阅读说明：本技术 一种自组装一体化纳米复合材料、制备方法及其应用 (Self-assembly integrated nano composite material, preparation method and application thereof ) 是由 李贺 谭啸峰 肖运帷 李琰 曾钰婷 宫金迪 庞小龙 周靖凯 于 2020-07-20 设计创作，主要内容包括：本发明涉及一种自组装一体化纳米复合材料,包括组分3,3’,5,5’-四甲基联苯胺(TMB)、葡萄糖氧化酶(GOD)和辣根过氧化物酶(HRP)。其中,TMB的负载量为700～800mg/g,GOD和HRP的质量比为1：0.5～5。该纳米复合材料通过疏水性的TMB分子从有机相转移到水相时,其能够在疏水作用下自发的组装成形貌均一纳米粒子,并且与水相中的葡萄糖氧化酶和辣根过氧化物酶通过氢键和范德华力与其形成一体化的纳米粒子,有效的实现了酶和显色底物的一体化整合。该纳米粒子中TMB的负载量高达700～800mg/g,能大幅提高检测体系对葡萄糖的响应信号,从而增加检测的准确性和稳定性以及灵敏度。线性范围宽为50μM-10mM(R<Sub>2</Sub>＝0.99),检测限低至15μM,具有实现简单、快速可视化、高灵敏和特异性检测等优点。(The invention relates to a self-assembly integrated nano composite material which comprises components of 3,3 ', 5, 5' -tetramethyl benzidine (TMB), Glucose Oxidase (GOD) and horseradish peroxidase (HRP). Wherein the loading amount of TMB is 700-800 mg/g, and the mass ratio of GOD to HRP is 1:0.5 to 5. When the nano composite material is transferred from an organic phase to an aqueous phase through hydrophobic TMB molecules, the nano composite material can be spontaneously assembled into nano particles with uniform appearance under the hydrophobic action, and forms integrated nano particles with glucose oxidase and horseradish peroxidase in the aqueous phase through hydrogen bonds and van der Waals force,effectively realizes the integrated integration of the enzyme and the chromogenic substrate. The loading amount of TMB in the nanoparticles is up to 700-800 mg/g, and the response signal of a detection system to glucose can be greatly improved, so that the accuracy, stability and sensitivity of detection are improved. The linear range is wide and is 50 mu M-10mM (R) 2 0.99), the detection limit is as low as 15 mu M, and the method has the advantages of simple realization, quick visualization, high sensitivity, specific detection and the like.)

1. An integrated composite nanoparticle, comprising the following components: 3,3 ', 5, 5' -tetramethylbenzidine, glucose oxidase and horseradish peroxidase.

2. The integrated composite nanoparticle according to claim 1, wherein the loading amount of the 3,3 ', 5, 5' -tetramethylbenzidine is 700-800 mg/g, and the mass ratio of the glucose oxidase to the horseradish peroxidase is 1:0.5 to 5.

3. A method for preparing the integrated composite nanoparticle according to claim 1, comprising the steps of:

a) preparing a TMB solution: 3,3 ', 5, 5' -tetramethyl benzidine is dissolved by an organic solvent, and the concentration is 1 mg/mL-10 mg/mL;

b) preparing a GOD/HRP mixed solution: dissolving glucose oxidase and horse radish peroxidase in phosphate buffer solution;

c) mixing and reacting: injecting the TMB solution into the GOD/HRP mixed solution through a peristaltic pump at the temperature of 4 ℃, and continuing to react for 2-12 h after the injection is finished;

d) and (3) centrifugal freeze-drying: and centrifuging and washing the mixed solution after the reaction, taking the precipitate, and freeze-drying to obtain the integrated composite nano particles, namely the TMB-GOD-HRP nano particles.

4. The method for preparing the integrated composite nanoparticle according to claim 3, wherein the organic solvent is one or more of dimethylsulfoxide, methanol, ethanol, isopropanol, N-methylpyrrolidone or N, N-dimethylformamide.

5. The method for preparing the integrated composite nanoparticle according to claim 3, wherein the glucose oxidase is derived from Aspergillus niger or Penicillium sp.

6. The preparation method of the integrated composite nanoparticle as claimed in claim 3, wherein the speed of the peristaltic pump is 0.05-0.2 mL/min.

7. The method for preparing the integrated composite nanoparticle as claimed in claim 3, wherein the centrifugation speed is 1000-5000 r/min.

8. The preparation method of the integrated composite nanoparticles according to claim 3, wherein the loading amount of the 3,3 ', 5, 5' -tetramethylbenzidine in the integrated composite nanoparticles is 700-800 mg/g.

9. Use of the integrated composite nanoparticle according to claim 1 or the integrated composite nanoparticle prepared by the preparation method according to claim 3 in glucose detection.

10. The method for detecting glucose as claimed in claim 8, comprising the steps of:

a) preparing a TMB-GOD-HRP solution: adding the integrated composite nanoparticles into a phosphate buffer solution, and performing ultrasonic treatment to obtain a stable white emulsion;

b) and (3) concentration detection: adding the TMB-GOD-HRP solution into the glucose solution, adding a surfactant, incubating for 1-4h, observing color change, detecting the change of absorbance by using a microplate reader, and judging the concentration of glucose in the sample according to a standard curve equation.

11. A glucose test strip or solution comprising the integrated composite nanoparticle according to claim 1 or the integrated composite nanoparticle prepared by the preparation method according to claim 3.

Technical Field

The invention belongs to the technical field of biosensing detection, relates to a self-assembly integrated nano composite material, a preparation method and application thereof, and particularly relates to an enzyme/chromogenic substrate signal molecule composite nano material prepared by the self-assembly method in an integrated manner, a preparation method and application thereof.

Background

Currently, the number of diabetic patients is continuously increasing worldwide, and it is estimated that 6.29 million people will be reached by 2045 years. According to recent research and research, the prevalence rate of diabetes of adults 18 years old and older in China is up to 11.6%, and the prevalence rate in the pre-diabetes period is up to 50.1% which is surprising. This means that 6 of every 10 adults in China had abnormal blood glucose. According to the proportion, the number of diabetic patients in China reaches 1.14 hundred million, and the number of diabetic patients in the early stage is close to 5 hundred million. Diabetes has become one of the most important and troublesome public health problems in China, so that rapid detection of glucose has great significance to human health.

At present, the traditional glucose colorimetric detection technology is adopted for rapid detection of glucose. The detection technology is generally based on two-step enzyme tandem reaction, namely glucose is oxidized into hydrogen peroxide by glucose oxidase, and then color change is generated by exogenously adding some chromogenic substrates (such as TMB, OPD, ABTS and the like) under the catalytic action of horseradish peroxidase to reflect the concentration of the glucose. Usually, these two steps are separated, which causes problems such as cumbersome steps, increased labor cost, and easy inactivation of enzyme.

Therefore, a rapid detection technique for glucose is needed to detect glucose or biomolecules with high sensitivity so as to obtain a rapid and simple detection result.

Disclosure of Invention

The invention aims to provide a self-assembly integrated nano composite material, which can be used for detecting glucose, wherein the nano composite material and the glucose generate a series reaction to cause a chromogenic substance to change color, so that the concentration of the glucose is judged according to the change degree of the color.

In order to achieve the purpose, the invention provides the following technical scheme: an integrated composite nanoparticle comprising the following components: 3,3 ', 5, 5' -tetramethylbenzidine, glucose oxidase and horseradish peroxidase.

The 3,3 ', 5, 5' -Tetramethylbenzidine (TMB), Glucose Oxidase (GOD) and horseradish peroxidase (HRP) form an integrated composite nanoparticle, namely TMB-GOD-HRP nanoparticles (TMB-GOD-HRP NPs), by intermolecular forces (hydrogen bonding and Van der Waals forces).

The loading amount of TMB in the TMB-GOD-HRP nano particles is 700-800 mg/g.

Preferably, the mass ratio of the glucose oxidase to the horseradish peroxidase is 1: 0.5-5.

The invention forms an integrated composite nanoparticle TMB-GOD-HRP NPs by using glucose oxidase, horseradish peroxidase and 3,3 ', 5, 5' -tetramethyl benzidine, the particle form of the TMB-GOD-HRP NPs and the detection reaction mechanism of the particle glucose are shown in figure 1, the nanoparticle can directly react with glucose, the glucose oxidase on the integrated composite nanoparticle firstly oxidizes the glucose into hydrogen peroxide, and then performs color development reaction with TMB molecules under the action of the horseradish peroxidase to change the color from colorless to blue-green, and the shade of the color, namely the absorbance, can positively correlate the concentration of the glucose in a reaction sample.

The glucose detection application of the nanoparticles can simplify the step of adding a chromogenic substrate in the traditional colorimetric glucose detection, and solves the problems of complex detection process, high detection cost and background interference in the existing glucose detection technology.

The invention also provides a preparation method of the TMB-GOD-HRP integrated composite nano particle, which comprises the following steps:

a) preparing a TMB solution: 3,3 ', 5, 5' -tetramethyl benzidine is dissolved by an organic solvent, and the concentration is 1 mg/mL-10 mg/mL;

b) preparing a GOD/HRP mixed solution: dissolving glucose oxidase and horse radish peroxidase in phosphate buffer solution;

c) mixing and reacting: injecting the TMB solution into the GOD/HRP mixed solution through a peristaltic pump at the temperature of 4 ℃, and continuing to react for 2-12 h after the injection is finished;

d) and (3) centrifugal freeze-drying: and centrifuging and washing the mixed solution after the reaction, taking the precipitate, and freeze-drying to obtain the integrated composite nanoparticles, namely TMB-GOD-HRP NPs.

When the hydrophobic TMB molecules are transferred from the organic phase to the aqueous phase, the TMB molecules can be spontaneously assembled into nano particles with uniform shapes under the hydrophobic action, and the nano particles are integrated with glucose oxidase and horseradish peroxidase in the aqueous phase through hydrogen bonds and van der Waals force.

Preferably, the organic solvent is one or more of dimethyl sulfoxide, methanol, ethanol, isopropanol, N-methylpyrrolidone or N, N-dimethylformamide.

Preferably, the glucose oxidase is derived from aspergillus niger or penicillium sp.

Preferably, the speed of the peristaltic pump is 0.05-0.2 mL/min.

Preferably, the speed of centrifugation is 1000-5000 r/min.

Preferably, the loading amount of the 3,3 ', 5, 5' -tetramethyl benzidine in the integrated composite nanoparticles is 700-800 mg/g.

The invention also provides an application of the integrated composite nano particles in glucose detection. The method for detecting the glucose comprises the following steps:

a) preparing a TMB-GOD-HRP solution: adding the integrated composite nanoparticles into a phosphate buffer solution, and performing ultrasonic treatment to obtain a stable white emulsion;

b) and (3) concentration detection: adding the TMB-GOD-HRP solution into the glucose solution, adding a surfactant, incubating for 1-4h, observing color change, detecting the change of absorbance by using a microplate reader, and judging the concentration of glucose in the sample according to a standard curve equation.

The TMB-GOD-HRP nano material with the high-load signal molecule TMB is prepared by utilizing the self-assembly function, and glucose is added to change the external condition, so that the stimulation signal molecule is rapidly released to generate a signal of color or fluorescence. The degree of the fluorescence signal and the stimulation is linearly related, so that the glucose concentration can be effectively determined.

The invention also provides glucose detection test paper or solution containing the integrated composite nanoparticles, so that the detection mode is more convenient, reagents do not need to be prepared on site, and the operation procedure is simplified.

The invention has the following advantages and beneficial effects:

1) the synthesized TMB-GOD-HRP effectively combines the TMB chromogenic substrate and two enzymes together, greatly simplifies the step of adding the chromogenic substrate in the traditional colorimetric detection, and can realize the instant detection of glucose.

2) The loading amount of TMB in the TMB-GOD-HRP nano particles is up to 700-800 mg/g, and the response signal of a detection system to glucose can be greatly improved, so that the accuracy, stability and sensitivity of detection are improved.

3) The invention utilizes the high specificity reaction of enzyme, thereby improving the high selectivity of the detection system.

4) The linear range of the glucose detection of the invention is 50 mu M-10mM (R)₂0.99), the detection limit can reach 15 μ M.

5) The method realizes the detection of the glucose by using the TMB-GOD-HRP nano particles, and has the advantages of mild reaction conditions, low cost, low detection limit, wide linear range, simple, quick and visual realization, high sensitivity and specificity detection and the like.

Drawings

FIG. 1 is a schematic diagram of the synthesis of an integrated composite nanoparticle TMB-GOD-HRP nanoparticle;

FIG. 2 is a schematic diagram of detection of glucose by TMB-GOD-HRP nanoparticles;

FIG. 3 is a schematic diagram of an ultraviolet-visible spectrum and color development for detecting glucose at different concentrations;

FIG. 4 is a fitted line graph of glucose sample concentration versus absorbance.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described with reference to the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.