CN102495047A - Method for determining trace glucose via fixing glucose oxidase through magnetic nanoparticle - Google Patents

Abstract

The invention discloses a method for measuring trace glucose by immobilizing glucose oxidase with magnetic nanoparticles. On the surface of the carbon paste electrode, the glucose oxidase fixed on the surface of the electrode is modified by magnetic nanoparticles to oxidize the glucose in the solution to generate hydrogen peroxide, and the hydrogen peroxide and luminol solution form an electrochemiluminescent system. The system generates very strong electrochemiluminescence signals and electrochemical signals under the action of electrode voltage. The intensity of the light signal is directly proportional to the concentration of glucose in the solution within a certain range. Based on this, an electrochemiluminescence analytical method for the determination of glucose was established. The cyclic voltammetry scan was carried out in the potential range of +0.2～+1.4V (vs.SCE), and the concentration of glucose in the range of 1×10-5～1.0×10-2mol/L had a good relationship with the peak intensity of electrochemiluminescence i _p linear relationship. The invention overcomes many disadvantages of the prior art such as being too complicated, has high sensitivity, and is easy to automate the detection of glucose.

Description

Method for Determination of Trace Glucose Using Magnetic Nanoparticles Immobilized Glucose Oxidase

technical field

The invention relates to a method for rapidly measuring trace glucose by using an electrochemiluminescent technology using magnetic nanoparticles to immobilize glucose oxidase, which can be used for measuring the glucose content in human blood.

Background technique

Glucose is an indispensable nutrient for the metabolism of living organisms. The heat released by its oxidation reaction is an important source of energy for human life activities. As the basic element of the human body and the most basic medical raw material, it has a wide range of functions and uses. It can be directly applied to the human body, and can also be used in food processing and pharmaceutical chemicals. It can rapidly increase human energy and endurance, and can be used as a supplement for hypoglycemia, cold and fever, dizziness, collapse, weakness of limbs and myocarditis, etc. It also has a certain therapeutic effect on cancer.

Realizing the rapid quantitative detection of glucose is of great significance in the fields of biochemistry, clinical chemistry and food analysis. So far, there have been many reports on glucose detection methods, such as electrochemical detection, surface-enhanced Raman scattering detection, photometric detection, chemiluminescence detection, and electroluminescence detection, etc. However, there is no report on the electroluminescent sensor for glucose oxidase (GOD) immobilized on magnetic nanoparticles. Electrochemiluminescence, also known as electrochemiluminescence, is a chemiluminescence phenomenon directly or indirectly induced by electrochemical reactions, and is a type of potential-controlled electrode oxidation-reduction reaction. The electrochemiluminescence analysis method developed in recent years is an analysis method combining electrochemical technology and chemiluminescence detection, which has the advantages of both: high sensitivity, wide linear range, and good controllability of reaction and time and space.

Contents of the invention

The purpose of the present invention is to provide a sensor with high sensitivity and good selectivity, and a method for measuring trace (eg 1 μmol/L) glucose.

The idea is as follows: the study found that the system of luminol and hydrogen peroxide can produce very strong electrochemiluminescence under the action of electrode voltage, and the concentration of luminol and hydrogen peroxide has a significant positive linear relationship to the luminescence. When the concentration of luminol is fixed and hydrogen peroxide is produced indirectly through the oxidation of glucose by glucose oxidase, the intensity of electrochemiluminescence is only linearly related to glucose within a certain concentration range. On this basis, magnetic nanoparticles are used to immobilize glucose oxidase ( GOD) established an electrochemiluminescence analysis method for the determination of trace glucose.

The invention relates to an enzymatic reaction and belongs to an enzyme sensor. When hydrogen peroxide is generated on the surface of the electrode, the luminol in the solution reacts electrochemically with it, thereby producing an excited state substance that can emit light of a certain wavelength and emit light. The peak intensity i _p of light is directly proportional to the concentration of glucose within a certain range.

Specific steps are as follows:

(1) Cut out the iron rod and the glass tube respectively, and grind and clean both ends of the iron rod and the glass tube; mix the solid paraffin and carbon powder in a mass ratio of 2 to 4:1, and heat slightly until the paraffin melts , stir evenly, and fill it into the glass tube; insert an iron rod into one end of the glass tube, fix it with paraffin, after cooling, remove the excess sundries outside the tube, and polish the surface on smooth paper to ensure sufficient magnetic force; The other end electrode end of the glass tube is a thin layer of paraffin carbon paste to obtain a solid paraffin carbon paste electrode; finally, the carbon paste electrode at one end of the thin layer of the glass tube paraffin carbon paste is sprayed with nitric acid and Wash with absolute ethanol, then rinse with double distilled water;

(2) Weigh FeCl ₃ 6H ₂ O and FeSO ₄ 7H ₂ O, dissolve them in twice distilled water at a molar ratio of Fe ²⁺ /Fe ³⁺ = 1:2 to obtain a mixed solution, and add a volume percentage of 25 % ammonia water to make the mixed solution pH = 9-12, stir at room temperature for 20-40 minutes, then raise the temperature to 80°C and keep the solution pH = 9-12, heat and mature for 20-40 minutes; the prepared black suspension is ultrasonically 10 ~30 minutes, under the magnetic separation, wash with hot water until neutral to obtain magnetic nano-Fe ₃ O ₄ particles;

(3) Weigh 45-55 mg of the magnetic nano Fe ₃ O ₄ particles obtained in step (2) and ultrasonically disperse them in 15-25 mL of absolute ethanol, and add 0.1 to 0.3 mL of 98% γ-aminopropane under stirring base triethoxysilane, stirred and reacted at room temperature for 9 to 20 hours, and the collected magnetic particles were ultrasonically cleaned with absolute ethanol and double-distilled water, respectively, and fixed to volume to obtain aminated magnetic nano-Fe ₃ O ₄ particles;

(4) hold the iron bar end of the solid paraffin carbon paste electrode that step (1) makes with magnet, get the magnetic nanometer Fe after the amination that step (3) makes ₃ O ₄ particles are added dropwise in step (1) After the surface of the prepared electrode is dried, add glutaraldehyde with a volume percentage concentration of 0.25% on the surface of the modified electrode, put it in a refrigerator at 4°C for 1 hour, rinse with water and dry it, and then add glucose oxidase dropwise solution, put it in a 4°C refrigerator overnight; before use, wash the electrode in agitated distilled water for 5 to 10 minutes; after each use, remove the magnet, rinse with distilled water, and wash away the magnetic nanocomposite glucose oxidase particles for Renew the electrode; store it in a 4°C refrigerator when the electrode is not in use;

(4) Detection method:

At room temperature, the scan rate is 50mV/s, the scan range is +0.2～+1.4V (vs. SCE), the high voltage of the photomultiplier tube is 600v, the sampling rate is 10T/S, the number of amplification stages is 3, and the measurement time is 60s for cyclic voltammetry Method test, measuring the intensity of electrochemiluminescence of different concentrations of glucose in 1mol/L sodium borate buffer solution containing 0.5mmol/L luminol at a pH of ^8.00 , and drawing a working curve; Within the concentration range of ×10 ^-2 mol/L, there is a good linear relationship with the peak intensity of electrochemiluminescence: i _p =65.4C+23.9, the correlation coefficient R=0.9987, and the detection limit is 1 μmol/L.

The invention overcomes many disadvantages of the prior art such as being too complicated, has high sensitivity, and is easy to automate the detection of glucose.

Description of drawings

Fig. 1 is a graph showing the relationship between the glucose content and the electrochemiluminescence peak intensity _ip in an example of the present invention.

Fig. 2 is the effect of the pH value of the solution of the embodiment of the present invention on the electrochemiluminescence peak intensity i _p .

Fig. 3 is a graph showing the relationship between the content of luminol and the peak intensity _ip of electrochemiluminescence in an embodiment of the present invention.

Detailed ways

Example:

(1) Cut out the iron rod and the glass tube respectively, and grind and clean both ends of the iron rod and the glass tube; mix the solid paraffin and carbon powder in a mass ratio of 3:1, heat slightly until the paraffin melts, and stir Evenly, fill it into the glass tube; insert an iron rod into one end of the glass tube, fix it with paraffin, after cooling, remove the excess debris outside the tube, and polish the surface on a smooth paper to ensure sufficient magnetic force; the glass tube The other end of the electrode end is a thin layer of paraffin carbon paste to obtain a solid paraffin carbon paste electrode; finally, the carbon paste electrode at one end of the thin layer of the glass tube paraffin carbon paste is treated with nitric acid and anhydrous water with a volume ratio of 1:1 before use. Washed with ethanol, then rinsed with double distilled water;

(2) Weigh FeCl ₃ 6H ₂ O and FeSO ₄ 7H ₂ O, dissolve them in twice distilled water at a molar ratio of Fe ²⁺ /Fe ³⁺ = 1:2 to obtain a mixed solution, and add a volume percentage of 25 % ammonia water to make the pH of the mixed solution = 11, stir at room temperature for 30 minutes, then heat up to 80°C and keep the pH of the solution = 11, heat and mature for 30 minutes; the prepared black suspension is ultrasonicated for 20 minutes, and separated by a magnet. Washing with hot water until neutral to obtain magnetic nano-Fe ₃ O ₄ particles;

(3) Weigh 48 mg of the magnetic nano Fe ₃ O ₄ particles obtained in step (2) and ultrasonically disperse them in 20 mL of absolute ethanol, and add 0.2 mL of 98% γ-aminopropyltriethoxysilane under stirring , stirred and reacted at room temperature for 12 hours, the collected magnetic particles were ultrasonically cleaned with absolute ethanol and double-distilled water respectively, and then fixed to volume to obtain aminated magnetic nano-Fe ₃ O ₄ particles;

(4) hold the iron bar end of the solid paraffin carbon paste electrode that step (1) makes with magnet, get the magnetic nanometer Fe after the amination that step (3) makes ₃ O ₄ particles are added dropwise in step (1) After the surface of the prepared electrode is dried, add glutaraldehyde with a volume percentage concentration of 0.25% on the surface of the modified electrode, put it in a refrigerator at 4°C for 1 hour, rinse with water and dry it, and then add glucose oxidase dropwise solution and placed in a 4°C refrigerator overnight. Before use, rinse the electrodes in agitated distilled water for 8 minutes. After each use, remove the magnet, rinse with distilled water, and wash away the magnetic nanocomposite glucose oxidase particles to refresh the electrodes. Store the electrodes in a 4°C refrigerator when not in use.

(4) Detection method:

At room temperature, select a scan rate of 50mV/s, a scan range of +0.2 to +1.4V (vs. SCE), a photomultiplier tube with a high voltage of 600v, a sampling rate of 10T/S, a magnification of 3, and a measurement time of 60s for cyclic voltammetry Test, measure the intensity of the electrochemiluminescence of different concentration glucose in the 0.1mol/L sodium borate buffer solution (pH 8.0) containing 0.5mmol/L luminol, draw working curve, its result is shown in accompanying drawing 1. Glucose has a good linear relationship with the electrochemiluminescence intensity in the concentration range of 1×10 ^-5 ~ 1.0×10 ^-2 mol/L: i _p =65.4C+23.9, the correlation coefficient R=0.9987, and the detection limit is 1 μmol/L L.

Effect of pH and temperature:

The ECL reaction of luminol needs to be carried out under alkaline conditions (pH 8.5-10.0). Considering that the biological activity of glucose oxidase is greatly affected by the pH value, this paper investigates the range of pH = 7.0-9.5. Changes in luminous intensity. When the concentration of luminol is 0.1mmol/L and the concentration of glucose is 1mmol/L, the results of the influence of the pH value are shown in Figure 2. It can be seen from the figure that when the pH=8.0, the ECL intensity of luminol is the largest, and the experiment was chosen to measure in the sodium borate buffer solution with pH=8.0.

At the same time, the catalytic activity of the enzyme also has a great relationship with the temperature. In the experiment, the temperature of the water bath was controlled by a heat-collecting constant temperature heating magnetic stirrer, and the current response of the enzyme electrode in the range of 20-60°C was investigated. It was found that when the temperature reached 40°C, the current The response is the largest, and the current tends to decrease as the temperature increases. Considering that the temperature is too high and the service life of the enzyme electrode is affected by the denaturation of glucose oxidase, the experiments are all carried out at room temperature of 25°C.

The effect of the concentration of luminol:

Keeping other conditions unchanged, the influence of the concentration of luminol on the intensity of electrochemiluminescence was investigated in the range of 0.01～1.2mmol/L. The results obtained are shown in Figure 3. It can be seen from the figure that when the glucose concentration is 1mmol/L, as the concentration of luminol increases from 0.01mmol/L to 0.5mmol/L, the ECL value of the solution increases rapidly. When the concentration of luminol is greater than 0.5mmol /L, the intensity of ECL tends to be stable. Therefore, the dosage of luminol in the experiment was 0.5mmol/L.

Application of electrodes in analytical testing:

The content of glucose in human serum was measured, and the results showed that the method was consistent with the results obtained by the clinical analysis method adopted by the hospital. In order to further verify the accuracy of the method, the standard addition method was used to measure the recovery rate of the samples, and the results are shown in Table 1. It can be seen that this method is used for the analysis and determination of clinical samples, and the results are satisfactory.

Table 1: Determination and recovery test results of glucose in human serum (n=5)

Claims (1)

1. method of measuring trace glucose is characterized in that concrete steps are:

(1) distinguishes intercepting iron staff and glass tube, and polish the two ends of iron staff and glass tube, clean; Is 2～4: 1 mixed with solid paraffin and carbon dust by mass ratio, is heated to melted paraffin wax slightly, stirs, and inserts in the glass tube; Iron staff is inserted in the end of glass tube, fixes, after the cooling, remove the outer unnecessary foreign material of pipe with paraffin, and on glossy paper polished surface, for guaranteeing enough big magnetic force; The other end electrode tip of glass tube is a paraffin carbon paste thin layer, makes the solid paraffin carbon paste electrode; It is that 1: 1 nitric acid and absolute ethyl alcohol cleans that one end carbon paste electrode of the thin layer of final glass pipe paraffin carbon paste uses volume ratio before use respectively, rinses well with redistilled water then;

(2) take by weighing FeCl ₃6H ₂O and FeSO ₄7H ₂O is with Fe ²⁺/ Fe ³⁺=1: 2 mol ratio is dissolved in and gets mixed solution in the redistilled water; Drip percent by volume and be 25% ammoniacal liquor and make mixed solution pH=9～12; Stirred under the room temperature 20～40 minutes, and be warmed up to 80 ℃ and keep pH value of solution=9～12 constant then, added thermal maturation 20～40 minutes; Ultrasonic 10～30 minutes of the black suspension of preparation under magnet separates, arrives the neutral magnetic Nano Fe of getting with hot wash ₃O ₄Particle;

(3) take by weighing the magnetic Nano Fe of 45～55mg step (2) gained ₃O ₄Particle is ultrasonic to be dispersed in the 15-25mL absolute ethyl alcohol; Stirring down, adding 0.1～0.3mL concentration expressed in percentage by volume is 98% gamma-aminopropyl-triethoxy-silane; Stirring reaction is 9～20 hours under the room temperature, and the magnetic particle of collection gets the magnetic Nano Fe after the amination with constant volume after absolute ethyl alcohol and the redistilled water ultrasonic cleaning respectively ₃O ₄Particle;

The iron staff end of the solid paraffin carbon paste electrode that (4) makes with magnet attracts step (1) is got the magnetic Nano Fe after the amination that step (3) makes ₃O ₄Particle drips the electrode surface that makes in step (1), after drying, drips concentration expressed in percentage by volume and be 0.25% glutaraldehyde on the modified electrode surface; Put into 4 ℃ of refrigerators after 1 hour; With water wash and dry up, drip glucose oxidase solution then, put into 4 ℃ of refrigerator overnight; Before the use, place electrode the distilled water of stirring to clean 5～10 minutes; After each the use, remove magnet, use distilled water flushing, the compound glucose oxidase enzyme particle of flush away magnetic Nano is so that upgrade electrode; Electrode do not put into 4 ℃ of refrigerators and preserve by the time spent;

(4) detection method: under the room temperature, choosing sweep speed is 50mV/s, sweep limit is+0.2～+ 1.4V (vs.SCE); Photomultiplier high pressure 600v; Sampling rate 10T/S, number of stages of amplification 3, Measuring Time 60s carry out the cyclic voltammetry test; Measure variable concentrations glucose and be the intensity of electrogenerated chemiluminescence of the 1mol/L sodium borate buffer solution of 8.00., the drawing curve at the pH that contains the 0.5mmol/L luminol; Glucose is 1.0 * 10 ^-5～1.0 * 10 ^-2Be good linear relationship with the electrogenerated chemiluminescence peak intensity in the mol/L concentration range: i _p=65.4C+23.9, coefficient R=0.9987 is detected and is limited to 1 μ mol/L.

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