CN103175884A - High-sensitivity glucose biosensor and preparation method thereof - Google Patents

Abstract

The invention relates to a high-sensitivity glucose biosensor and a preparation method thereof. The polished platinum electrode is placed in a mixed solution of K ₂ PdCl ₄ and sulfuric acid to obtain a platinum electrode modified by palladium nanoparticles, and glucose oxidase and bovine serum Dissolve albumin in phosphate buffer solution, then add glutaraldehyde solution and acid-treated single-walled carbon nanotube solution to mix to obtain an enzyme solution, dip the platinum electrode modified by palladium nanoparticles in the enzyme solution to obtain glucose biological sensor. The invention has the advantages of simple electrode fabrication and modification methods, high sensitivity, good stability and lower detection limit than other electrochemical glucose detection methods.

Description

A high-sensitivity glucose biosensor and its preparation method

technical field

The invention mainly relates to a biosensor and a preparation method thereof, belonging to the technical field of electroanalysis and chemical detection.

Background technique

Glucose is an extremely important substance in the organism. In recent years, there have been many literatures on the detection of glucose content, but many of their detection objects are serum, interstitial fluid and cell fluid. There are many methods widely used to detect glucose, such as spectrophotometry, fluorescence detection, chemiluminescence, electrochemical analysis, etc., and these methods have been successfully used in capillary electrophoresis to detect glucose.

The electrochemical analysis method has high sensitivity, less sample amount, simple instrument and relatively cheap price, so it has been widely used. In recent years, many electrochemical methods have been used to detect glucose. Electrochemical detection of glucose usually uses glucose dehydrogenase or glucose oxidase to make an enzyme biosensor. Enzyme biosensors use enzymes as biological sensitive elements, and capture the measurable signals that are proportional to the concentration of the target substance generated by the reaction between the target substance and the sensitive element through various physical and chemical signal converters. Analytical instruments for the quantitative determination of substances. Compared with other analytical methods, electrochemical biosensors have the advantages of portability, low cost, high sensitivity, and good stability. Coupled with the catalytic and sensitizing effects of CNTs themselves, enzyme biosensors based on CNTs have broad application prospects. .

Chemically modified electrodes are currently one of the most active research fields in electrochemistry and electroanalytical chemistry, endowing electrodes with certain properties, which can perform the desired reaction with high selectivity, and overcome the overpotential that occurs when unmodified electrodes are measured It can even be used to detect substances without electrical activity, and has been widely used in the field of analytical chemistry, especially in the preparation of biosensors. Commonly used chemically modified electrodes include Hg modified microelectrodes, chemically modified carbon paste microelectrodes, micro metal particles modified microelectrodes, surface molecular film modified microelectrodes, powder microelectrodes, enzyme electrodes, etc.

In the prior art, the mixture of TiO2-MWNTs-CS is modified on the glassy carbon electrode, then Prussian blue is electrodeposited on the surface of the modified electrode, and finally glucose oxidase and gold nanoparticles are adsorbed on the Prussian blue. Glucose biosensor based on titanium dioxide-multiwall carbon nanotubes-chitosan composite and sysfunctionalized gold nanoparticles[J].Bioprocess . Eng., 2011, 34:1143-1150.). There are Pt and Pd nanoparticles fixed on MWCNTs by electrodeposition to make glucose biosensors to detect glucose, (Cui H.F, Ye J.S, Zhang W.D, Li C.M, Luong J.H.T., Sheu F.S. Selective and sensitive electrochemical detection of glucose in neutral solution using platinum-lead alloy nanoparticle/carbon nanotubenanocomposites[J]. Analytica Chimica Acta2007,594:175-183.). In the literature (Reitz E, Jia W, Gentile M, Wang Y, Lei Y. CuO Nanospheres Based Nonenzymatic Glucose Sensor[J]. Electroanalysis, 2008, 20:2482.), the CuO nanospheres were immobilized on the glassy carbon electrode, and the Nafion membrane was prepared as an enzyme-free biosensor. These biosensors are either complicated to prepare, or have insufficient sensitivity and stability.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a high-sensitivity glucose biosensor and its preparation method. The method is simple and easy to make, and the prepared sensor has high sensitivity, low detection limit and strong anti-interference ability.

The technical scheme that the present invention takes is:

A high-sensitivity glucose biosensor comprises a palladium-platinum electrode, carbon nanotubes distributed on the surface of the palladium-platinum electrode and interwoven into a network structure, and glucose oxidase loaded on the electrode and the carbon nanotube.

A method for preparing a high-sensitivity glucose biosensor, comprising the following steps:

(1) Put the polished platinum electrode in the mixed solution of K ₂ PdCl ₄ and sulfuric acid, set the electrodeposition potential to -0.1 to -0.5V (vs. SCE), electrodeposit for 10-15s, rinse and dry to obtain Platinum electrodes decorated with palladium nanoparticles;

(2) Prepare enzyme solution: dissolve glucose oxidase and bovine serum albumin in phosphate buffer solution, then add glutaraldehyde solution and acid-treated single-wall carbon nanotube solution, mix the enzyme solution evenly and set aside The concentration of glucose oxidase in the enzyme solution is 908U/mL (according to the activity of glucose oxidase is 109U/mg, equivalent to 8.3mg/mL), the mass ratio of glucose oxidase and bovine serum albumin is 1:1-10, The concentration of glutaraldehyde in the enzyme solution is 8-10 mg/mL, the pH of the phosphate buffer solution is 6.0-8.0, and the concentration of the acid-treated single-wall carbon nanotube solution in the enzyme solution is 0.2-1.0 mg/mL;

(3) Dip the platinum electrode modified with palladium nanoparticles in the enzyme solution for 3-5 times, each dipping time is 10-15s.

In the mixed solution of K2PdCl4 and sulfuric acid described in step (1), the concentration of K2PdCl4 is 1×10 ^-3 mol/L, and the concentration of H ₂ SO ₄ is 0.5 mol/L. The preferred potential of the electrodeposition is set to -0.2V (vs. SCE), and the electrodeposition is 15s.

The preparation method of the acid-treated single-walled carbon nanotube solution described in step (2): Add each gram of single-walled carbon nanotubes into 400mL6mol/L nitric acid, heat and reflux for 6-6.5h, filter after cooling, and wash with double distilled water To neutrality, put it in an oven to dry, and then use double distilled water to make it into an aqueous solution with a concentration of 10mg/mL.

The phosphate buffer solution is prepared by preparing 0.2754～2.1202g Na ₂ HPO ₄ ·12H ₂ O and 0.0517～0.8553g NaH ₂ PO ₄ ·2H ₂ O in a 250mL volumetric flask.

The present invention forms very regular and orderly arranged palladium particles on the surface of the platinum electrode by electrochemical deposition, and the surface of the palladium-plated platinum electrode has many porous structures. The loading on the surface greatly increases the catalytic active sites, promotes the electron transfer rate, and improves the sensitivity of the current response. The surface of the electrode is relatively flat, and the carbon nanotubes on the surface of the sensor are cross-linked together to form a huge network structure, which is conducive to the conduction of electrons. The load has a certain contribution to the improvement of the current response sensitivity.

The palladium nanoparticles and carbon nanotubes on the surface of the sensor can promote electron transfer, increase the specific surface area of the electrode, enhance the catalytic activity, and improve the detection sensitivity. Utilizing the high selectivity of the enzyme in the biosensor and avoiding the interference of other substances, the rapid and accurate detection of glucose in human blood is realized. Meanwhile, the detection limit of the glucose sensor of the present invention is 5×10 ^-7 mol/L, which is low. The invention has the advantages of simple electrode fabrication and modification methods, high sensitivity, good stability and lower detection limit than other electrochemical glucose detection methods.

Description of drawings

Fig. 1 is the scanning electrochemical microscope picture of the palladium-platinum electrode of the present invention.

Fig. 2 is a scanning electrochemical microscope image of the glucose biosensor of the present invention.

Fig. 3 is a diagram of a device for capillary electrochemical detection of glucose in the present invention.

Fig. 4 is a detection cell for capillary electrochemical detection of glucose in the present invention.

Fig. 5 is the electrophoresis spectrogram of interfering substance mixed sample of the present invention, a-DA (0.2mM), b-glucose (0.6mM), c-cysteine (0.5mM), d-AA (0.5mM), e - UA (0.8 mM).

Fig. 6 is the electrophoretic spectrogram of glucose in human serum measured by the standard addition method of the present invention, a.0, b.0.6mmol/L glucose, c.1.0mmol/L glucose.

Among them, 1. Working electrode, 2. Reference electrode, 3. Counter electrode, 4. Quartz glass capillary, 5. Reference cell, 6. Detection cell, 7. Connecting tube, 8. High voltage power supply, 9. Sampling system, 10. Collection system, 11. Detection pool, 12. Capillary, 13. Negative pole, 14. Positive pole.

Detailed ways

Below in conjunction with embodiment further illustrate.

Embodiment 1 is the best embodiment

(1) Preparation of platinum electrode modified by palladium nanoparticles: A platinum wire with a diameter of 200 μm and a length of about 5 cm was inserted into a section of quartz capillary (250 μm ID, 375 μm O.D.) about 3 cm long, so that the quartz capillary Both ends of the platinum wire are exposed at the same time, one end is covered with epoxy resin glue, and slowly withdraw from the other end, so that the platinum wire is just exposed to the end of the capillary, and filled with epoxy resin glue. After 24 hours, the glue is cured. Cut a section of 5cm copper wire, smooth it on metallographic sandpaper, wind the exposed platinum wire on the copper wire, coat the winding place with conductive silver glue, put it in an oven at 70°C for 30 minutes, and finally use A glass tube (400 μm ID, 700 μm OD) about 5 cm long is placed at the interface between the platinum wire and the copper wire, and the two ends of the glass tube are glued with epoxy resin, and it can be used after 24 hours of drying. Polish the electrode smooth with fine metallographic sandpaper, so that the cross section of the platinum wire is flush with the cross section of the capillary tube. Ultrasonic the polished platinum electrode in secondary water, absolute ethanol, and secondary water for 5 minutes respectively, and place the cleaned platinum electrode in In the mixed solution of 1×10 ^-3 mol/L K ₂ PdCl ₄ and 0.5 mol/L H ₂ SO ₄ , the electrodeposition potential was set to -0.2V (vs. SCE), and the electrodeposition was 15s. After the electrodeposition is completed, the working electrode is rinsed with secondary water to obtain a palladium nanoparticle-modified microelectrode, which is then dried naturally at room temperature.

(2) Prepare enzyme solution: prepare enzyme solution: dissolve glucose oxidase and bovine serum albumin in phosphate buffer solution, then add glutaraldehyde solution and acid-treated single-walled carbon nanotube solution, and dissolve the enzyme solution Mix well and set aside; the mass ratio of glucose oxidase and bovine serum albumin is 1:5, the concentration of glucose oxidase in the enzyme solution is 8.3mg/mL, the concentration of glutaraldehyde in the enzyme solution is 9mg/mL, phosphoric acid The concentration of the salt buffer solution is 0.025mol/L, the pH is 7.4, the phosphate buffer solution is 1.8131g Na ₂ HPO ₄ 12H ₂ O and 0.1853g NaH ₂ PO ₄ 2H ₂ O in a 250mL volumetric flask, acid The concentration of the treated single-walled carbon nanotube solution in the enzyme solution is 0.5mg/mL;

(3) Dip the platinum electrode modified with palladium nanoparticles in the enzyme solution for 3 times, each dipping time is 10-15s.

Figure 1 and Figure 2 are scanning electron micrographs of the palladium-plated platinum electrode and the glucose biosensor, respectively. It can be seen from Figure 1 that the palladium nanoparticles electrochemically deposited on the surface of the platinum electrode are arranged in an orderly manner on the surface of the platinum electrode, which promotes the electron transfer rate and improves the sensitivity of the current response. It can be seen from Figure 2 that the surface of the electrode is relatively flat, and the carbon nanotubes on the surface of the sensor are cross-linked together to form a huge network structure, which is conducive to the conduction of electrons, and the large specific surface area of carbon nanotubes It also increases the load of the enzyme on the surface of the electrode, which contributes to the improvement of the sensitivity of the current response.

Example 2

Change the mass ratios of glucose oxidase and bovine serum albumin in step (2) to 1:1, 1:3, 1:8, and 1:10 respectively, and the other steps are the same as in Example 1.

Example 3

Change the concentration of the acid-treated single-walled carbon nanotube solution in the enzyme solution in step (2) to 0.2 mg/mL, 0.8 mg/mL, and 1.0 mg/mL, and other steps are the same as in Example 1.

Example 4

Change the concentration of glutaraldehyde in the enzyme solution in step (2) to 4 mg/mL, 13 mg/mL, and 17 mg/mL, and other steps are the same as in Example 1.

Capillary Electrophoresis Amperometric Detection of Glucose

(1) Instrument

Self-assembled capillary electrophoresis system, 0-30kV high-voltage power supply (Shandong Normal University Instrument Factory, Jinan); separation capillary (25μm I.D, 375μm O.D, length 60cm, Yongnian Ruifeng Chromatography Device Co., Ltd.); CHI832b electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.); filter (0.20 μm, Pall Corporation); three-electrode system: saturated calomel electrode (reference electrode), platinum electrode (counter electrode), palladium nanoparticle modified microelectrode (working electrode), metallographic sandpaper (Sandpaper WAW5 (06), Shanghai Grinding Wheel Factory, Shanghai); Ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu); Quartz sub-boiling high-purity water distiller (Jintan Jingbo Experimental Instrument Factory, Jiangsu); High-speed centrifuge (Jintan City Medical Instrument Factory, Jiangsu).

(2) Detection method steps and optimal conditions

Detection method steps: use the glucose sensor of Example 1, wash the capillary with sodium hydroxide solution, secondary water, and buffer solution before using the capillary, then use the three-dimensional manipulator to align the processed capillary with the working electrode, and adjust the high voltage to 18kV, conduct an experiment to see if the baseline current is stable, if the baseline current is stable, then adjust the high voltage to 5kV, inject the prepared glucose standard solution for 10s, adjust the voltage to 18kV, start the experiment, and get the electropherogram for analysis.

Optimum detection conditions: separation voltage is 18kV, detection voltage is -0.8V, concentration of buffer solution is 0.025mol/L, and pH is 7.4.

Reproducibility and detection limit

Under the above-mentioned detection method and optimal detection conditions, the relative standard deviations of the glucose peak current and migration time were 3.8% and 1.4%, respectively, when the 1×10 ^-3 mol/L glucose standard solution was measured in parallel 10 times. Under this experimental condition, when the signal-to-noise ratio S/N=3, the detection limit of glucose is 5×10 ^-7 mol/L. The invention has the advantages of simple experimental operation, electrode production and modification method, high sensitivity and good stability, and the detection limit is lower than other electrochemical detection glucose methods.

Table 1 Comparison of working electrode, linear range and detection limit for electrochemical detection of glucose by capillary electrophoresis

modified electrode[J].Talanta,2005,66:1281-1286.[4] Mattos I L, Areias M C C. Automated determination of glucose in soluble coffee using PrussianBlue-glucose

modified electrode[J].Talanta,2005,66:1281-1286.

[5] Tan X, Tian Y X, Cai P X, Zou X Y. Glucose biosensor based on glucose oxidase immobilized insol–gel chitosan/silica hybrid composite film on Prussian blue modified glass carbon electrode[J]. Analytical, Chemical and Bioanalytical 2005,381(2):500-507.

[6]Cui H F, Ye J S, Zhang W D, Li C M, Luong J H T, Sheu F S. Selective and sensitive electrochemical detection of glucose in neutral solution using platinum–leadalloy nanoparticle/carbon nanotube nanocomposites[J]. Chimica Acta,2007,594(2):175-183.

[7]Pang X Y,He D M,Luo S L,Cai Q Y.An amperometric glucose biosensor fabricatd with Ptnanoparticle decorated carbon nanotubes/TiO ₂ nanotubes arrays composite[J].Sensors and Actuators B,2009,137:134–138.

[8]Wang X, Zhang Y, Cheng C, Dong R, Hao J.Glucose in human serum determined by capillaryelectrophoresis with glucose micro-biosensor[J].Analyst,2011,136,1753-1759.

interference experiment

Actual samples often contain some electroactive substances such as ascorbic acid (AA), cysteine (L-Cys), uric acid (UA), etc. These substances will generate peak currents during the determination of glucose and may interfere with the detection of glucose , therefore, the interference determination of these possible substances is carried out, a mixed standard sample containing glucose, DA, L-Cys, AA, and UA is configured, and the mixed sample is detected simultaneously, and the obtained electrophoretic spectrum is shown in Figure 5. It can be seen from the figure that their migration time is different from that of glucose, and the electrophoretic peak can be well separated from the electrophoretic peak of glucose, so the detection of glucose will not be affected.

Detection of Glucose in Human Serum

The above-mentioned capillary electrophoresis electrochemical detection can be used for the detection of glucose in human blood samples. The concentration of glucose in the blood of two hypertensive and two normal people was detected respectively. The blood samples were diluted 100 times with 0.025mol/L PB, and then directly Carry out capillary sampling, measure the glucose content in 4 serum samples by standard addition method, the result of 1 sample among them is shown in Figure 6.

The result of the content determination of glucose in table 2 human serum

Sample 1, 3---serum of diabetic person; sample 2, 4---serum of healthy person.

It can be seen from Table 2 that the detection of glucose in blood by this method is basically consistent with the detection results of the hospital, but the overall result is slightly smaller, which may be because the blood sample is placed for a long time, resulting in a small amount of glucose decomposition.

Claims (6)

1. a high sensitivity glucose biological sensor, is characterized in that, comprises plating palladium platinum electrode, is distributed in plating palladium platinum electrode surface and hands over the carbon nano-tube that reticulates structure, and the glucose oxidase of electrode and the load of carbon nano-tube institute.

2. the preparation method of a high sensitivity glucose biological sensor, is characterized in that, comprises that step is as follows:

(1) the good platinum electrode of polishing is placed in K ₂PdCl ₄In the mixed solution of sulfuric acid, the electro-deposition current potential is made as-0.1 to-0.5V, and electro-deposition 10-15s rinses, dries and to get the platinum electrode modified of palladium nano-particles;

(2) preparation enzyme solutions: glucose oxidase and bovine serum albumin(BSA) are dissolved in phosphate buffered solution, and then add the Single Walled Carbon Nanotube solution of glutaraldehyde solution and acid treatment, mix this enzyme solutions stand-by; The mass ratio of glucose oxidase and bovine serum albumin(BSA) is 1:1-10, the concentration 8.3mg/mL of glucose oxidase in enzyme solutions, the concentration of glutaraldehyde in enzyme solutions is 8-10mg/mL, phosphate buffered solution pH6.0～8.0, the concentration of Single Walled Carbon Nanotube solution in enzyme solutions of acid treatment is 0.2-1.0mg/mL;

(3) platinum electrode of palladium nano-particles being modified dips 3-5 time in enzyme solutions, and the time that at every turn dips is 10～15s.

3. the preparation method of a kind of high sensitivity glucose biological sensor according to claim 2, is characterized in that, the described K of step (1) ₂PdCl ₄In the mixed solution of sulfuric acid, K ₂PdCl ₄Concentration is 1 * 10 ^-3Mol/L, H ₂SO ₄Concentration is 0.5mol/L.

4. the preparation method of a kind of high sensitivity glucose biological sensor according to claim 2, is characterized in that, described electro-deposition selects current potential to be made as-0.2V, electro-deposition 15s.

5. the preparation method of a kind of high sensitivity glucose biological sensor according to claim 2, it is characterized in that, the preparation method of the Single Walled Carbon Nanotube solution of the described acid treatment of step (2) adds every gram Single Walled Carbon Nanotube and adds hot reflux 6-6.5h in 400mL6mol/L nitric acid, cooled and filtered, be washed to neutrality with second distillation, put into baking oven dry, then with redistilled water, it be mixed with the aqueous solution that concentration is 10mg/mL..

6. the preparation method of a kind of high sensitivity glucose biological sensor according to claim 2, is characterized in that, phosphate buffered solution is every 0.2754～2.1202g Na ₂HPO ₄12H ₂O adds 0.0517～0.8553g NaH ₂PO ₄2H ₂O constant volume in the volumetric flask of 250mL makes.

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