and the reproducibility is good.
4. The adopted reducing agent, reaction medium and reactant have low cost and no pollution.
5. The detection cost is low without the help of expensive detection instruments, and the visual detection of the glucose detection can be realized through the preliminary observation of naked eyes.
Drawings
FIG. 1 shows PEI-Pt obtained in example 1 of the present invention25Transmission Electron Microscope (TEM) images of (a).
FIG. 2 shows PEI-Pt obtained in example 1 of the present invention25Histogram of particle size distribution of (1).
FIG. 3 is a graph of a glucose standard obtained in example 1 of the present invention.
FIG. 4 is a graph comparing the selectivity of different sugars obtained in example 1 of the present invention.
FIG. 5 shows PEI-Pt obtained in example 2 of the present invention50A TEM image of (a).
FIG. 6 shows PEI-Pt obtained in example 2 of the present invention50Histogram of particle size distribution of (1).
FIG. 7 is a graph of a glucose standard obtained in example 2 of the present invention.
FIG. 8 is a comparison of the different sugar selectivities obtained in example 2 of the present invention.
FIG. 9 shows PEI-Pt obtained in example 3 of the present invention100A TEM image of (a).
FIG. 10 shows PEI-Pt obtained in example 3 of the present invention100Histogram of particle size distribution of (1).
FIG. 11 is a graph of a glucose standard obtained in example 3 of the present invention.
FIG. 12 is a comparison of the different sugar selectivities obtained in example 3 of the present invention.
FIG. 13 shows PEI-Pt obtained in example 4 of the present invention150A TEM image of (a).
FIG. 14 shows PEI-Pt obtained in example 4 of the present invention150Histogram of particle size distribution of (1).
FIG. 15 is a graph of a glucose standard obtained in example 4 of the present invention.
FIG. 16 is a comparison of the different sugar selectivities obtained in example 4 of the present invention.
Detailed Description
Example 1
Polyethyleneimine-stabilized nano platinum particles (PEI-Pt)25) The preparation of (1):
dissolving 1mg of PEI with the molecular weight of 70000 in 1mL of deionized water, and adding 20 mu L of 1mol/L hydrochloric acid solution to adjust the solution to be an acidic solution with the pH value of 4; then 178.5 mul of potassium tetrachloroplatinate solution with the concentration of 2mmol/L is added, mixed and put into a metal bath, the rotating speed is controlled to be 500 r/min, and the incubation is carried out for 15min at the reaction temperature of 20 ℃; then adding 67 mu L of 1mg/mL sodium borohydride dissolved in 0.3M sodium hydroxide into the incubated solution for reduction, and incubating for 10min under the same metal bath condition; finally adding 22 mu L hydrochloric acid solution to make the pH of the solution neutral, and incubating for 2h in a metal bath under the same conditions to make the solution slowly turn brown, i.e. preparing PEI-Pt25。
TEM vs. PEI-Pt as shown in FIG. 125The morphology of (1) is shown in FIG. 2 is PEI-Pt25The diameter of the nano platinum particles is 3.72 +/-0.89 nm.
Establishing PEI-Pt25Standard curve for glucose detection:
mixing 50 μ L GOx 5mg/mL water solution with 200 μ L glucose (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1mmol/L) at different concentrations, reacting at 50 deg.C in metal bath for 30min to generate H2O2(ii) a Adding 200 mu L of 4 mu mol/L PEI-Pt into another centrifugal tube25，600μL,6
The reaction was carried out in a buffer solution of 400. mu.L, 0.2mol/L, pH 4, acetic acid-sodium acetate in mmol/L TMB at 30 ℃ for 10 min. Mixing the above PEI-Pt25TMB and acetic acid-sodium acetate mixed buffer solution are used as color developing agents and are respectively added into each glucose solution to react for 20min at the temperature of 30 ℃, and an ultraviolet visible spectrophotometer is used for measuring absorbance at 652nm to obtain a glucose concentration standard curve. As shown in FIG. 3, the degree of increase in absorbance and the glucose concentration are in a linear relationship of 0.1 to 1.0mmol/L,the standard working curve equation is
0.22183+0.08826x, standard deviation R2＝0.98692。
To verify the selectivity of the method of the present invention for glucose detection, glucose was replaced with other sugars (maltose, lactose, fructose) according to the same method, at concentrations of 10mmol/L, and the test results are shown in FIG. 4. Besides strong absorption on glucose, the absorbance on other sugars is very small, which indicates that the method constructed by the invention has good selectivity on glucose and can be used for selective detection of glucose in a complex system.
Example 2
Polyethyleneimine-stabilized nano platinum particles (PEI-Pt)50) The preparation of (1):
dissolving 1mg of PEI with the molecular weight of 70000 in 1mL of deionized water, and adding 20 mu L of 1mol/L hydrochloric acid solution to adjust the solution to be an acidic solution with the pH value of 4; then adding 178.5 mu L of 2mmol/L potassium tetrachloroplatinate solution, mixing and putting into a metal bath, controlling the rotating speed at 500 r/min, and incubating for 20min at the reaction temperature of 20 ℃; then adding 135 mul of 1mg/mL sodium borohydride dissolved in 0.3M sodium hydroxide into the incubated solution for reduction, and incubating for 10min under the same metal bath condition; finally adding 45 mu L of hydrochloric acid solution to make the pH of the solution neutral, and incubating the solution in a metal bath under the same conditions for 2.5h to make the solution slowly turn brown, namely preparing the PEI-Pt50。
TEM vs. PEI-Pt as shown in FIG. 550FIG. 6 shows PEI-Pt50The diameter of the nano platinum particles is 4.09 +/-0.72 nm.
Establishing PEI-Pt50Standard curve for glucose detection:
mixing 50 μ L GOx 5mg/mL water solution with 200 μ L glucose (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1mmol/L) at different concentrations, reacting at 50 deg.C in metal bath for 30min to generate H2O2(ii) a Adding 200 mu L of 6 mu mol/L PEI-Pt into another centrifugal tube50600 μ L of TMB (6 mmol/L) and 400 μ L of acetic acid-sodium acetate buffer solution (0.2 mol/L) at pH 4, and reacted at 30 ℃ for 10 min. Mixing the above PEI-Pt50TMB and acetic acid-sodium acetate mixed buffer solution are used as color developing agents and are respectively added into each glucose solution to react for 20min at the temperature of 30 ℃, and an ultraviolet visible spectrophotometer is used for measuring absorbance at 652nm to obtain a glucose concentration standard curve. As shown in FIG. 7, the degree of the increase of absorbance and the glucose concentration are in a linear relationship, the standard working curve equation is that y is 0.20244+0.12766x, and the standard deviation R is2＝0.99588。
To verify the selectivity of the method of the present invention for glucose detection, glucose was replaced with other sugars (maltose, lactose, fructose) according to the same method, at concentrations of 10mmol/L, and the test results are shown in FIG. 8. Besides strong absorption on glucose, the absorbance on other sugars is very small, which indicates that the method constructed by the invention has good selectivity on glucose and can be used for selective detection of glucose in a complex system.
Example 3
Polyethyleneimine-stabilized nano platinum particles (PEI-Pt)100) The preparation of (1):
dissolving 0.5mg of PEI with the molecular weight of 70000 in 1mL of deionized water, and adding 10 mu L of 1mol/L hydrochloric acid solution to adjust the solution to be an acidic solution with the pH value of 4; adding 357 mu L of 2mmol/L potassium tetrachloroplatinate solution, mixing, placing into a metal bath, controlling the rotating speed at 500 rpm, and incubating for 20min at the reaction temperature of 20 ℃; then adding 135 mul of 1mg/mL sodium borohydride dissolved in 0.3M sodium hydroxide into the incubated solution for reduction, and incubating for 15min under the same metal bath condition; finally adding 45 mu L of hydrochloric acid solution to make the pH of the solution neutral, and incubating the solution in a metal bath under the same conditions for 2.5h to make the solution slowly turn brown, namely preparing the PEI-Pt100。
TEM vs. PEI-Pt as shown in FIG. 9100FIG. 10 shows PEI-Pt100The diameter of the nano platinum particles is 4.30 +/-0.79 nm.
Establishing PEI-Pt100Standard curve for glucose detection:
taking 50 μ L GOx 5mg/mL water solution, and mixing with 200 μ L glucose (0.1, 0.2, 0.3, 0.4) with different concentrations0.5, 0.6, 0.7, 0.8, 1mmol/L) and reacting at 50 ℃ in a metal bath for 30min to produce H2O2(ii) a Adding 200 mu L of 8 mu mol/L PEI-Pt into another centrifugal tube100600 μ L of TMB (6 mmol/L) and 400 μ L of acetic acid-sodium acetate buffer solution (0.2 mol/L) at pH 4, and reacted at 30 ℃ for 10 min. Mixing the above PEI-Pt100TMB and acetic acid-sodium acetate mixed buffer solution are used as color developing agents and are respectively added into each glucose solution to react for 20min at the temperature of 30 ℃, and an ultraviolet visible spectrophotometer is used for measuring absorbance at 652nm to obtain a glucose concentration standard curve. As shown in FIG. 11, the degree of absorbance increase and the glucose concentration are in a linear relationship, the standard working curve equation is that y is 0.30044+0.13932x, and the standard deviation R is2＝0.99813。
To verify the selectivity of the method of the present invention for glucose detection, glucose was replaced with other sugars (maltose, lactose, fructose) according to the same method, at concentrations of 10mmol/L, and the test results are shown in FIG. 12. Besides strong absorption on glucose, the absorbance on other sugars is very small, which indicates that the method constructed by the invention has good selectivity on glucose and can be used for selective detection of glucose in a complex system.
Example 4
Polyethyleneimine-stabilized nano platinum particles (PEI-Pt)150) The preparation of (1):
dissolving 0.5mg of PEI with the molecular weight of 70000 in 0.5mL of deionized water, and adding 10 mu L of 1mol/L hydrochloric acid solution to adjust the solution to be an acidic solution with the pH value of 4; adding 535.5 mu L of 2mmol/L potassium tetrachloroplatinate solution, mixing, placing into a metal bath, controlling the rotation speed at 500 rpm, and incubating for 25min at the reaction temperature of 20 ℃; then adding 202 mu L of 1mg/mL sodium borohydride dissolved in 0.3M sodium hydroxide into the incubated solution for reduction, and incubating for 15min under the same metal bath condition; finally adding 67 mu L of hydrochloric acid solution to make the pH of the solution neutral, and incubating for 3h in a metal bath under the same conditions to make the solution slowly turn brown, namely preparing the PEI-Pt150。
TEM vs PEI-Pt as shown in FIG. 13150FIG. 14 shows PEI-Pt150The diameter of the nano platinum particles is 5.22 +/-1.18 nm.
Establishing PEI-Pt150Standard curve for glucose detection:
mixing 50 μ L GOx 5mg/mL water solution with 200 μ L glucose (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1mmol/L) at different concentrations, reacting at 50 deg.C in metal bath for 30min to generate H2O2(ii) a Adding 200 mu L of 10 mu mol/L PEI-Pt into another centrifugal tube150600 μ L of TMB (6 mmol/L) and 400 μ L of acetic acid-sodium acetate buffer solution (0.2 mol/L) at pH 4, and reacted at 30 ℃ for 10 min. Mixing the above PEI-Pt150TMB and acetic acid-sodium acetate mixed buffer solution are used as color developing agents and are respectively added into each glucose solution to react for 20min at the temperature of 30 ℃, and an ultraviolet visible spectrophotometer is used for measuring absorbance at 652nm to obtain a glucose concentration standard curve. As shown in FIG. 15, the degree of absorbance increase and the glucose concentration are in a linear relationship, the standard working curve equation is that y is 0.31239+0.14303x, and the standard deviation R is2＝0.98342。
To verify the selectivity of the method of the present invention for glucose detection, glucose was replaced with other sugars (maltose, lactose, fructose) according to the same method, at concentrations of 10mmol/L, and the test results are shown in FIG. 16. Besides strong absorption on glucose, the absorbance on other sugars is very small, which indicates that the method constructed by the invention has good selectivity on glucose and can be used for selective detection of glucose in a complex system.
Claims (2)
1. A method for detecting glucose by using polyethyleneimine-stabilized nano platinum particles is characterized by comprising the following steps:
(1) preparation of polyethyleneimine PEI stabilized nano platinum particles: adding 0.5-1.0 mg of PEI (polyetherimide) into 1mL of deionized water, adding the PEI into the deionized water, adding 1mol/L of hydrochloric acid to adjust the pH to 4, and mixing the PEI and the potassium tetrachloroplatinate according to a molar ratio of 1: adding potassium tetrachloroplatinate into a PEI aqueous solution according to a proportion of 25-150, and then incubating for 15-25 min in a metal bath at a temperature of 20 ℃ and a rotating speed of 500 rpm; and then, according to the mol ratio of PEI to sodium borohydride of 1:125-750, dissolving sodium borohydride in 0.3mol/L sodium hydroxide aqueous solution to prepare 1mg/mL solution, adding the sodium borohydride solution into the incubated solution, and incubating for 10-15 min under the same metal bath condition; then, according to the molar ratio of 1: 1, adding 1mol/L hydrochloric acid into the solution which is incubated again, adjusting the pH of the solution to be neutral, changing the color of the solution, and finally incubating in a metal bath for 2-3 h to prepare polyethyleneimine-stabilized nano platinum particles, namely PEI-PtnN represents the number of stable platinum atoms per PEI molecule on average;
(2) and (3) glucose detection: according to the molar ratio of 1: 12-120, mixing glucose oxidase (GOx) with glucose solution with concentration of 0.1-1.0 mmol/L, reacting at 50 deg.C for 30min to generate hydrogen peroxide (H)2O2) (ii) a Taking another centrifuge tube, and preparing PEI-Pt with the concentration of 4-10 mu mol/LnSolution and 6 mmol/L3, 3’,5,5’Tetramethylbenzidine (TMB) solution, based on PEI-PtnThe molar ratio of TMB is 1: 450-180 of PEI-PtnAdding TMB into an acetic acid-sodium acetate buffer solution with the pH value of 4.0 of 400 mu L and 0.2mol/L, and reacting for 10min at the temperature of 30 ℃; mixing the above PEI-PtnTMB and acetic acid-sodium acetate mixed buffer solution are used as color developing agents and are respectively added into each glucose solution to react for 20min at the temperature of 30 ℃, the color developing product solution is blue, the color becomes dark along with the increase of the glucose concentration, an ultraviolet visible spectrophotometer is used for measuring the absorbance at 652nm, the absorbance is used as a vertical coordinate, the glucose concentration is used as a horizontal coordinate, a standard curve is drawn, a standard curve equation is obtained, and the linear relation is formed between the increase degree of the absorbance and the glucose concentration at 0.1-1.0 mmol/L.
2. The method for detecting glucose by using the nano platinum particles stabilized by polyethyleneimine as claimed in claim 1, wherein the method comprises the following steps: the molecular weight of the polyethyleneimine is 70000.
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